The promoter of rbcS in a C3 plant (rice) directs organ-specific, light-dependent expression in a C4 plant (maize), but does not confer bundle sheath cell-specific expression.

The small subunit of ribulose-bisphosphate carboxylase (Rubisco), encoded by rbcS, is essential for photosynthesis in both C3 and C4 plants, even though the cell specificity of rbcS expression is different between C3 and C4 plants. The C3 rbcS is specifically expressed in mesophyll cells, while the C4 rbcS is expressed in bundle sheath cells, and not mesophyll cells. Two chimeric genes were constructed consisting of the structural gene encoding beta-glucuronidase (GUS) controlled by the two promoters from maize (C4) and rice (C3) rbcS genes. These constructs were introduced into a C4 plant, maize. Both chimeric genes were specifically expressed in photosynthetic organs, such as leaf blade, but not in non-photosynthetic organs. The expressions of the genes were also regulated by light. However, the rice promoter drove the GUS activity mainly in mesophyll cells and relatively low in bundle sheath cells, while the maize rbcS promoter induced the activity specifically in bundle sheath cells. These results suggest that the rice promoter contains some cis-acting elements responding in an organ-specific and light-inducible regulation manner in maize but does not contain element(s) for bundle sheath cell-specific expression, while the maize promoter does contain such element(s). Based on this result, we discuss the similarities and differences between the rice (C3) and maize (C4) rbcS promoter in terms of the evolution of the C4 photosynthetic gene.

Genomic organization and transcriptional regulation of maize ZmRR1 and ZmRR2 encoding cytokinin-inducible response regulators.

Maize genomic clones encoding cytokinin-inducible response regulators, ZmRR1 and ZmRR2, have been isolated. In comparison with the corresponding cDNAs, ZmRR2 was found to be interrupted in the translated region by an intron whereas ZmRR1 was not. The 5'-flanking regions of the two genes shared conserved regions and putative cis-elements, which had been identified in maize or other plant species. The run-on transcription assay and the analysis of stable maize transformants of ZmRR1 promoter-beta-glucuronidase fusion gene revealed that the accumulation of the transcripts in response to cytokinins is, at least in parts, attributed by transcriptional activation.

Flower-predominant expression of a gene encoding a novel class I chitinase in rice (Oryza sativa L.).

A flower-predominant cDNA for a gene, termed OsChia 1;175, was isolated from a cDNA library of rice pistils. Northern blot and RT-PCR analyses revealed that the OsChia 1;175 gene is highly expressed in floral organs (pistils, stamens and lodicules at the heading stage) but not or at an extremely low level in vegetative organs. OsChia 1;175 encodes a protein that consists of 340 amino acid residues, and the putative mature protein shows 52% to 63% amino acid identity to class I chitinases of rice or other plants. The phylogenetic tree shows that the OsChia 1;175 protein is a new type of plant class I chitinase in rice. The expression of OsChia 1;175 in vegetative organs is not induced by several chemicals, UV, and wounding. The soluble putative mature OsChia 1;175 protein expressed in Escherichia coli exhibited chitinase activity in the assay with colloidal chitin as a substrate. Genomic Southern analysis revealed that the OsChia 1;175 gene was organized as a low-copy gene family. The rice genomic library was screened and a genome clone corresponding to OsChia 1;175 was isolated. The transcription start sites of the OsChia 1;175 gene were mapped by primer extension analysis. The 1.2 kb putative promoter region of the OsChia 1;175 gene was fused to the GUS (beta-glucuronidase) gene, and this chimeric gene was introduced to rice by Agrobacterium-mediated transformation. The flower-predominant gene expression was identified also in the transgenic rice plants. The high promoter activity was detected in the stigmas, styles, stamens and lodicules in transgenic plants. The possible functions of OsChia 1;175 are discussed.

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.

The promoter for the maize C4 pyruvate, orthophosphate dikinase gene directs cell- and tissue-specific transcription in transgenic maize plants.

