Kunihiko Syono

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).

Expression of a foreign gene in callus derived from DNA-treated protoplasts of rice (Oryza sativa L.)

SummaryProtoplasts isolated from suspension cultures of rice cells were treated with bacterial plasmid DNA carrying a chimaeric gene consisting of the nopaline synthase promoter, the aminoglycoside phosphotransferase II (APH(3′)II) structural gene from bacterial transposon Tn5 and the terminator region from cauliflower mosaic virus DNA. Colonies capable of proliferating in medium containing kanamycin (100 μg/ml) were selected. A transformation frequency of approximately 2% to 3% was recorded in several experiments. The enzyme (APH(3′)II) was also detected in kanamycin-resistant callus, which had survived after repeated selection. There was some variation in the APH(3′)II activity in the transformants which paralleled the copy number of the inserted genes.

Transformation of Vinca protoplasts mediated by Agrobacterium spheroplasts

SummaryVinca rosea protoplasts and Agrobacterium tumefaciens spheroplasts harboring octopine-type Ti plasmid were mixed and treated with polyethylene glycol or polyvinyl alcohol, which facilitated the introduction of spheroplasts into plant protoplasts. After the protoplasts had been kept at 40° C for 4 days, bacteria were found to be completely eliminated from the medium. Among treated protoplasts 1–2 per 1,000 formed colonies on the Murashige and Skoog medium (1962) lacking hormones. When the colonies were isolated and subcultured, they could be maintained as clones. Octopine, an amino acid specific to crown gall, was detected in half of these clones. The phenotypic features of these putative transformants were compared but did not show any coincidental tendencies in relation to color, hardness, form, growth rate, or octopine production. The significance of this system in transformation of higher plant cells is discussed.

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.

Rearrangement of Cortical Microtubules in Elongating Cells derived from Tobacco Protoplasts — A Time-course Observation by Immunofluorescence Microscopy —

Summary Protoplasts derived from a tobacco cell culture (BY-2), Nicotiana tabacum L. cv. Bright Yellow, could be elongated by cultivation in a medium containing α-naphthaleneacetic acid and benzyladenine (Hasezawa and Syono, 1983). The transition of cortical microtubules (MTs) in the elongating cells were closely observed by immunofluorescence microscopy. The arrays of MTs were random in the protoplasts, but they were gradually rearranged with cell elongation to form lines at right angles to the elongating axis. After 10 days of culture, the MT arrays became sparse and began to slant towards the axis. Finally, MT arrays collapsed and the cells lost dividing abilities. The significance of this system in the study of the cytoskeleton of higher plant cells will be discussed.

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

Abstract. 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.

Evidence for the expression of the rol genes of Nicotiana glauca in genetic tumors of N. glauca x N. langsdorflli

SummaryThus 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.

Changes of Actin Filaments and Cellulose Fibrils in Elongating Cells Derived from Tobacco Protoplasts

Summary Using single cells derived from tobacco protoplasts, changes of actin filaments (AFs) and cellulose fibrils (CFs) in relation to the arrangement of cortical microtubules (MTs) were observed by fluorescent microscopy. The pattern of AFs were random in the protoplasts. After 3 days of culture, winding thick filaments of AFs run along the axis in the central cytoplasm of elongating cells. In the cortical cytoplasm, arrays and meshes of fine AFs were also observed. These arrays seemed to follow the transition of cortical MTs, which became rearranged from a random pattern to arrays at right angles to the axis of the cells during cell elongation (Hasezawa et al., 1988a). The CFs of reformed cell wall showed the pattern of arrays at right angles to the axis similar to those of MTs from the first to the 7th day of culture.

Cell-free biosynthesis of cytokinins in cultured tobacco cells

Summary Cell-free biosynthesis of free cytokinins not related to the degradation of t-RNA was demonstrated by using enzyme preparation extracted from cultured tobacco ( Nicotiana tabacum cv. Xanthi) cells. The reaction mixture for cytokinin biosynthesis contained 20 mM Tris-HCl buffer (pH 7.6), 5 mM magnesium acetate, 0.5 mM isopentenylpyrophosphate, 50 μ M adenosine as a substrate and dialyzed enzyme extract. After 2.5 hours of reaction at 27°C, N 6 -( Δ 2 -isopentenyl)adenosine, N 6 -( Δ 2 -isopentenyl)adenine and trans-zeatin were biosynthesized. The radioactivity of [U- 14 C]adenosine was incorporated into these three cytokinins after 30 minutes of reaction in the cell-free system. From the time course of biosynthesis of cytokinins and the incorporation of [U- 14 C]adenosine into cytokinins, we concluded that adenosine accepts the isopentenyl group directly to form N 6 -( Δ 2 -isopentenyl) adenosine. This is converted to trans-zeatin via N 6 -( Δ 2 -isopentenyl)adenine by several enzymes.