SD3, an Arabidopsis thaliana homolog of TIM21, affects intracellular ATP levels and seedling development.

It is poorly understood how plants control their growth by cell division, elongation, and differentiation. We have characterized a seedling-lethal mutant segregation distortion 3 (sd3) that showed a very dwarf phenotype when grown in the light and, in the dark, had short hypocotyls with reduced ploidy levels. The corresponding gene of SD3 encodes a protein with high similarity to yeast translocase on the inner mitochondrial membrane 21 (TIM21), which is a component of the TIM23 complex. Indeed, SD3 protein fused to GFP localized in the mitochondria. SD3 overexpression increased cotyledon size in the light and hypocotyl thickness in the dark. The expression of genes for several subunits of the respiratory-chain complexes III and IV was up-regulated in SD3-overexpressing plants. Furthermore, these plants showed high levels of ATP whereas those of sd3 were low. These results suggested that SD3 induced an increase in cell size by raising the expression of the respiratory-chain subunit genes and hence increased the intracellular ATP levels. We propose that intracellular ATP levels regulated by mitochondria control plant organ size.

Homologous chromosome pairing is completed in crossover defective atzip4 mutant.

In transposon-tagged lines of Arabidopsis, we found a mutant that was defective in meiotic chromosome segregation. This mutant, named atzip4-4, was due to a novel mutant allele of AtZIP4, which has sequence similarity to yeast ZIP4/SPO22, which codes a ZMM protein that is a proposed unit of the synapsis initiation complex. The chiasma distribution in atzip4-4 differed from that in the wild-type, involved in a deficiency of interfering crossovers in the mutant genome. On the other hand, FISH staining of loci on two independent chromosomes in mutant meiocytes indicated that homologous chromosome pairing to synapse progresses normally until the pachytene stage, yet homologous chromosomes often separated abruptly at diplotene and diakinesis. These results suggest that AtZIP4 plays an important role in normal crossover formation and meiotic chromosome segregation, but not in homolog search. The relationship of AtZIP4 and other related proteins in meiotic events is discussed and compared with that in yeast.

Cytological and biochemical analysis of COF1, an Arabidopsis mutant of an ABC transporter gene.

In transposon-tagged lines of Arabidopsis we found two new mutants, cof1-1 and cof1-2 (cuticular defect and organ fusion), that show the phenotype of wilting when grown in soil, organ fusion of rosette leaves and infertility. Toluidine blue testing and scanning electron microscopy observation revealed that these mutants had cuticular defects in the stems and adult leaves, but not in cotyledones. Transmission electron microscopy observation revealed thinner cuticle layers in the mutants, and cuticular materials interspersed between the two fused epidermal layers were observed in the mutant rosette leaves. These two mutants had a transposon insertion in the coding regions of WBC11, which was classified as a member of ABC transporter genes in Arabidopsis. WBC11 showed high sequence similarity to CER5 (also called WBC12), which was involved in cuticular lipid export. Gas chromatographic analysis revealed that C29 alkane extracted from the stem surface of cof1 mutants was reduced whereas C29 ketone was accumulated, which was different from the case of cer5 mutants. While cer5 mutants had fairly normal morphology, cof1 mutants had pleiotropic phenotypes so that COF1/WBC11 could have important roles different from those of CER5/WBC12. We also found that C29 alkane was accumulated in the intracellular extract of cof1 mutants, suggesting a function for WBC11 in the direct transport of lipid molecules. Pollen observation showed that mutant pollen grains were irregularly shaped. The function of COF1/WBC11 in lipid transport for the construction of cuticle layers and pollen coats for normal organ formation is discussed.

Loss of Necrotic Spotted Lesions 1 associates with cell death and defense responses in Arabidopsis thaliana.

We isolated a lesion mimic mutant, necrotic spotted lesions 1 (nsl1), from Ds-tagged Arabidopsis thaliana accession No-0. The nsl1 mutant exhibits a growth retardation phenotype and develops spotted necrotic lesions on its rosette and cauline leaves. These phenotypes occur in the absence of pathogens indicating that nsl1 mutants may constitutively express defense responses. Consistent with this idea, nsl1 accumulates high levels of callose and autofluorescent phenolic compounds localized to the necrotic lesions. Furthermore RNA gel blot analysis revealed that genes associated with disease resistance activation are upregulated in the nsl1 mutants and these plants contain elevated levels of salicylic acid (SA). Crossing nsl1 with an SA deficient mutant, eds16-1, revealed that the nsl1 lesions and growth retardation are dependent upon SA. The nsl1 phenotypes are not suppressed under either the rar1-10 or sgt1b-1 genetic background. NSL1 encodes a novel 612aa protein which contains a membrane-attack complex/perforin (MACPF) domain, which is conserved in bacteria, fungi, mammals and plants. The possible modes of action of NSL1 protein in negative regulation of cell death programs and defense responses are discussed.

A trial of phenome analysis using 4000 Ds-insertional mutants in gene-coding regions of Arabidopsis.

Mutant lines covering all Arabidopsis genes allow us to pursue systematic functional genomics. A comprehensive phenotype description, called a phenome, is highly sought after in the profiling of -omics data. We selected 4000 transposon-insertional lines with transposon insertions in their gene-coding regions, and systematically observed the visible phenotype of each line. For the first 3 weeks after germination, plants were grown on agar plates and the juvenile phenotypes were recorded. Then the plants were transferred to soil and their phenotypes were recorded at each growth stage. About 140 lines showed clear and reproducible visible phenotypes, including novel phenotypic mutants as well as previously reported ones. All descriptions of the mutants showing visible phenotypes were classified into eight primary categories (seedling, leaves, flowering and growth, stems, branching, flowers, siliques and seed yield) and 43 secondary categories of morphological phenotypes. Phenotypic images have been entered into a searchable database (http://rarge.gsc.riken.jp/phenome/). One example investigated through the use of plural alleles was a mutant of a novel gene related to glycerolipid biosynthesis, with a unique visible phenotype of sepal opening. Our results suggest that we can find more novel visible phenotypes and their corresponding genes, and that phenotypic mutants of gene knockouts are not exhausted yet. This study provides basic data on large-scale phenotyping of gene knockout lines in plants, and will contribute to the completion of an international effort to develop a phenome database of all the functional genes in Arabidopsis.