The pyruvate,orthophosphate dikinase (PPDK) gene coding the chloroplast enzyme involved in C4 photosynthesis has a dual promoter system. The first promoter is responsible for the transcription of a larger transcript and its product is targeted to the chloroplast (hence, it is designated as C4Pdk promoter) while the second promoter is responsible for the transcription of a smaller transcript and its product remains in the cytosol. In this study, chimeric maize C4Pdk promoter (0.9 or 1.5 kb)-beta-glucuronidase or luciferase fusion genes were introduced into maize plants by Agrobacterium-mediated transformation. The cell- and tissue-specificities of the maize C4Pdk promoter in the transgenic maize plants were examined by histochemical and enzymic activity analyses of the reporters in different photosynthetic cells and tissues. The results showed that the reporter proteins are almost exclusively localized in leaf mesophyll cells. Among the tissues tested, leaf blade had the highest reporter activities with sheath exhibiting about 10% of the activities in blade. Husk, stem, tassel and root had no or very little reporter activities. Taken together, these results suggest that the maize C4Pdk promoter is specifically transcribed in the mesophyll cells of leaf blade and to a much less extent in the mesophyll cells of sheath, but not in leaf bundle sheath cells or other tissues. Furthermore, the 0.9 kb maize C4Pdk promoter sequences appear to contain the necessary cis-acting elements for its cell- and organ-specific expression.

Binding of cell type-specific nuclear proteins to the 5'-flanking region of maize C4 phosphoenolpyruvate carboxylase gene confers its differential transcription in mesophyll cells.

C4-type phosphenolpyruvate carboxylase (C4PEPC) acts as a primary carbon assimilatory enzyme in the C4 photosynthetic pathway. The maize C4PEPC gene (C4Ppc1) is specifically expressed in mesophyll cells (MC) of light-grown leaves, but the molecular mechanism responsible for its cell type-specific expression has not been characterized. In this study, we introduced a chimeric maize C4Ppc1 5'-flanking region/beta-glucuronidase (GUS) gene into maize plants by Agrobacterium-mediated transformation. Activity assay and histochemical staining showed that GUS is almost exclusively localized in leaf MC of transgenic maize plants. This observation suggests that the introduced 5' region of maize C4Ppc1 contains the necessary cis element(s) for its specific expression in MC. Next, we investigated whether the 5' region of the maize gene interacts with nuclear proteins in a cell type-specific manner. By gel shift assays with nuclear extracts prepared from MC or bundle sheath cells (BSC), cell type-specific DNA-protein interactions were detected: nuclear factors PEP(Ib) and PEP(Ic) are specific to MC whereas PEP(Ia) and PEP(IIa) are specific to BSC. Light alters the binding activity of these factors. These interactions were not detected in the assay with nuclear extract prepared from root, or competed out by oligonucleotides corresponding to the binding sites for the maize nuclear protein, PEP-I, which is known to bind specifically to the promoter region of C4Ppc1. The results suggest that novel cell type-specific positive and negative nuclear factors bind to the maize C4Ppc1 5'-flanking region and regulate its differential transcription in MC in a light-dependent manner.

Advances in cereal gene transfer.

Over the past five years, transgenic strains of various major cereals have been produced, with transformation of rice and maize being most common. A majority of the cereal transformants obtained to date has been generated by the particle bombardment technique, but Agrobacterium-mediated transformation is rapidly becoming the method of choice. Rice, the plant in which transformation-related technology is most advanced, appears to be the model monocotyledon for basic and applied studies.

Transformation of rice mediated by Agrobacterium tumefaciens.

