Two types of pea leghemoglobin genes showing different O2-binding affinities and distinct patterns of spatial expression in nodules.

Five distinct cDNA clones for leghemoglobin (Lb) were isolated from pea (Pisum sativum) nodules. They were classified into two groups designated PsLbA and PsLbB according to sequence homology, O(2)-binding affinities of the recombinant proteins, and in situ localization of the mRNAs. The PsLbB group was comprised of four cDNA clones: PsLb120-1, -8, -29, and -34. They showed a high similarity of deduced amino acid sequences and O(2)-binding affinities of their recombinant proteins. Among them, the spatial expression pattern of PsLb120-1 was investigated in great detail, indicating that its transcripts were localized in the region from infection zone II to the distal part of nitrogen fixation zone III in effective nodules. PsLb5-10, which is the only cDNA clone of the PsLbA type, differed considerably from the PsLbB type in amino acid sequence, and the recombinant protein had a higher O(2)-binding affinity than those of the PsLbB type. The transcripts of PsLb5-10 were detected throughout the central tissue of effective nodules. However, in ineffective nodules on the pea mutant E135 (sym13), transcripts of PsLb5-10 were restricted to the distal portion of the central tissue as well as those of PsLb120-1. These findings indicate that the pea genome contains two types of Lb genes and suggest that they have different roles in the development of nitrogen-fixing symbiosis in pea nodules.

Over-expression of tobacco knotted1-type class1 homeobox genes alters various leaf morphology.

We compared the phenotypes of transgenic tobacco plants over-expressing various knotted1-type class1 homeobox genes. All transformants showed abnormal leaf morphology, with the degree of abnormality depending upon the Nicotiana tabacum homeobox (NTH) gene that was over-expressed. Tobacco plants over-expressing NTH1 or NTH9 showed a relatively weak phenotype, while NTH15 and NTH20 over-expressing plants exhibited severe alterations, with occasional ectopic shoot formation on the leaves. Plants over-expressing NTH22 had a relatively severe phenotype, but did not form any ectopic shoots. These results indicate that all of the NTH genes can influence leaf development from the shoot apical meristem, but that the effect varies with the gene. Based on phylogenetic analysis of the NTH genes and comparison of the phenotypes of plants over-expressing them, we suggest that the kn1-type class1 family can be divided into two subgroups, and that the differences in their ability to induce the abnormal phenotype corresponds to the structures of their conserved domains.

Role of malate synthesis mediated by phosphoenolpyruvate carboxylase in guard cells in the regulation of stomatal movement.

To clarify the pathway and role of malate synthesis in guard cells, epidermal strips isolated from Vicia faba L. leaflets were treated with 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate (DCDP), a specific inhibitor of phosphoenolpyruvate carboxylase (PEPC). When dark-closed stomata were illuminated, malate accumulated in guard cells and stomata opened; these were inhibited by 60% and 30%, respectively, by 5 mM DCDP treatment. When light-opened stomata were treated with DCDP, both malate level in guard cells and stomatal aperture decreased. Treatment with 5 mM DCDP partially inhibited CO2 incorporation into malate in guard cells. Treatment with mannitol at 0.4 M (osmotic stress) in the light increased malate level in guard cells and closed stomata. DCDP treatment decreased both malate level and stomatal aperture under stressed condition. These results show that malate synthesis in the light under both non-stressed and stressed conditions is dependent on PEPC activity. The extent of the decrease in malate level by DCDP treatment was larger under stressed condition than under nonstressed condition, suggesting that osmotic stress may enhance the activity of this pathway of malate synthesis which is induced by light. Role of malate synthesis in guard cells is discussed.

Transgenic tobacco over-expressing a homeobox gene shows a developmental interaction between leaf morphogenesis and phyllotaxy.

The tobacco gene, NTH1, encodes a polypeptide of 326 amino acids and is a member of the class1 KN1-type family of homeobox genes. Expression of NTH1 has mainly been observed in vegetative and reproductive shoot apices, not observed in roots or expanded leaves. Over-expression of NTH1 in transgenic plants caused abnormal leaf morphology, consisting of wrinkling and curvature. Interestingly, the direction of leaf curvature tended to be conserved among almost all of the leaves in any given transformant. In transgenic plants exhibiting clockwise or anticlockwise phyllotaxy, leaves curved to the right or left, respectively, when looking from the shoot apex toward the base. Micro-surgical experiments demonstrated that the presence of the shoot apex is necessary for the development of leaf curvature, indicating that the order of formation of leaves on the stem (the generative spiral) affects leaf development. We found a correlation between the severity of leaf curvature and the value of the plastochron ratio, a parameter of phyllotaxy. Transformants with more severe phenotypes had larger plastochron ratios. From these findings, we discuss the possibility that an increase in the plastochron ratio, caused by over-expression of NTH1 in the shoot apex, may be involved in leaf curvature.

