Analysis of ENOD40 expression in alb1, a symbiotic mutant of Lotus japonicus that forms empty nodules with incompletely developed nodule vascular bundles.

The alb1 mutant of Lotus japonicus (Ljsym74) forms empty nodules in which most of the bacteria remain in abnormally enlarged infection threads and fail to enter the host plant cells. The alb1 mutant was also found to be defective in differentiation of ramified nodule vascular bundles; only a single vascular bundle differentiates at the proximal end of the alb1 nodules and it fails to differentiate further. Histochemical analysis using fluorescein-conjugated wheat-germ agglutinin (F-WGA) indicated that the mutation in the ALB1 gene specifically affects the differentiation of vascular bundles in nodules. Analysis of nodulin gene expression revealed that the expression of an early nodulin gene, ENOD40, was very low in alb1 nodules. At early developmental stages of alb1 nodules, the pattern of ENOD40 transcription was essentially the same as that in wild-type nodules; transcripts were localized in dividing cortical cells and in the pericycle of the root stele opposite nodule primordia, as in wild-type nodules. However, mature alb1 nodules exhibited very weak or no expression of ENOD40 in the peripheral cells of the undeveloped nodule vascular bundle. The ENOD40 expression pattern in alb1 nodules is distinct from that in another ineffective mutant, fen1 (Ljsym76), in which ENOD40 expression persists prior to premature senescence. These findings lead us to speculate that ENOD40 may play a role in the differentiation of nodule vascular bundles.

PIS1, a negative regulator of the action of auxin transport inhibitors in Arabidopsis thaliana.

In order to clarify the mechanism underlying the polar auxin transport system, the pis1 mutant in Arabidopsis thaliana that is hypersensitive to N-1-naphthylphthalamic acid (NPA), an auxin transport inhibitor was isolated and characterized. Whereas the pis1 mutant is normally sensitive to phytohormones, auxins, cytokinin and ethylene precursor, this mutant is hypersensitive to NPA over the broad spectrum of its effects such as growth of seedlings, root elongation, root gravitropism, root phototropism and root curling. This result indicates that the pis1 mutant is specifically affected in the polar auxin transport system. This result also defines a genetic factor controlling both gravitropism and phototropism, and strongly indicates the involvement of auxin transport during both tropic responses. NPA, 2,3,5-triiodobenzoic acid (TIBA) and 9-hydroxyfluorene-9-carboxylic acid (HFCA) represent different classes of auxin transport inhibitors. The pis1 mutation conferred hypersensitivity to both NPA and TIBA but not to HFCA. These results show the genetic separation of the actions of NPA/TIBA and of HFCA. The PIS1 gene product might be specifically involved in the response pathway of NPA/TIBA, leading to interference with auxin-efflux carriers, and might act as a negative regulator of the action of NPA/TIBA.

The excessive production of indole-3-acetic acid and its significance in studies of the biosynthesis of this regulator of plant growth and development.

Because of the importance of indole-3-acetic acid (IAA) in the growth and development of plants, extensive studies of the biosynthesis of IAA have been performed during the four decades since the discovery of IAA as a plant hormone. The pathway for the biosynthesis of IAA in plants remains, however, to be unelucidated, even though studies within the past decade have revealed unexpected aspects of such biosynthesis. By contrast, two pathways to IAA have been characterized in bacteria at the molecular level: the indole-3-acetamide (IAM) pathway (L-tryptophan-->IAM-->IAA); the indole-3-pyruvic acid pathway (L-tryptophan-->indole-3-pyruvic acid-->indole-3-acetaldehyde-->IAA) (Fig. 1). In both pathways, the details of the biosynthesis of IAA were clarified using IAA-overproducing bacteria. After a description of recent advances of the studies of the biosynthesis of IAA in plants, this review focuses on the excessive production of IAA in several organisms and its significance in the studies of the biosynthesis of IAA.

Genetic analysis of the effects of polar auxin transport inhibitors on root growth in Arabidopsis thaliana.

Polar auxin transport inhibitors, including N-1-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), have various effects on physiological and developmental events, such as the elongation and tropism of roots and stems, in higher plants. We isolated NPA-resistant mutants of Arabidopsis thaliana, with mutations designated pir1 and pir2, that were also resistant to TIBA. The mutations specifically affected the root-elongation process, and they were shown ultimately to be allelic to aux1 and ein2, respectively, which are known as mutations that affect responses to phytohormones. The mechanism of action of auxin transport inhibitors was investigated with these mutants, in relation to the effects of ethylene, auxin, and the polar transport of auxin. With respect to the inhibition of root elongation in A. thaliana, we demonstrated that (1) the background level of ethylene intensifies the effects of auxin transport inhibitors, (2) auxin transport inhibitors might act also via an inhibitory pathway that does not involve ethylene, auxin, or the polar transport of auxin, (3) the hypothesis that the inhibitory effect of NPA on root elongation is due to high-level accumulation of auxin as a result of blockage of auxin transport is not applicable to A. thaliana, and (4) in contrast to NPA, TIBA itself has a weak auxin-like inhibitory effect.

