Analysis of soybean chloroplast DNA replication by two-dimensional gel electrophoresis

Chloroplast DNA replication was studied in the green, autotrophic suspension culture line SB-1 of Glycine max. Three regions (restriction fragments Sac I 14.5, Pvu II 4.1 and Pvu II 14.8) on the plastome were identified that displayed significantly higher template activity in in vitro DNA replication assays than all other cloned restriction fragments of the organelle genome, suggesting that these clones contain sequences that are able to direct initiation of DNA replication in vitro. In order to confirm that the potential in vitro origin sites are functional in vivo as well, replication intermediates were analyzed by two-dimensional gel electrophoresis using cloned restriction fragments as probes. The two Pvu II fragments that supported deoxynucleotide incorporation in vitro apparently do not contain a functional in vivo replication origin since replication intermediates from these areas of the plastome represent only fork structures. The Sac I 14.5 chloroplast DNA fragment, on the other hand, showed intermediates consistent with a replication bubble originating within its borders, which is indicative of an active in vivo origin. Closer examination of cloned Sac I 14.5 sub-fragments confirmed high template activity in vitro for two, S/B 5 and S/B 3, which also seem to contain origin sites utilized in vivo as determined by two-dimensional gel electrophoresis. The types of replication intermediate patterns obtained for these sub-fragments are consistent with the double D-loop model for chloroplast DNA replication with both origins being located in the large unique region of the plastome [17, 18]. This is the first report of a chloroplast DNA replication origin in higher plants that has been directly tested for in vivo function.     

Structural changes in the plastid DNA of rice (Oryza sativa L.) during tissue culture

To investigate the rearrangement of the plastid genome during tissue culture, DNA from rice callus lines, which had been derived individually from single protoplasts isolated from seed or pollen callus (protoclones), was analyzed by Southern hybridization with rice chloroplast DNA (ctDNA) clones as probes. Among 44 long-term cultured protoclones, maintained for 4, 8 or 11 years, 28 contained plastid DNA (ptDNA) from which portions had been deleted. The ptDNA of all protoclones that had been maintained for 11 years had a deletion that covered a large region of the plastid genome. The deletions could be classified into 15 types from their respective sizes and positions. By contrast, no deletions were found in the ptDNA of 38 protoclones that had been maintained for only 1 month. These results indicate that long-term culture causes deletions in the plastid genome. Detailed hybridization experiments revealed that plastid genomes with deletions in several protoclones were organized as head-to-head or tail-to-tail structures. Furthermore, ptDNAs retained during long-term culture all had a common terminus at one end, where extensive rearrangement is known to have occurred during the speciation of rice and tobacco. Morphological analysis revealed the accumulation of starch granules in plastids and amyloplasts in protoclones in which the plastid genome had undergone deletion. Our observations indicated that novel structural changes in the plastid genome and morphological changes in the plastid had occurred in rice cells during long-term tissue culture. Moreover, the morphological changes in plastids were associated with deletions in the plastid genome.     

Molecular analysis of organelle DNA of different subspecies of rice and the genomic stability of mtDNA in tissue cultured cells of rice

Summary Chloroplast (ct) and mitochondrial (mt) DNAs were isolated from two subspecies of rice (Oryza sativa), japonica (Calrose 76) and indica (PI353705) and compared by restriction endonuclease fragment pattern analysis. Similarly, PI353705 (A5) mtDNA was also compared with the mtDNA of its long term tissue cultured line, BL2. Variation in the ctDNA of the 2 subspecies was detected with two (AvaI and BglI) of the 11 restriction endonucleases tested, whereas their mtDNAs showed considerable variation when restricted by PstI, BamHI, HindIII and XhoI endonucleases. Thus, the chloroplast DNA was more highly conserved than the mtDNA in the subspecies comparisons. Only minor variation was observed between the restriction endonuclease patterns of the mtDNAs of BL2 and A5. Southern blots of mtDNA were hybridized with heterologous probes from maize and spinach organelle genes. Differences were found in the hybridization patterns of the two subspecies for six of the eight (mitochondrial and chloroplast) probes tested. Two of the seven (mitochondrial) probes (coxII and 26S rRNA) detected tissue culture generated variation in mtDNA. The relative values of restriction endonuclease and hybridization patterns for studying phylogenetic and genetic relationships in rice are discussed.Florida Agricultural Experiment Station Journal Series No. 8807. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA, and does not imply its approval to the exclusion of other products that may also be suitable     

Comparison of pressure chamber and psychrometer estimates of leaf water potential in rice