The conserved KNOX domain mediates specificity of tobacco KNOTTED1-type homeodomain proteins.

Overproduction of the tobacco KNOTTED1-type homeodomain proteins NTH1, NTH15, and NTH23 in transgenic tobacco plants causes mild, severe, and no morphological alterations, respectively. The deduced amino acid sequences of the homeodomains and adjacent ELK domains are highly conserved, and the N-terminal KNOX domains also are moderately conserved. To investigate the contributions of both the conserved and divergent regions to the severity of morphological alterations, we generated chimeric proteins by exchanging different regions of NTH1, NTH15, and NTH23. The severity of the abnormal phenotype was dependent upon the synergistic action of both the N terminus, containing the KNOX domain, and the C terminus, containing the ELK homeodomain. Detailed analysis focusing on the C terminus revealed that the C-terminal half of the ELK domain is more effective in inducing the abnormal phenotypes than are the homeodomains. For the N terminus, severe morphological alterations were induced by exchanging a part of the KNOX domain of NTH1 with the corresponding region of NTH15. This limited region in the KNOX domain of all homeodomain proteins includes a predicted alpha-helical region, but only that in NTH15 is predicted to form a typical amphipathic structure. We discuss the possibility, based on these results, that the secondary structure of the KNOX domain is important for the induction of abnormal morphology in transgenic tobacco plants.

The expression of tobacco knotted1-type class 1 homeobox genes correspond to regions predicted by the cytohistological zonation model.

We have isolated and characterized four tobacco homeobox genes, NTH1, NTH9, NTH20, NTH22 (Nicotiana tabacum homeobox) which belong to the class 1 knotted1-type family of homeobox genes. Comparison of the inferred amino acid sequences of the ELK homeodomains of these genes and previously reported kn1-type class 1 proteins has revealed that the four new tobacco genes belong to distinct subclasses, suggesting that each NTH gene may have distinct functions. Using in situ hybridization and by analysing the distribution of GUS activity in tobacco plants transformed with NTH promoter::GUS constructs, localized expression of the three NTH genes was observed in the shoot apical meristem (SAM). In the vegetative SAM, NTH1 and NTH15 showed overlapping expression in the corpus, NTH20 was expressed in the peripheral zone, and NTH9 was predominantly expressed in the rib zone. The expression patterns of the different NTH genes correspond to regions predicted by the cytohistological zonation model, suggesting that each NTH gene specifies the function of the SAM zone with which it is associated.

The homeobox gene NTH23 of tobacco is expressed in the basal region of leaf primordia.

We reported isolation and characterization of a homeobox gene from tobacco, NTH23. The homeodomain structure of NTH23 was highly homologous to the same regions of class 2 genes of the KN1-type homeobox (sharing more than 85% amino acid identity), but was less similar to class 1 genes of KN1-type. RNA gel blot analysis revealed that NTH23 was expressed in all organs we tested although the gene is primarily expressed in young leaves. To determine more precisely the spatial expression pattern of NTH23 in tobacco, a chimeric NTH23::GUS fusion gene was introduced into tobacco. The signal of GUS activity was observed at the basal part of leaf blade primordia in the NTH23::GUS transgenic tobacco plants. This observation suggests the possibility that NTH23 may be important for the lateral growth of leaf blades.

Alteration of hormone levels in transgenic tobacco plants overexpressing the rice homeobox gene OSH1.

The rice (Oryza sativa L.) homeobox gene OSH1 causes morphological alterations when ectopically expressed in transgenic rice, Arabidopsis thaliana, and tobacco (Nicotiana tabacum L.) and is therefore believed to function as a morphological regulator gene. To determine the relationship between OSH1 expression and morphological alterations, we analyzed the changes in hormone levels in transgenic tobacco plants exhibiting abnormal morphology. Levels of the plant hormones indole-3-acetic acid, abscisic acid, gibberellin (GA), and cytokinin (zeatin and trans-zeatin [Z]) were measured in leaves of OSH1-transformed and wild-type tobacco. Altered plant morphology was found to correlate with changes in hormone levels. The more severe the alteration in phenotype of transgenic tobacco, the greater were the changes in endogenous hormone levels. Overall, GA1 and GA4 levels decreased and abscisic acid levels increased compared with wild-type plants. Moreover, in the transformants, Z (active form of cytokinin) levels were higher and the ratio of Z to Z riboside (inactive form) also increased. When GA3 was supplied to the shoot apex of transformants, internode extension was restored and normal leaf morphology was also partially restored. However, such GA3-treated plants still exhibited some morphological abnormalities compared with wild-type plants. Based on these data, we propose the hypothesis that OSH1 affects plant hormone metabolism either directly or indirectly and thereby causes changes in plant development.