The regulatory functions of the rolB and rolC genes of Agrobacterium rhizogenes are conserved in the homologous genes (Ngrol) of Nicotiana glauca in tobacco genetic tumors.

To compare patterns of expression between the Ngrol genes of N. glauca and the Rirol genes of Agrobacterium rhizogenes, we performed fluorometric and histochemical analysis of transgenic genetic tumors on the hybrid of Nicotiana glauca x N. langsdorffii (F1) that harbored a beta-glucuronidase (GUS) reporter gene fused to the promoter of NgrolB, NgrolC, RirolB or RirolC. The promoters of NgrolB and NgrolC had 2- to 3-fold lower activity than those of RirolB and RirolC. However, the changes in patterns of GUS activity caused by deletion of NgrolB and NgrolC promoters were similar to those of RirolB and RirolC promoters. This result suggests that the cis-acting sequences that regulate the level of expression of RirolB and RirolC are conserved in the NgrolB and NgrolC promoters. Furthermore, an auxin dependent (NAA-dependent) increase in GUS activity was observed in the case of NgroB-GUS and RirolB-GUS. Histochemical analysis showed GUS activity encoded by both NgrolB-GUS and RirolB-GUS in normal-type F1 transgenic plants was located in meristematic zones, while that encoded by NgrolC-GUS and RirolC-GUS was detected mainly in vascular systems of various organs. Thus, the patterns of expression of the Ngrol genes were the same as those of the Rirol genes in terms of promotion by auxin and tissue-specificity, indicating that regulatory mechanisms for both sets of genes have been conserved during the evolution of the genus Nicotiana after transfer from a progenitor of Agrobacterium to that of Nicotiana.

Involvement of L-tryptophan aminotransferase in indole-3-acetic acid biosynthesis in Enterobacter cloacae.

L-Tryptophan aminotransferase (L-tryptophan:2-oxoglutarate aminotransferase; EC 2.6.1.27) from Enterobacter cloacae was purified 62-fold and characterized to determine its role in indole-3-acetic acid biosynthesis. The enzyme reversibly catalyzed the transamination of L-tryptophan with 2-oxoglutarate as the amino acceptor to yield indole-3-pyruvic acid and L-glutamate, and the Km values for L-tryptophan and indole-3-pyruvic acid were 3.3 mM and 24 microM, respectively. In the indole-3-acetaldehyde synthesis experiments in vitro, 94% of L-tryptophan was efficiently converted to indole-3-acetaldehyde by the purified L-tryptophan aminotransferase plus indolepyruvate decarboxylase. Furthermore, the amounts of L-tryptophan decreased with increases in the indolepyruvate decarboxylase activity, while the amounts of indole-3-acetaldehyde increased with increases in this activity. In genetic experiments, the amounts of L-tryptophan produced by Enterobacter and Pseudomonas strains harboring the gene for indolepyruvate decarboxylase were lower than those produced by these same strains without the gene, while the amounts of indole-3-acetic acid produced by Enterobacter and Pseudomonas strains harboring the gene for indolepyruvate decarboxylase were higher than those produced by these same strains without the gene. These results clearly show that L-tryptophan aminotransferase is involved in the indole-3-acetic acid biosynthesis and that indolepyruvate decarboxylase is the rate-limiting step in this pathway.

Sequence of the cellular T-DNA in the untransformed genome of Nicotiana glauca that is homologous to ORFs 13 and 14 of the Ri plasmid and analysis of its expression in genetic tumours of N. glauca x N. langsdorffii.

A region homologous to the TL-DNA of Agrobacterium rhizogenes was previously detected in the genome of untransformed Nicotiana glauca and designated cellular T-DNA (cT-DNA). Subsequently, part of this region was sequenced and two genes, which corresponded to rolB and rolC and were named NgrolB and NgrolC, were found. We have now sequenced a region of the cT-DNA other than the region that includes NgrolB and C and we have found two other open reading frames (ORFs), NgORF13 and NgORF14. These ORFs correspond to ORFs 13 and 14 of the TL-DNA of A. rhizogenes and exhibit a high degree of homology to these ORFs, without having a nonsense codon. We have not found any sequence homologous to rolD (ORF15). The two genes, NgORF13 and 14, as well as the NgrolB and C genes, are expressed in genetic tumors of hybrids between N. glauca and N. langsdorffii but not in leaf tissues of the hybrid.