Agrobacterium tumefaciens has been routinely utilized in gene transfer to dicotyledonous plants, but monocotyledonous plants including important cereals were thought to be recalcitrant to this technology as they were outside the host range of crown gall. Various challenges to infect monocotyledons including rice with Agrobacterium had been made in many laboratories, but the results were not conclusive until recently. Efficient transformation protocols mediated by Agrobacterium were reported for rice in 1994 and 1996. A key point in the protocols was the fact that tissues consisting of actively dividing, embryonic cells, such as immature embryos and calli induced from scutella, were co-cultivated with Agrobacterium in the presence of acetosyringonc, which is a potent inducer of the virulence genes. It is now clear that Agrobacterium is capable of transferring DNA to monocotyledons if tissues containing 'competent' cells are infected. The studies of transformation of rice suggested that numerous factors including genotype of plants, types and ages of tissues inoculated, kind of vectors, strains of Agrobacterium, selection marker genes and selective agents, and various conditions of tissue culture, are of critical importance. Advantages of the Agrobacterium-mediated transformation in rice, like on dicotyledons, include the transfer of pieces of DNA with defined ends with minimal rearrangements, the transfer of relatively large segments of DNA, the integration of small numbers of copies of genes into plant chromosomes, and high quality and fertility of transgenic plants. Delivery of foreign DNA to rice plants via A. tumefaciens is a routine technique in a growing number of laboratories. This technique will allow the genetic improvement of diverse varieties of rice, as well as studies of many aspects of the molecular biology of rice.

Identification of the amino acid residues responsible for cold tolerance in Flaveria brownii pyruvate,orthophosphate dikinase.

Pyruvate,orthophosphate dikinase (PPDK), an enzyme important in C4 photosynthesis, is typically a cold-sensitive enzyme. However, a cold-tolerant form of the enzyme has been isolated from the leaves of Flaveria brownii. Using an E. coli expression system and the PPDK cDNAs from F. brownii (cold-tolerant), F. bidentis (cold-sensitive) and maize (intermediately cold-tolerant), site-directed mutagenesis studies indicated that as few as three amino acids residues (of 880 residues) strongly influence the cold sensitivity of Flaveria PPDK. Gel filtration analysis of the PPDK expressed in E. coli showed that subunit association and cold tolerance are closely linked.

Identification of the amino acid residues responsible for cold tolerance in Flaveria brownii pyruvate,orthophosphate dikinase.

Pyruvate,orthophosphate dikinase (PPDK), an enzyme important in C4 photosynthesis, is typically a cold-sensitive enzyme. However, a cold-tolerant form of the enzyme has been isolated from the leaves of Flaveria brownii. Using an Escherichia coli expression system and the PPDK cDNAs from F. brownii (cold-tolerant), F. bidentis (cold-sensitive) and maize (intermediate cold tolerance), site-directed mutagenesis studies indicated that as few as three amino acids residues (of 880 residues) strongly influence the cold sensitivity of Flaveria PPDK. Gel filtration analysis of the PPDK expressed in E. coli showed that subunit association and cold tolerance are closely linked.

Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers.

Novel 'super-binary' vectors that carried two separate T-DNAs were constructed. One T-DNA contained a drug-resistance, selection-marker gene and the other contained a gene for beta-glucuronidase (GUS). A large number of tobacco (Nicotiana tabacum L.) and rice (Oryza sativa L.) transformants were produced by Agrobacterium tumefaciens LBA4404 that carried the vectors. Frequency of co-transformation with the two T-DNAs was greater than 47% GUS-positive, drug-sensitive progeny were obtained from more than half of the co-transformants. Molecular analyses by Southern hybridization and polymerase chain reactions confirmed integration and segregation of the T-DNAs. Thus, the non-selectable T-DNA that was genetically separable from the selection marker was integrated into more than a quarter of the initial, drug-resistant transformants. Since various DNA fragments may be inserted into the non-selectable T-DNA by a simple procedure, these vectors will likely be very useful for the production of marker-free transformants of diverse plant species. Delivery of two T-DNAs to plants from mixtures of A. tumefaciens was also tested, but frequency of co-transformation was relatively low.

High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens.

Transformants of maize inbred A188 were efficiently produced from immature embryos cocultivated with Agrobacterium tumefaciens that carried "super-binary" vectors. Frequencies of transformation (independent transgenic plants/embryos) were between 5% and 30%. Almost all transformants were normal in morphology, and more than 70% were fertile. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. Between one and three copies of the transgenes were integrated with little rearrangement, and the boundaries of T-DNA were similar to those in transgenic dicotyledons and rice. F1 hybrids between A188 and five other inbreds were transformed at low frequencies.

