Arabidopsis PIS1 encodes the ABCG37 transporter of auxinic compounds including the auxin precursor indole-3-butyric acid.

Differential distribution of the plant hormone auxin within tissues mediates a variety of developmental processes. Cellular auxin levels are determined by metabolic processes including synthesis, degradation, and (de)conjugation, as well as by auxin transport across the plasma membrane. Whereas transport of free auxins such as naturally occurring indole-3-acetic acid (IAA) is well characterized, little is known about the transport of auxin precursors and metabolites. Here, we identify a mutation in the ABCG37 gene of Arabidopsis that causes the polar auxin transport inhibitor sensitive1 (pis1) phenotype manifested by hypersensitivity to auxinic compounds. ABCG37 encodes the pleiotropic drug resistance transporter that transports a range of synthetic auxinic compounds as well as the endogenous auxin precursor indole-3-butyric acid (IBA), but not free IAA. ABCG37 and its homolog ABCG36 act redundantly at outermost root plasma membranes and, unlike established IAA transporters from the PIN and ABCB families, transport IBA out of the cells. Our findings explore possible novel modes of regulating auxin homeostasis and plant development by means of directional transport of the auxin precursor IBA and presumably also other auxin metabolites.

Morphological effects of sinefungin, an inhibitor of S-adenosylmethionine-dependent methyltransferases, on Anabaena sp. PCC 7120.

Anabaena cells develop regular one-dimensional filaments through cell division in planes parallel to each other. A gcvP mutant displayed morphological defects such as filaments with sharp bends and/or branching and irregular cell clumps. The defects probably result from depletion of S-adenosylmethionine (AdoMet), because they were rescued by the application of methionine, an AdoMet precursor, and because sinefungin, a strong inhibitor of AdoMet-dependent methyltransferases, caused morphological abnormalities in wild-type Anabaena similar to those of the mutant. AdoMet-dependent methylation is involved in the spatial regulation of cell polarity in Anabaena.

Genetic variations in rice in vitro cultures at the EPSPs-RPS20 region.

In vitro cultures of plant cells have often been utilized to generate genetic variations, which are designated somaclonal variations. Little is known about the major genetic alterations in the cultured cells and the nature of these genetic changes. Here, we examined different lines of rice Oc cells that have been cultured for more than 20 years on agar media or in liquid media. We surveyed 35 clones obtained from PCR amplification of the 3-kb EPSPs-RPS20 region. The sequence divergence among the Oc cells was even greater than that between Japonica and Indica rice cultivars. The divergent sequences appeared to be maintained as multiple copies in a single cell. Surprisingly, the nucleotide substitutions in the Oc cells were characterized by an extremely high frequency of transition mutations of A/T-to-G/C, a feature which is similar to that of the mutations caused by chemical mutagens such as 5-bromouracil and 2-aminopurine. Although no replacements in the exons of this region were observed among the AA-genome Oryza species, our results revealed that the nucleotide substitutions of the cultured cell lines occurred more frequently at replacement sites in the exons than at synonymous sites. These distinct mutation biases found in rice in vitro cultures might contribute importantly to somaclonal variations.

Expression analysis of the NgORF13 promoter during the development of tobacco genetic tumors.

We investigated the expression pattern of the promoter of Nicotiana glauca (Ng) ORF13 in the hybrids between N. glauca and N. langsdorffii harboring the NgORF13-beta-glucuronidase (GUS) chimeric gene. The promoter of NgORF13 of N. glauca had lower activities than the promoter of RiORF13 of Agrobacterium rhizogenes agropine-type root-inducing (Ri) plasmid. However, the localization of GUS activity in the NgORF13 transgenic plants was similar to that in the RiORF13 transgenic plants. The GUS activity of NgORF13-GUS was high in genetic tumors cultured in vitro or developed spontaneously on F1 plants with aging or by wounding. The GUS activity in tumors was observed in bud primordia, vascular bundles and leaves in the buds. While the activity was lower than in tumors, NgORF13-GUS was also expressed in vascular bundles and the parenchymatous tissues in plants regenerated from tumors. Furthermore, the promoter activity of NgORF13 was induced by wounding and activated by exogenous application of methyl jasmonate. During tumorization, NgORF13 was induced at an early stage and showed expression patterns similar to both NgrolB and NgrolC whose expression were investigated by Nagata et al. (1996) Plant Cell Physiol. 37: 489-498. It is thought that Ngrol genes might be involved in the formation of genetic tumors, and, moreover, NgORF13 might work in cooperation with NgrolB and NgrolC.

Partial purification of an enzyme hydrolyzing indole-3-acetamide from rice cells.

The activity of indole-3-acetamide (IAM) hydrolase from rice cells was enriched ca. 628-fold by gel filtration and anion exchange column chromatography. The molecular masses of the IAM hydrolase estimated by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis were approximately 50.5 kD and 50.0 kD, respectively. The enzyme exhibited maximum activity at pH 6.0-6.5. The enzyme was stable against heat treatments between 4 and 50 degrees C and works optimally at 52 degrees C. The activity remained constant at 4 degrees C for at least 143 days. The purified enzyme fraction hydrolyzed indoleacetic acid ethyl ester (Et-IAA) in addition to IAM and its homologue, 1-naphthalene-acetamide, but not indole-3-acetonitrile. Km values of the enzyme were 0.96 mM and 0.55 mM for IAM and Et-IAA, respectively. Although the molecular mass of the enzyme was very similar to that of IAM hydrolase of Agrobacterium tumefaciens involved in tumor formation, the biochemical properties of the enzyme including its high Km value were considerably different from those of the A. tumefaciens enzyme. Based on these enzyme properties, we will discuss whether the amidohydrolase is involved in auxin biosynthesis in rice cells.

Isolation and characterization of four cell wall purple acid phosphatase genes from tobacco cells.

Four full-length cDNAs were isolated from a cDNA library prepared from tobacco cultured cells and designated NtPAP4, NtPAP12, NtPAP19 and NtPAP21, which could correspond to purple acid phosphatase (PAP). Levels of both NtPAP12 and NtPAP21 mRNA in the protoplasts immediately increased after the protoplasts were transferred to a medium for cell wall regeneration, and the accumulation of the mRNA was correlated with cell wall regeneration for 3 h. It is likely that the NtPAP12 and NtPAP21 gene products are wall-bound PAPs at the early stage of regenerating walls in tobacco protoplasts.

Root, root hair, and symbiotic mutants of the model legume Lotus japonicus.

To gain an overview of plant factors controlling nodule number and organogenesis, an extensive screening using model legume Lotus japonicus was carried out. This screening involved 40,000 M2 seeds, and 32 stable mutant lines were isolated. From these, 16 mutant lines maintaining the phenotypic variation were selected and genetically analyzed. With respect to nodule number, four loci were identified, Ljsym77, Ljsym78, slippery root (slp), and radial organization1 (rdo1). The former two mutants have an increased number of nodules, while the latter two have a decreased number. Ljsym78-1 and Ljsym78-2 are hypernodulating mutants with a branched root system and were found to be allelic to Ljsym16. The phenotype of the Ljsym77 mutant was highly pleiotropic, being deficient in light and gravity responses. The slp mutant was isolated as a low-nodulating mutant lacking root hairs. Concerning nodule organogenesis, nine symbiotic loci were identified, including the two loci alb1 and fen1. Mutants affecting the developmental process of nodule organogenesis were placed in three phenotypic categories: Nod- (Ljsym70 to Ljsym73), Hist- (alb1-1, alb1-2, and Ljsym79), and Fix- (fen1, Ljsym75, and Ljsym81).