Cloning of cDNAs for genes that are specifically or preferentially expressed during the development of tobacco genetic tumors.

To identify genes involved in the formation of genetic tumors in interspecific hybrids (F1) between Nicotiana glauca and N. langsdorffii, genetic tumor-related cDNA probes were obtained by a subtractive hybridization procedure and used to screen libraries of genetic tumor cDNAs. As a result, 17 distinct cDNA clones were isolated for genes that are specifically or preferentially expressed in genetic tumor tissues but are not expressed at all or are barely expressed in normal F1 plants. Among the isolated clones were genes that encoded so-called stress proteins, such as glucan endo-1,3-beta-glucosidase, osmotin, pathogenesis-related proteins and proteinase inhibitor I. Transcripts corresponding to two of the isolated cDNA clones accumulated to a significant extent only in genetic tumor tissues and were not present in callus tissues from parental plants or in the stems and leaves from normal F1 plants. Analysis of genomic DNA revealed that four of these clones hybridized only to genomic sequences from N. langsdorffii and one hybridized only to a genomic sequence from N. glauca. Eight apparently novel clones were further analyzed to determine the kinetics of accumulation of the corresponding mRNAs during development of genetic tumors. The patterns of accumulation of the mRNAs after induction of tumors by cutting of F1 stems could be divided into three groups, an indication that at least three distinct regulatory mechanisms are operative at the transcriptional level to control the expression of these tumor-related genes during the formation of genetic tumors.

Isolation and characterization of a cDNA that encodes a novel proteinase inhibitor I from a tobacco genetic tumor.

We have isolated a cDNA clone, designated GTI, by screening a tobacco genetic tumor cDNA library with a tumor-specific "subtracted" cDNA probe. The cDNA contained the entire coding sequence for a 94-amino-acid polypeptide that exhibited significant homology to members of the proteinase inhibitor I family from tomato and potato. The predicted protein has a pre-sequence of 22 amino acids but lacks a pro-sequence, unlike genes for proteinase inhibitor I isolated to date. Furthermore, the protein encoded by GTI cDNA has a novel reactive site, having glutamine as the P1 reactive residue. These results suggest that the GTI protein is a novel member of the proteinase inhibitor I family. The mRNA for GTI accumulated at a high level but only transiently after the wounding of tobacco plants. Thus, it appears that the GTI protein has a function that is related to the protection of tissues against damage due to wounding.

Evidence for the expression of the rol genes of Nicotiana glauca in genetic tumors of N. glauca X N. langsdorffii.

Thus far, no evidence has been presented that the rol genes (Ng rol) of Nicotiana glauca (Furner et al. 1986) are expressed in this plant. However, we found that the Ng rol genes were transcribed in genetic tumors formed in hybrids of N. glauca X N. langsdorffii. During the culture of such genetic tumors, the level of transcription of Ng rol B increased while that of Ng rol C decreased in parallel with an increase in the endogenous auxin level in the tissues. Moreover, the transcription of these genes was completely suppressed by the application of exogenous auxin. Since the measured endogenous level of auxin was rather low, the formation of tumors in these hybrids can be tentatively explained as a consequence of the expression of rol genes that is responsible for the increased sensitivity to auxin of the hybrids. This novel finding is discussed in relation to the cause of genetic tumors in Nicotiana.

Molecular cloning of a gene for indole-3-acetamide hydrolase from Bradyrhizobium japonicum.

A pLAFR1 cosmid genomic library of wild-type Bradyrhizobium japonicum J1063 was constructed. A cosmid clone designated pBjJ4, containing a 26-kilobase (kb) DNA insert, was identified as being able to confer the ability to convert alpha-naphthaleneacetamide acid on B. japonicum J1B7 Rifr, which cannot perform this conversion. The gene coding for the enzyme that converts alpha-naphthaleneacetamide to alpha-naphthaleneacetic acid was localized in the 3.5-kb region of pBjJ4 by recloning in plasmid pSUP202. The gene coding for the enzyme was also mapped by Tn5 insertion mutagenesis to a region of ca. 2.3 kb. When the gene was placed behind the lacZ promoter and used to transform Escherichia coli, a high level of expression of indole-3-acetamide hydrolase activity was found. Since there have been no reports of this activity in E. coli, we have thus confirmed that the gene cloned here is a structural gene for indole-3-acetamide hydrolase and have designated it as the bam (Bradyrhizobium amidehydrolase) gene. Southern hybridization with the central region of the bam gene indicated that a high degree of similarity exists among the bam gene, the iaaH gene from Pseudomonas savastonoi, and the tms-2 gene from Agrobacterium tumefaciens. The result suggests that there is a common origin for the gene that encodes the enzyme that catalyzes the biosynthesis of indoleacetic acid.

