Isolation and expression analysis of gibberellin 20-oxidase homologous gene in apple.

To characterize the gibberellin (GA) 20-oxidase gene in apple, the genomic and cDNA clone from "Fuji" apple (accession no. AB037114) was isolated. The deduced amino acid sequence of this cDNA showed 71% and 66% identity to those of GA 20-oxidase cloned from French bean and Arabidopsis, respectively. The transcript of this gene was detected mainly in immature seeds between 1-3 months after full bloom. These results suggested that this apple GA 20-oxidase gene might be involved in GA biosynthesis in developing apple seed.

The evolution of C4 plants: acquisition of cis-regulatory sequences in the promoter of C4-type pyruvate, orthophosphate dikinase gene.

In a previous study, we identified the C4-like pyruvate, orthophosphate dikinase gene (Pdk) in the C3 plant rice, with a similar structure to the C4-type Pdk in the C4 plant maize. In order to elucidate the differences between C4-type and C4-like Pdk genes in C4 and C3 plants, we have produced chimeric constructs with the beta-glucuronidase (GUS) reporter gene under the control of the Pdk promoters. In transgenic rice, both rice and maize promoters directed GUS expression in photosynthetic organs in a light-dependent manner. However, the maize promoter exhibited a much higher transcriptional activity than the rice promoter did. These results indicate that the rice C4-like Pdk gene resembles the maize C4-type Pdk gene in terms of regulation of expression. We also tested the activity of the rice promoter in transgenic maize. GUS activity was seen in both photosynthetic and non-photosynthetic organs. Thus, the rice promoter does not confer a strict organ-specific gene expression, as the maize promoter does. Moreover, the rice promoter directed GUS expression not only in mesophyll cells but also in bundle sheath cells, whereas the maize promoter directed expression only in mesophyll cells. Taken together, the results obtained from both transgenic maize and rice demonstrate that the rice and maize promoters differ not only quantitatively, but also qualitatively, in terms of their cell- and organ-specificity. Experiments with swapped promoters using the rice and maize promoters further demonstrated that a limited sequence region from -330 to -76 of the maize promoter confers light-regulated, high-level expression to the rice promoter in maize mesophyll protoplasts. We conclude the gain of cis-acting elements conferring high-level expression and mesophyll cell specificity was necessary for establishment of a C4-type Pdk gene during the course of evolution from C3 to C4 plants.

Decreased GA1 content caused by the overexpression of OSH1 is accompanied by suppression of GA 20-oxidase gene expression.

We previously reported that overexpression of the rice homeobox gene OSH1 led to altered morphology and hormone levels in transgenic tobacco (Nicotiana tabacum L.) plants. Among the hormones whose levels were changed, GA1 was dramatically reduced. Here we report the results of our analysis on the regulatory mechanism(s) of OSH1 on GA metabolism. GA53 and GA20, precursors of GA1, were applied separately to transgenic tobacco plants exhibiting severely changed morphology due to overexpression of OSH1. Only treatment with the end product of GA 20-oxidase, GA20, resulted in a striking promotion of stem elongation in transgenic tobacco plants. The internal GA1 and GA20 contents in OSH1-transformed tobacco were dramatically reduced compared with those of wild-type plants, whereas the level of GA19, a mid-product of GA 20-oxidase, was 25% of the wild-type level. We have isolated a cDNA encoding a putative tobacco GA 20-oxidase, which is mainly expressed in vegetative stem tissue. RNA-blot analysis revealed that GA 20-oxidase gene expression was suppressed in stem tissue of OSH1-transformed tobacco plants. Based on these results, we conclude that overexpression of OSH1 causes a reduction of the level of GA1 by suppressing GA 20-oxidase expression.

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.

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).

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.

The promoters of two carboxylases in a C4 plant (maize) direct cell-specific, light-regulated expression in a C3 plant (rice).