Ectopic expression of a tobacco homeobox gene, NTH15, dramatically alters leaf morphology and hormone levels in transgenic tobacco.

The shoot apical meristem functions to generate the lateral organs of a plant throughout the vegetative and reproductive phases. Homeobox genes play key roles in controlling such developmental programs, but their modes of action have not been well defined. Here we describe isolation and biological functions of a novel tobacco homeobox gene, designated NTH15 (Nicotiana tabacum homeobox 15), from a tobacco shoot apex cDNA library. NTH15 encodes a polypeptide of 342 amino acids, its homeodomain is very similar to the class 1 KNOTTED-type homeodomains. NTH15 mRNA is mainly localized in corpus cells in the tobacco shoot apical meristem, but not in tunica layers nor in differentiated lateral organs. The NTH15 cDNA was fused to the cauliflower mosaic virus 35S promoter and used to generate transgenic tobacco plants. Almost all transgenic tobacco plants showed abnormal leaf and/or flower morphology, and were categorized into three groups depending on severity of the leaf phenotype. In transgenic leaves, drastic decrease of GA1 and increase of cytokinin were observed, while the levels of other phytohormones were only slightly changed. Taken together, our results suggest NTH15 is involved in tobacco morphogenesis and abnormal leaf morphology in transgenic plants results from altered hormone levels.

Isolation and characterization of cDNA clones with enhanced expression in tobacco plants expressing the rice homeobox gene, OSH1.

We have used subtractive hybridization to isolate cDNA clones whose expression were up-regulated in transgenic tobacco ectopically expressing the rice homeobox gene, OSH1. Thirty-nine distinct cDNA clones, which we term HRGs (Homeobox Regulated Genes), were identified. Some of them were specifically expressed in transformants, indicating that their expression was possibly regulated by transgene.

Abnormal cell divisions in leaf primordia caused by the expression of the rice homeobox gene OSH1 lead to altered morphology of leaves in transgenic tobacco.

Transgenic tobacco plants were generated carrying a rice homeobox gene, OSH1, controlled by the promoter of a gene encoding a tobacco pathogenesis-related protein (PR1a). These lines were morphologically abnormal, with wrinkled and/or lobed leaves. Histological analysis of shoot apex primordia indicates arrest of lateral leaf blade expansion, often resulting in asymmetric and anisotrophic growth of leaf blades. Other notable abnormalities included abnormal or arrested development of leaf lateral veins. Interestingly, OHS1 expression was undetectable in mature leaves with the aberrant morphological features. Thus, OSH1 expression in mature leaves is not necessary for abnormal leaf development. Northern blot and in situ hybridization analyses indicate that PR1a-OSH1 is expressed only in the shoot apical meristem and in very young leaf primordia. Therefore, the aberrant morphological features are an indirect consequence of ectopic OSH1 gene expression. The only abnormality observed in tissues expressing the transgene was periclinal (rather than anticlinal) division in mesophyll cells during leaf blade initiation. This generates thicker leaf blades and disrupts the mesophyll cell layers, from which vascular tissues differentiate. The OSH1 product appears to affect the mechanism controlling the orientation of the plane of cell division, resulting in abnormal periclinal division of mesophyll cell, which in turn results in the gross morphological abnormalities observed in the transgenic lines.

Two transcripts with different sizes derived from a rice homeobox gene, OSH1.

A rice homeobox gene, OSH1, contains two functionally independent promoters which generate a larger transcript and a smaller transcript. In Arabidopsis, each promoter can drive the expression of a reporter gene in a different manner, indicating that the expression of different sized transcripts is independently regulated by each promoter. Over-expression of the larger transcript in transformed plants caused altered morphologies (Matsuoka et al., Plant Cell, 1993, 5, 1039-1048); in contrast, over-expression of the smaller transcript did not cause any morphological changes. The results suggest that the product of the smaller transcript fails to alter the expression of its target gene(s) in the transformants, while that of the larger transcript is capable of altering the expression of its target gene(s).