Summary The need to compare pressure-chamber estimates of leaf water potential with a psychrometric method has been established for several crop species. We investigated this relationship for rice (Oryza sativa L.) as well as the need to protect leaves from water loss during sampling and measuring period in the pressure chamber. Two rice cultivars grown in containers on a clay-loam soil were stressed to varying degrees by withholding water. Fully expanded leaves were sampled for estimation of leaf water potential by the dew point hygrometer and pressure-chamber techniques. The same leaf was used in both methods allowing direct comparison. Additionally, two alternative methods of leaf handling for measurement by the pressure chamber technique were compared. Protection of leaf samples against water loss during excision, transport and handling was found to be more important at higher leaf water potentials (>−1.0 MPa). The two cultivars used appeared to differ in their response to protection of the leaf sample. These results serve to further caution pressure chamber users on extrapolating comparisons between the two measurement methods and between tissue handling techniques even within a crop species.     

Distribution of mitochondrial plasmid-like DNA in cultivated rice (Oryza sativa L.) and its relationship with varietal groups

Summary Mitochondrial (mt) plasmid-like DNA was found in most of more than 100 rice cultivars (Oryza sativa L.) by the use of 0.7% agarose gel electrophoresis (AGE). The DNA varied in molecular weight and number. By electron microscopy, small circular DNAs of different sizes could be detected in addition to the DNAs of high molecular weight, even in cultivars in which mt plasmid-like DNA was not detected by AGE. The detection of the mt plasmid-like DNAs by AGE did not depend on their presence or absence, but on their high stoichiometry. The relationship between cytoplasms with mt plasmid-like DNAs and varietal (for example, Indica rice) groups was close. The geographical distribution of cytoplasms is discussed.     

High transmission of paternal plastid DNA in alfalfa plants demonstrated by restriction fragment polymorphic analysis

Summary A high frequency of paternal plastid transmission occurred in progeny from crosses among normal green alfalfa plants. Plastid transmission was analyzed by hybridization of radiolabeled alfalfa plastid DNA (cpDNA) probes to Southern blots of restriction digests of the progeny DNA. Each probe revealed a specific polymorphism differentiating the parental plastid genomes. Of 212 progeny, 34 were heteroplastidic, with their cpDNAs ranging from predominantly paternal to predominantly maternal. Regrowth of shoots from heteroplasmic plants following removal of top growth revealed the persistence of mixed plastids in a given plant. However, different shoots within a green heteroplasmic plant exhibited paternal, maternal, or mixed cpDNAs. Evidence of maternal nuclear genomic influence on the frequency of paternal plastid transmission was observed in some reciprocal crosses. A few tetraploid F₁ progeny were obtained from tetraploid (2n=4x=32) Medicago sativa ssp. sativa x diploid (2n=2x=16) M. sativa ssp. falcata crosses, and resulted from unreduced gametes. Here more than the maternal genome alone apparently functioned in controlling plastid transmission. Considering all crosses, only 5 of 212 progeny cpDNAs lacked evidence of a definitive paternal plastid fragment.Contribution No. 89-524-J from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan     

Transgenic plant production mediated by Agrobacterium in Indica rice

Summary A reproducible system has been developed for the production of transgenic plants in indica rice using Agrobacterium-mediated gene transfer. Three-week-old scutella calli served as an excellent starting material. These were infected with an Agrobacterium tumefaciens strain EHA101 carrying a plasmid pIG121Hm containing genes for -glucuronidase (GUS) and hygromycin resistnace (HygR). Hygromycin (50 mg/l) was used as a selectable agent. Inclusion of acetosyringone (50M) in the Agrobacterium suspension and co-culture media proved to be indispensable for successful transformation. Transformation efficiency of Basmati 370 was 22% which was as high as reported in japonica rice and dicots. A large number of morphologically normal, fertile transgenic plants were obtained. Integration of foreign genes into the genome of transgenic plants was confirmed by Southern blot analysis. GUS and HygR genes were inherited and expressed in R1 progeny. Mendelian segregation was observed in some R1 progeny.Abbreviations GUS ß-glucuronidase - HygR hygromycin-resistance - AS acetosyringone     

DNA replication in soybean protoplasts and suspension-cultured cells: Comparison of exponential and fluorodeoxyuridine synchronized cultures