C4 plants have two carboxylases which function in photosynthesis. One, phosphoenolpyruvate carboxylase (PEPC) is localized in mesophyll cells, and the other, ribulose bisphosphate carboxylase (RuBPC) is found in bundle sheath cells. In contrast, C3 plants have only one photosynthetic carboxylase, RuBPC, which is localized in mesophyll cells. The expression of PEPC in C3 mesophyll cells is quite low relative to PEPC expression in C4 mesophyll cells. Two chimeric genes have been constructed consisting of the structural gene encoding beta-glucuronidase (GUS) controlled by two promoters from C4 (maize) photosynthetic genes: (i) the PEPC gene (pepc) and (ii) the small subunit of RuBPC (rbcS). These constructs were introduced into a C3 cereal, rice. Both chimeric genes were expressed almost exclusively in mesophyll cells in the leaf blades and leaf sheaths at high levels, and no or very little activity was observed in other cells. The expression of both genes was also regulated by light. These observations indicate that the regulation systems which direct cell-specific and light-inducible expression of pepc and rbcS in C4 plants are also present in C3 plants. Nevertheless, expression of endogenous pepc in C3 plants is very low in C3 mesophyll cells, and the cell specificity of rbcS expression in C3 plants differs from that in C4 plants. Rice nuclear extracts were assayed for DNA-binding protein(s) which interact with a cis-regulatory element in the pepc promoter. Gel-retardation assays indicate that a nuclear protein with similar DNA-binding specificity to a maize nuclear protein is present in rice.(ABSTRACT TRUNCATED AT 250 WORDS)

A rice homeotic gene, OSH1, causes unusual phenotypes in transgenic tobacco.

A rice gene, OSH1, which shares homology with animal homeobox genes, has been isolated. We have introduced the OSH1 cDNA into tobacco in order to examine its function. Expression of the OSH1 cDNA in tobacco induced morphological abnormalities in the leaves, petals and stems of the transformants suggesting that OSH1 functions as a morphological regulator. OSH1 cDNA expression was analyzed under the control of three different promoters. This work revealed that not only the level of OSH1 expression but also the site and timing of the expression affect the morphology of the plant.

Tissue-specific light-regulated expression directed by the promoter of a C4 gene, maize pyruvate,orthophosphate dikinase, in a C3 plant, rice.

Pyruvate,orthophosphate dikinase (PPDK; EC 2.7.9.1) activity is abundant in leaves of C4 plants, while it is difficult to detect in leaves of C3 plants. Recent studies have indicated that C3 plants have a gene encoding PPDK, with a structure similar to that of PPDK in C4 plants. However, low expression makes PPDK detection difficult in C3 plants. This finding suggests that high PPDK expression in C4 plants is due to regulatory mechanisms which are not operative in C3 plants. We have introduced a chimeric gene consisting of the gene encoding beta-glucuronidase (GUS; EC 3.2.1.31) controlled by the PPDK promoter from a C4 plant, maize, into a C3 cereal, rice. The chimeric gene was exclusively expressed in photosynthetic organs, leaf blades and sheaths, and not in roots or stems. Histochemical analysis of GUS activity demonstrated high expression of the chimeric gene in photosynthetic organs, localized in mesophyll cells, and no or very low activity in other cells. GUS expression was also regulated by light in that it was low in etiolated leaves and was enhanced by illumination. These observations indicate that the mechanisms responsible for cell-specific and light-inducible regulation of PPDK observed in C4 plants are also present in C3 plants. We directly tested whether rice has DNA-binding protein(s) which interact with a previously identified cis-acting element of the C4-type gene. Gel retardation assays indicate the presence in rice of a protein which binds this element and is similar to a maize nuclear protein which binds PPDK in maize. Taken together, these results indicate that the regulatory system which controls PPDK expression in maize is not unique to C4 plants.

Expression of a rice homeobox gene causes altered morphology of transgenic plants.