Purification and characterization of phospholipase D (PLD) from rice (Oryza sativa L.) and cloning of cDNA for PLD from rice and maize (Zea mays L.).

Phospholipase D (PLD) was purified to high homogeneity from rice bran (Oryza sativa L.). Two peaks of PLD activity were resolved by Mono Q anion-exchange chromatography. The molecular mass of PLD in both peaks was 82 kDa on SDS-PAGE and 78 kDa in gel filtration. Antibodies raised against the protein in one of the peaks precipitated the enzyme activities in both peaks. Enzymatic characteristics of PLD in the two peaks were identical except for a difference of 0.1 in the isoelectric points. Sequence analysis covering more than 10% of the amino acids of the proteins and peptide mapping did not detect any difference in the primary structure of the proteins. A cDNA for PLD was isolated from rice and it encoded a protein of 812 residues. The N-terminal sequences of purified PLDs matched the deduced amino acid sequence starting from residue 47. A Northern blot showed this gene was expressed in leaves, roots, developing seeds and cultured cells, and a Southern blot detected a single band of rice genomic DNA hybridizing to the cDNA. A cDNA for PLD was also isolated from maize. The similarity of the deduced amino acid sequences of PLD was 90% between rice and maize, 73% between the cereals and castor bean.

Cold stability of pyruvate, orthophosphate dikinase of Flaveria brownii.

The nucleotide sequences of the complementary DNA of pyruvate, Pi dikinase (PPDK) from Flaveria bidentis, a C4 plant which possesses a cold-sensitive form of PPDK, and Flaveria brownii, a 'C4-like' plant which possesses a cold-tolerant form of PPDK, were determined. PPDK was isolated from the leaves of both Flaveria species and purified and the N-terminal amino acid sequences characterised. Together with a maize PPDK cDNA, cDNA inserts which code for the mature form of PPDK of F. bidentis and of F. brownii were expressed in bacteria and the cold sensitivity of the expressed PPDK studied. The cold sensitivity of the PPDK expressed in bacteria mimics the cold sensitivity of PPDK found in vivo in all three plant species. This study indicates that the cold sensitivity of plant PPDK is controlled by the primary structure of the enzyme.

Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.

A large number of morphologically normal, fertile, transgenic rice plants were obtained by co-cultivation of rice tissues with Agrobacterium tumefaciens. The efficiency of transformation was similar to that obtained by the methods used routinely for transformation of dicotyledons with the bacterium. Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the R0, R1 and R2 generations. Sequence analysis revealed that the boundaries of the T-DNA in transgenic rice plants were essentially identical to those in transgenic dicotyledons. Calli induced from scutella were very good starting materials. A strain of A. tumefaciens that carried a so-called 'super-binary' vector gave especially high frequencies of transformation of various cultivars of japonica rice that included Koshihikari, which normally shows poor responses in tissue culture.

Cucumber mosaic virus-tolerant transgenic tomato plants expressing a satellite RNA.

A satellite RNA (T73-satRNA) gave reduced symptom severity on tomato plants when coinoculated with an ordinary strain of cucumber mosaic virus (CMVO). cDNA for T73-satRNA was introduced into a binary vector (pTOK162) through a homologous recombination in an Agrobacterium tumefaciens cell, and then transferred to leaf disks of tomato. Stable integration and transcription of the cDNA in the regenerants were verified by Southern and northern blot hybridizations, respectively. Upon inoculation with CMV-O, the transformants exhibited very slight symptoms of CMV, grew normally, and finally set fruits, whereas untransformed wildtype tomato plants showed very severe symptoms, and their growth was retarded and formed few fruits. Agarose gel electrophoresis of total RNA from CMV-O-inoculated transformants detected RNA molecules corresponding to T73-satRNA.

Genetic characterization of a double-flowered tobacco plant obtained in a transformation experiment.

