SummaryFlavonoid compounds such as anthocyanins and proanthocyanidins (PAs; so-called condensed tannins) have a multitude of functions in plants. They must be transported from the site of synthesis in the cytosol to their ®nal destination, the vacuoles. Three models have been proposed for sequestering anthocyanins in vacuoles, but the transport machinery for PAs is poorly understood. Novel Arabidopsis mutants, transparent testa 19 (tt19), which were induced by ion beam irradiation, showed a great reduction of anthocyanin pigments in the vegetative parts as well as brown pigments in the seed coat. The TT19 gene was isolated by chromosome walking and a candidate gene approach, and was shown to be a member of the Arabidopsis glutathione S-transferase (GST) gene family. Heterologous expression of a putative ortholog, petunia anthocyanin 9 (AN9 ), in tt19 complemented the anthocyanin accumulation but not the brown pigmentation in the seed coat. This suggests that the TT19 gene is required for vacuolar uptake of anthocyanins into vacuoles, but that it has also a function different from that of AN9. The depositional pattern of PA precursors in the mutant was different from that in the wild type. These results indicate that TT19 participates in the PA pathway as well as the anthocyanin pathway of Arabidopsis. As involvement of GST in the PA pathway was previously considered unlikely, the function of TT19 in the PA pathway is also discussed in the context of the putative transporter for PA precursors.

After bacterial invasion, ubiquitin is conjugated to host endosomal proteins and recognized by the autophagic machinery independent of LC3.

Since the isolation and characterization of dwarf1-1 (dwf1-1) from a T-DNA insertion mutant population, phenotypically similar mutants, including deetiolated2 (det2), constitutive photomorphogenesis and dwarfism (cpd ), brassinosteroid insensitive1 (bri1), and dwf4, have been reported to be defective in either the biosynthesis or the perception of brassinosteroids. We present further characterization of dwf1-1 and additional dwf1 alleles. Feeding tests with brassinosteroid-biosynthetic intermediates revealed that dwf1 can be rescued by 22␣-hydroxycampesterol and downstream intermediates in the brassinosteroid pathway. Analysis of the endogenous levels of brassinosteroid intermediates showed that 24-methylenecholesterol in dwf1 accumulates to 12 times the level of the wild type, whereas the level of campesterol is greatly diminished, indicating that the defective step is in C-24 reduction. Furthermore, the deduced amino acid sequence of DWF1 shows significant similarity to a flavin adenine dinucleotide-binding domain conserved in various oxidoreductases, suggesting an enzymatic role for DWF1. In support of this, 7 of 10 dwf1 mutations directly affected the flavin adenine dinucleotide-binding domain. Our molecular characterization of dwf1 alleles, together with our biochemical data, suggest that the biosynthetic defect in dwf1 results in reduced synthesis of bioactive brassinosteroids, causing dwarfism.T-DNA-insertion mutagenesis has proven to be useful for the isolation of many important genes controlling plant growth and development (Choe and Feldmann, 1998). The Arabidopsis dwarf1 (dwf1) mutant was originally isolated from a T-DNA mutant population, and was the first mutant shown to cosegregate with the selectable marker in the T-DNA (Feldmann et al., 1989). The dwf1 mutant was identified because of its short stature, dark-green leaves, reduced fertility, and robust stems when grown in the light. Physiologically, dwf1 was not rescued by any of the known growth-promoting phytohormones such as GA 3 or auxin (Feldmann et al., 1989). Using the plant DNA flanking the T-DNA as a probe, DWF1 was cloned and sequenced (accession no. U12400).Independently, Takahashi et al. (1995) isolated a morphologically similar mutant, diminuto (dim), from a different T-DNA mutant collection. Cloning and sequencing revealed that dim is disrupted in the DWF1 sequence, indicating that it is an allele of dwf1. One year later, Kauschmann et al. (1996) isolated another allele of DWF1 from a transposon-tagged population. They identified three tiny mutants named cabbage1, cabbage2, and cabbage3 (cbb1, cbb2, and cbb3). Altmann et al. (1995) found the sequence of genomic DNA flanking the transposon in cbb1 to be identical to that of DWF1. Kauschmann et al. (1996) originally found that cbb1 (dwf1-6) could be rescued by exogenous application of brassinosteroids, suggesting that cbb1 (dwf1-6) is defective in brassinosteroid biosynthesis. They also analyzed the expression of genes known to be involved in cell elongation, such as ␥-tonoplast intri...

Recently, heavy ions or ion beams have been used to generate new mutants or varieties, especially in higher plants. It has been found that ion beams show high relative biological effectiveness (RBE) of growth inhibition, lethality, and so on, but the characteristics of ion beams on mutation have not been clearly elucidated. To understand the effect of ion beams on mutation induction, mutation rates were investigated using visible known Arabidopsis mutant phenotypes, indicating that mutation frequencies induced by carbon ions were 20-fold higher than by electrons. In chrysanthemum and carnation, flower-color and flower-form mutants, which are hardly produced by gamma rays or X rays, were induced by ion beams. Novel mutants and their responsible genes, such as UV-B resistant, serrated petals and sepals, anthocyaninless, etc. were induced by ion beams. These results indicated that the characteristics of ion beams for mutation induction are high mutation frequency and broad mutation spectrum and therefore, efficient induction of novel mutants. On the other hand, PCR and sequencing analyses showed that half of all mutants induced by ion beams possessed large DNA alterations, while the rest had point-like mutations. Both mutations induced by ion beams had a common feature that deletion of several bases were predominantly induced. It is plausible that ion beams induce a limited amount of large and irreparable DNA damage, resulting in production of a null mutation that shows a new mutant phenotype.

SummaryThe brassinosteroid (BR) biosynthetic pathway, and the sterol pathway which is prerequisite to the BR pathway, are rapidly being characterized because of the availability of a large number of characteristic dwarf mutants in Arabidopsis. Here we show that the Arabidopsis dwarf5 mutants are disrupted in a sterol D 7 reduction step. dwf5 plants display the characteristic dwarf phenotype typical of other BR mutants. This phenotype includes small, round, dark-green leaves, and short stems, pedicels, and petioles. Metabolite tracing with 13 C-labeled precursors in dwf5 veri®ed a de®ciency in a sterol D 7 reductase activity. All six independent alleles contain loss-of-function mutations in the sterol D 7 reductase gene. These include a putative mRNA instability mutation in dwf5-1, 3¢ and 5¢ splice-site mutations in dwf5-2 and dwf5-6, respectively, premature stop codons in dwf5-3 (R400Z) and dwf5-5 (R409Z), and a mis-sense mutation in dwf5-4 (D257N). The dwf5 plant could be restored to wild type by ectopic overexpression of the wild-type copy of the gene. Both the Arabidopsis dwf5 phenotype and the human Smith±Lemli±Opitz syndrome are caused by loss-of-function mutations in a sterol D 7 reductase gene, indicating that it is required for the proper growth and development of these two organisms.