We have isolated a cDNA clone encoding a homeobox sequence from rice. DNA sequence analysis of this clone, which was designated as Oryza sativa homeobox 1 (OSH1), and a genomic clone encoding the OSH1 sequence have shown that the OSH1 gene consists of five exons and encodes a polypeptide of 361 amino acid residues. Restriction fragment length polymorphism analysis has shown that OSH1 is a single-copy gene located near the phytochrome gene on chromosome 3. Introduction of the cloned OSH1 gene into rice resulted in altered leaf morphology, which was similar to that of the maize morphological mutant Knotted-1 (Kn1), indicating that OSH1 is a rice gene homologous to the maize Kn1 gene. RNA gel blot analysis has shown that the gene is primarily expressed in the shoot apices of young rice seedlings. This finding is supported by results of transformation experiments in which the 5' flanking region of the gene directed expression of a reporter gene in the shoot apex, particularly in stipules, of transgenic Arabidopsis. To elucidate the biological function of the OSH1 gene product, the coding region was introduced into Arabidopsis under the control of the cauliflower mosaic virus 35S promoter. Almost all transformants showed abnormal morphology. The typical phenotype was the formation of clumps of abundant vegetative and reproductive shoot apices containing meristems and leaf primordia, which did not form elongated shoots. Some transformants with a less severe phenotype formed elongated shoots but had abnormally shaped leaves and flowers with stunted sepals, petals, and stamens. The abnormal phenotypes were inherited, and the level of expression of the introduced OSH1 correlates with the severity of the phenotype. These findings indicate that the abnormal morphologies of the transgenic plants are caused by the expression of the OSH1 gene product and, therefore, that OSH1 is related to the plant development process.

Light-Independent Expression of cab and rbcS Genes in Dark-Grown Pine Seedlings.

In angiosperms, light has been shown to induce the expression of cab and rbcS genes, which encode the apoprotein of light-harvesting chlorophyll a/b binding protein (LHCP) and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), respectively. By contrast, chlorophylls are synthesized in the cotyledons of pine seedlings even if seeds are germinated in the dark. We have examined the expression of cab and rbcS genes in the cotyledons of pine (Pinus thunbergii) seedlings grown in darkness. The proteins of LHCP and the large subunit and the small subunit of Rubisco were detected in the cotyledons of dark-grown seedlings. The transcripts of cab and rbcS genes were present at substantial levels in dark-grown seedlings. However, the transcripts and the translated products of the genes were not found in the embryos of dry seeds. These results indicate that light is not required for the expression of cab and rbcS genes during the course of development of the cotyledons of pine seedlings. The processing of the precursor polypeptides of the mature proteins also appears to take place even in the dark.

Sequence-specific interactions of a maize factor with a GC-rich repeat in the phosphoenolpyruvate carboxylase gene.

A plant nuclear protein PEP-I, which binds specifically to the promoter region of the phosphoenolpyruvate carboxylase (PEPC) gene, was identified. Methylation interference analysis and DNA binding assays using synthetic oligonucleotides revealed that PEP-I binds to GC-rich elements. These elements are directly repeated sequences in the promoter region of the PEPC gene and we have suggested that they may be cis-regulatory elements of this gene. The consensus sequence of the element is CCCTCTCCACATCC and the CTCC is essential for binding of PEP-I. PEP-I is present in the nuclear extracts of green leaves, where the PEPC gene is expressed. However, no binding was detected in tissues where the PEPC gene is not expressed in vivo, such as roots or etiolated leaves. Thus, PEP-I is the first factor identified in plants which has different binding activity in light-grown compared with dark-grown tissue. PEP-I binding is also tissue-specific, suggesting that PEP-I may function to coordinate PEPC gene expression with respect to light and tissue specificity. This report describes the identification and characterization of the sequences required for PEP-I binding.

A metal-dependent DNA-binding protein interacts with a constitutive element of a light-responsive promoter.