A leaf-disk transformation experiment was performed with tobacco (Nicotiana tabacum L.) using a binary vector and a strain of Agrobacterium tumefaciens that carried a wild-type Ti-plasmid, pTiBo542. Although the majority of kanamycin-resistant, transgenic plants was morphologically normal, one of the plants was double-flowered and had a slightly wavy stem and leaves whose edges were bent slightly upwards. The abnormal morphology was controlled by a single, dominant Mendelian gene. Young plants that carried this gene were distinguishable from normal plants at the stage of cotyledons. The homozygotes, with respect to this gene, were more seriously deformed than the heterozygotes. DNA segments derived from the binary vector and from the TL-and TR-DNA of pTiBo542 were detected in the double-flowered plant, but the T-DNA genes involved in biosynthesis of phytohormones were absent from the plant. The abnormal morphology, the resistance to kanamycin, and the segments of foreign DNA were genetically linked, and the linkage was very tight, at least between the abnormal morphology and the resistance to kanamycin; the meiotic recombination frequency was less than 0.02%, if recombination occurred at all.

Transformation of cultured cells of Chenopodium quinoa by binary vectors that carry a fragment of DNA from the virulence region of pTiBo542.

A 15.2-kb KpnI fragment from the virulence region of pTiBo542, the Ti plasmid harbored by Agrobacterium tumefaciens strain A281, was introduced into binary vectors. The fragment contained the virB, virC and virG genes, and it is known to have the ability to increase the virulence of strains of A. tumefaciens. The strains of A. tumefaciens that carried the resulting plasmids were able to transform cells in a suspension culture of Chenopodium quinoa Willd cells which were not transformable by common vectors. Although the sizes of the plasmids was very large, a foreign segment of DNA was introduced into one of the plasmids by homologous recombination in A. tumefaciens cells, and the segment was subsequently transferred to plant cells.

Efficient selection of somatic hybrids in Nicotiana tabacum L. using a combination of drug-resistance markers introduced by transformation.

Kanamycin resistance and chloramphenicol resistance were introduced separately to two different tobacco plants (Nicotiana tabacum L.) by Agrobacterium-mediated transformation. Leaf mesophyll protoplasts were prepared from the progeny of these transformants and were subjected to electrofusion. On the 10th day and the 20th day after the fusion treatment, respectively, kanamycin (100 mg/l) and chloramphenicol (30 mg/l) were added to the suspension of protoplasts. The parental protoplasts and an unfused mixture of these protoplasts failed to form colonies when this selection procedure was employed. However, three independent fusion experiments yielded more than 7000 doubleresistant colonies with a frequency between 0.30% and 0.54%. All of the surviving colonies showed continuous growth in the presence of the two antibiotics. The majority of regenerated plants resembled, morphologically, a tetraploid tobacco plant, and their somatic chromosome numbers were 2n=96. The leaf segments from these putative somatic hybrids formed calli and proliferated vigorously on a medium that contained both antibiotics. Southern hybridization permitted the detection of the DNA fragments that conferred kanamycin and chloramphenicol resistance on these somatic hybrid plants. The system described here can be considered to be a universal system for the selection of somatic hybrids and is applicable to various combinations of protoplasts in which pre-selected genetic markers are absent.

Genes responsible for the supervirulence phenotype of Agrobacterium tumefaciens A281.

Agrobacterium tumefaciens A281 induces large, rapidly appearing tumors on a variety of plants and has a wider host range than other strains of A. tumefaciens. By using Tn3HoHo1 transposon mutagenesis and complementation analysis, a 2.5-kilobase DNA fragment which is responsible for the supervirulence phenotype was identified in the virulence (vir) region of the Ti plasmid. This fragment contains the virG locus, as well as the 3' end of the virB operon. A clone of this fragment conferred the supervirulence phenotype on A348, a nonsupervirulent strain. The increased virulence was correlated with an increased expression of vir genes, which could be achieved by introducing an extra copy of the transcriptional activator virG or the supervirulence region for maximum virulence. The virulence of the supervirulent strain A281 could be increased even further if the entire virB operon was added in addition to the virG operon. A plasmid, pToK47, containing virB and virG increased the virulence of all A. tumefaciens strains into which the plasmid was introduced. These data suggest that a highly virulent binary vector system can be constructed which might prove especially useful in the transformation of certain higher plants.