Light-Induced Alkalinization of the Suspending Medium of Guard Cell Protoplasts from Vicia faba L.

The light-dependent pH changes in the suspending medium of guard cell protoplasts (GCP) from Vicia faba were studied. Upon illumination, the medium was initially slightly alkalinized and then acidified. The extent of alkalinization was lower in CO(2)-free air than in normal air. This initial alkalinization was inhibited by DCMU. Acidification in CO(2)-free air became observable in shorter duration of light exposure than that in normal air. The rate of acidification was higher in CO(2)-free air than in normal air. The CO(2) level of the medium decreased in the light, and increased in the dark. (14)CO(2) uptake was enhanced 2- to 3-fold by light, but not in the presence of DCMU. These results indicate that photosynthetic CO(2) fixation does take place in GCP and that the initial alkalinization is due to this photosynthetic CO(2) uptake. Diethylstilbestrol, a nonmitochondrial membrane-bound ATPase inhibitor, inhibited the acidification, suggesting that the acidification resulted from H(+) extrusion by GCP. The acidification in light was also prevented by KCN, and partly by DCMU. Possible mechanisms of alkalinization and acidification are discussed in relation to guard cell metabolism.

Light Activation of NADP-Malate Dehydrogenase in Guard Cell Protoplasts from Vicia faba L.

Light-induced swelling of guard cell protoplasts (GCP) from Vicia faba was accompanied by increases in content of K(+) and malate. DCMU inhibited the increase of K(+) and malate, and consequently swelling.Effect of light on the activity of selected enzymes that take part in malate formation was studied. When isolated GCP were illuminated, NADP-malate dehydrogenase (NADP-MDH) was activated, and the activity reached a maximum within 5 minutes. The enzyme activity underwent 5- to 6-fold increase in the light. Upon turning off the light, the enzyme was inactivated in 5 minutes NAD-MDH and phosphoenolpyruvate carboxylase (PEPC) were not influenced by light. The rapid light activation of NADP-MDH was inhibited by DCMU, suggesting that the enzyme was activated by reductants from the linear electron transport in chloroplasts. An enzyme localization study by differential centrifugation indicates that NADP-MDH is located in the chloroplasts, NAD-MDH in the cytosol and mitochondria, and PEPC in the cytosol. After light activation, the activity of NADP-MDH in guard cells was 10 times that in mesophyll cells on a chlorophyll basis. The physiological significance of light-dependent activation of NADP-MDH in guard cells is discussed in relation to stomatal movement.

Further evidence for the transformation ofVinca rosea protoplasts byAgrobacterium tumefaciens spheroplasts.

Protoplasts ofVinca rosea were transformed by spheroplasts ofAgrobacterium tumefaciens harboring nopalinetype Ti plasmids according to the procedure of Hasezawa et al. (1981). These transformants frequently differentiated tracheids, but further differentiation to teratomata has not so far been observed. Transformation was confirmed by the improved detection of nopaline synthase, where the sensitivity and specificity of the enzyme reaction was increased by employing(14)C-α-ketoglutaric acid and(3)H-arginine as substrates. The nopaline synthase activity was identified by the comigration of these two radioisotopes in the cnromatogram. Furthermore, the T-DNA structure of one of these transformants was examined by Southern hybridization according to Thomashow et al. (1980) and compared with that ofVinca rosea crown gall.

Introduction of Escherichia coli cells and spheroplasts into Vinca protoplasts.

In the presence of 10% polyvinyl alcohol (PVA), Escherichia coli cells or spheroplasts can be easily introduced into Vinca protoplasts by endocytosis. Uptake proceeded quite rapidly; bacterial cells or spheroplasts were found within the cytoplasm of Vinca protoplasts after 10 min of incubation with PVA.

Changes in endogenous cytokinin levels in partially synchronized cultured tobacco cells.

During the course of partially synchronized cell divisions in cultured tobacco (Xanthi) cells the amount of endogenous cytokinins in the butanol-soluble fraction increased 5 to 10 times in 3 hours and paralleled the increase in frequency of mitosis. Among three cytokinins detected in tobacco cells, the activity corresponding to the R(F) of authentic zeatin in thin layer chromatography changed in parallel with the mitotic index.

Identification of Cytokinins of Root Nodules of the Garden Pea, Pisum sativum L.

Five cytokinin activities which induced soybean callus proliferation were detected in ethanol extracts of root nodules of the garden pea (Pisum sativum L., cv. Little Marvel). The most active factors among them were identified as zeatin and its riboside on the basis of their mobility on thin layer chromatography in three solvent systems. Smaller activities of zeatin ribotide, isopentenyladenine and its riboside were also detected. Cytokinin activity gradually decreased with the cultivation period, but no qualitative change in the active compounds was found.