We have used DNase I footprinting to characterize nuclear factors that bind to the light-responsive promoter of pea rbcS-3A, one member of the gene family encoding the small subunit of ribulose-1,5-bisphosphate carboxylase. A sequence-specific binding activity, designated 3AF1, binds to an AT-rich sequence present at the -45 region of the rbcS-3A promoter. A tetramer of the 3AF1 binding site, designated as Box VI, can form multiple complexes with tobacco leaf and root nuclear extracts. Mutations of 3 base pairs in Box VI severely reduce DNA-protein complex formation in vitro. The wild-type Box VI tetramer, but not the mutant tetramer, is active in transgenic tobacco plants when placed upstream of the cauliflower mosaic virus 35S promoter truncated at -90. These results correlate binding of 3AF1 to the in vivo function of Box VI. The Box VI tetramer/35S chimeric construct confers expression in diverse cell types and organs and its activity is not dependent on light. By using the Box VI tetramer as a probe to screen a cDNA expression library, we have obtained a putative cDNA clone for the 3AF1 DNA-binding activity. Lysogen extracts of Escherichia coli expressing the cDNA clone give sequence-specific complexes with Box VI. The deduced amino acid sequence of the protein encoded by the cDNA contains two stretches of about 100 residues that are 80% homologous. Moreover, in each of the two repeats, there is an arrangement of histidines and cysteines, which may be related to the two known types of zinc-finger motifs found in many DNA-binding proteins. Consistent with the expectation that metal coordination plays an important role in DNA binding by this protein, we found that 1,10-phenanthroline can abolish the formation of DNA-protein complexes. Interestingly, we found that the same treatment did not abolish the DNA binding activity of 3AF1 in crude nuclear extracts of tobacco. These data indicate that the nuclear 3AF1 activity is likely due to multiple DNA-binding proteins all interacting with Box VI in vitro. RNA gel blot analysis shows that multiple transcripts homologous to this cDNA clone are expressed in different tobacco organs.

Structure and characterization of a cDNA clone for phenylalanine ammonia-lyase from cut-injured roots of sweet potato.

A cDNA clone for phenylalanine ammonia-lyase (PAL) induced in wounded sweet potato (Ipomoea batatas Lam.) root was obtained by immunoscreening a cDNA library. The protein produced in Escherichia coli cells containing the plasmid pPAL02 was indistinguishable from sweet potato PAL as judged by Ouchterlony double diffusion assays. The M(r) of its subunit was 77,000. The cells converted [(14)C]-l-phenylalanine into [(14)C]-t-cinnamic acid and PAL activity was detected in the homogenate of the cells. The activity was dependent on the presence of the pPAL02 plasmid DNA. The nucleotide sequence of the cDNA contained a 2121-base pair (bp) open-reading frame capable of coding for a polypeptide with 707 amino acids (M(r) 77, 137), a 22-bp 5'-noncoding region and a 207-bp 3'-noncoding region. The results suggest that the insert DNA fully encoded the amino acid sequence for sweet potato PAL that is induced by wounding. Comparison of the deduced amino acid sequence with that of a PAL cDNA fragment from Phaseolus vulgaris revealed 78.9% homology. The sequence from amino acid residues 258 to 494 was highly conserved, showing 90.7% homology.

Binding site requirements for pea nuclear protein factor GT-1 correlate with sequences required for light-dependent transcriptional activation of the rbcS-3A gene.

Nuclear protein factor GT-1 binds to sequence boxes II, III, II* and III* upstream of the light-responsive pea rbcS-3A gene. We have shown previously that box II and box III are required for expression of rbcS-3A when redundant elements upstream of -170 (relative to the transcription start site) are removed. Here we present evidence that deletion and substitution mutations downstream of -170 which eliminate expression also decrease binding. Using a series of 2 bp substitution mutations we have defined a core of six residues (GGTTAA) within box II (GTGTGGTTAATATG) that are critical for binding. The most detrimental mutation for binding, which changes the double Gs to Cs, is sufficient to eliminate detectable expression in vivo when only 170 bp of 5' flanking sequences are present. The simplest interpretation of these data is that GT-1 is an activator of rbcS-3A transcription. Footprinting experiments show that GT-1 from both light-grown and dark-adapted plants binds to the same sequences in vitro. Therefore, the lack of expression of rbcS-3A in the dark is not due to the absence of GT-1. In our analysis of the sequence elements upstream of -170, we have mapped two additional GT-1 sites (boxes II** and III**) between -330 and -410. The similarities and differences among the GT-1 sites located upstream and downstream of -170 are discussed in terms of the different sequence requirements for rbcS-3A expression during development.