Expression of nodulin genes in plant-determined ineffective nodules of pea.

The mutant E135 (sym 13) of pea (Pisum sativum L.) forms a normal number of small white nodules that contain bacteroids, but these bacteroids lack nitrogenase activity. To evaluate the effects of the sym 13 gene on the expression of nodulin genes, cDNA clones for nodulins were isolated from pea nodules and the expression of nodulin genes in ineffective E135 nodules was compared with that in nitrogen-fixing nodules on the wild-type parent, cv. Sparkle. Nineteen cDNA clones for nodulins, including ENOD2 and cDNAs for two distinct leghemoglobins (Lbs), were isolated from Sparkle nodules by a subtractive hybridization procedure. All the nodulin genes examined were expressed in nodules on both E135 and Sparkle plants. However, the level of expression of seven genes, one of which was an Lb gene that corresponded to PsN5, was significantly lower in E135 nodules. The levels of Lb apo-proteins, with the exception of Lb-III and Lb-IV, in E135 nodules resembled those in Sparkle nodules, but the level of heme in E135 nodules was lower than that in Sparkle nodules. Although the expression of the two Lb genes that corresponded to PsN5 and PsN120 in E135 nodules was slightly depressed by exogenous ammonia, the level of the PsN5 transcript was still lower than the control level in Sparkle nodules. Our results indicate that the plant gene sym 13 does not influence the induction of nodulin genes but does influence the level of the expression of some genes, one of which is a gene for Lb, as well as the level of heme.

Alternative RNA products from a rice homeobox gene.

Three cDNA clones were isolated from rice, OSH42, OSH44 and OSH45, which encode homeodomain sequences in the C-terminal region. The sequences of these cDNAs differ in the N- and C-termini, but they share an identical homeodomain and an acidic amino acid-rich region. The transcripts corresponding to these cDNAs are encoded by a single gene on rice chromosome 8. Differential transcription initiation results in a large transcript comprised of exons 1 and 3-7 and a smaller transcript comprised of exons 2-7. The larger transcript is constitutively expressed in all tissues tested, while the smaller transcript is expressed in leaves, stems and rachis but not in roots, flowers, or suspension callus cells. Alternative splicing also occurs at three different acceptor sites in intron 6 in all tissues tested. The GAL4 DNA-binding domain of yeast was used to study the function of various protein domains. The acidic amino acid-rich region activates the expression of a reporter gene controlled by the GAL4 target sequence, indicating that it functions as a transactivation domain. The larger transcript encodes a unique alanine and glycine-rich region on the N-terminal side of the acidic region, which is not encoded by the smaller transcript. This region completely suppresses the transactivation activity of the acidic region. This suggests that the product of the larger transcript fails to activate the expression of the target gene(s) while the product of the smaller transcript activates the expression of its target gene(s).

Expression of rice OSH1 gene is localized in developing vascular strands and its ectopic expression in transgenic rice causes altered morphology of leaf.

Transgenic rice plants (Oryza sativa cv. Nipponbare) carrying 1 or 2 copies of a rice homeobox gene, OSH1, under the control of the CaMV 35S promoter were generated. The transgene caused altered morphology of leaf, such as ligule-replacement and abnormal division of sclerenchyma cells. The phenotype of these leaves resembles that of maize leaf morphological mutant, Knotted 1, which is caused by duplication of the KN1 gene (Veit et al., 1990). The in situ hybridization analysis has revealed that the expression of endogenous OSH1 is mainly localized in developing vascular strands of stem. We have discussed the biological roles of OSH1 in rice based on these results.

Micronucleus test with cyclophosphamide administered by intraperitoneal injection and oral gavage.

The effect of route of administration on the micronucleus test was examined in 2 laboratories: cyclophosphamide (CYP) was administered by intraperitoneal injection (i.p.) or oral gavage (p.o.) to 2 strains of mice. MS/Ae and CD-1. On the basis of a small-scale acute toxicity study and a pilot micronucleus experiment, the final micronucleus test was performed with a 48-h sampling time at doses of 25-200 mg/kg i.p. and 50-400 mg/kg p.o. CYP via the i.p. route was more toxic and induced more micronucleated polychromatic erythrocytes (MNPCEs) in MS/Ae mice than in CD-1 mice. Administration-route-related differences were not distinctly shown in the MS/Ae strain. In CD-1, however, higher doses were required for the p.o. route than for the i.p. route to induce about equal amounts of clastogenic damage.