Cell-suspension cultures of soybean (Glycine max (L.) Merr., line SB-1) have been used to study DNA replication. Cells or protoplasts incorporate either radioactive thymidine or 5-bromodeoxyuridine (BUdR) into DNA. The DNA has been extracted as large molecules which can be visualized by autoradiography. Nuclei were isolated and lysed on slides thus avoiding degradation of DNA by a cytoplasmic endonuclease. The autoradiograms demonstrated that DNA synthesis occurs at several sites tandemly arranged on single DNA molecules separated by center to center distances ranging from 10 to 30 m. Velocity sedimentations through alkaline gradients confirm the lengths of the replicated regions seen in autoradiograms. By using velocity sedimentation it also has been possible to demonstrate that replication proceeds by the synthesis of very small (4–6S) DNA intermediates which join to form the larger, replicon-size pieces seen in autoradiograms. Both small (4–6S) and large (20–30S) intermediates are observed in synchronized and exponential cultures. However, after synchronization with fluorodeoxyuridine (FUdR) the rate of DNA synthesis is reduced. Since the size of intermediates is not reduced by FUdR treatment, it is concluded that the slower rate of replication results from a reduction in the number of tandem replication units but not in the rate at which they are elongated. After FUdR treatment, the density analogue of thymidine, BUdR, can be substituted for almost all of the thymidine residue in DNA, resulting in a buoyant density increase (in CsCl) from 1.694 to 1.747 g/cm³. Using this density analogue it is possible to estimate the amount of template DNA attached to new replication sites. When this is done, it can be shown that synchronized cells initiate replication at about 5,000 different sites at the beginning of S. (Each such site will replicate to an average length of 20 m.) Use of BUdR also substantiates that at early stages of replication, very small replicated regions (<8S) exist which are separated by unreplicated segments of DNA which replicate at a later time. Most of these conclusions agree with the pattern of DNA replication established for animal cells. However, a major difference appears to be that after prolonged inhibition of soybean cell replication with FUdR, very small, as well as replicon-size intermediates accumulate when replication is restored. This indicates that regulation of replication in these cells may be different from animal cells.Abbreviations BUdR 5-Bromodeoxyuridine - FUdR 5-Fluorodeoxyuridine     

Targeted inactivation of the tobacco plastome origins of replication A and B

According to the Kolodner and Tewari model [Kolodner, R.D. and Tewari, K.K. (1975) Nature, 256, 708.], plastid DNA replication involves displacement-loop and rolling-circle modes of replication, which are initiated on a pair of origins of replication (ori). In accordance with the model, such a pair of oris -oriA and oriB- was described in Nicotiana tabacum [Kunnimalaiyaan, M. and Nielsen B.L. (1997b) Nucl. Acids Res. 25, 3681.]. However, as reported previously, both copies of oriA can be deleted without abolishing replication. Deletion of both oriBs was not found [Mühlbauer, S.K. et al. (2002) Plant J. 32, 175.]. Here we describe new ori inactivation lines, in which one oriB is deleted and the other copy is strongly mutated. In addition, lines oriA and oriB were deleted from the same inverted repeat. In contrast to the expectations of the model, neither oriA nor oriB is essential. Some of the deletions led to reduced growth of plants and reduced plastid DNA copy number in later stages of leaf development. The gross structure of plastid DNA was unchanged; however, the location of the ends of branched plastid DNA complexes was different in the inactivation mutants. Taken together, the results indicate that there are additional mechanisms of plastid DNA replication and/or additional origins of replication. These mechanisms seem to be different from those found in eubacteria, which, according to the endosymbiont theory, are the progenitors of plastids.     

Genotypic variability for callus formation and plant regeneration in rice (Oryza sativa L.)

Summary Sixty rice varieties (Oryza sativa L.), belonging to three subspecies, japonica, indica and javanica (some japonicaXindica hybrids were included), were compared for their capacity for callus growth and plant regeneration. Tissue cultures initiated from mature seeds on Murashige and Skoog's (1962) medium with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) were transferred to a medium containing 0.02 mg/l 2,4-D and 10 mg/l kinetin, from which plantlets were regenerated. Large variabilities in callus growth and plant regeneration potentials were revealed among the varieties tested. Most japonica varieties formed a callus that weighed more than 100 mg per seed 30 days after inoculation, and showed a relatively high regenerative potential, whereas indica varieties, japonica-indica hybrids and javanica varieties showed poor callus growth and plant regeneration, although considerable varietal variation was observed in each subspecies. The callus growth potential was not correlated with the plant regeneration potential. Histological observations revealed that the epithelium cells of the scutellum mainly proliferated to form a callus, from which shoot and root primordia were differentiated independently from each other. The shoot primordia developed into plantlets when roots were formed adventitiously. In a few cases, shoots and roots were bilaterally initiated from a single primordium.     

Changes in protein and amino acid contents in two cultivars of rice seedlings with different apparent tolerance to cadmium

Changes in protein and amino acid contents in Cd-treated rice (Oryza sativa L.) seedlings of two cultivars were investigated. By assessing the decrease in chlorophyll content in the second leaves as an indicator of Cd toxicity, it was seen that cv. Tainung 67 (TNG 67) seedlings were apparently more tolerant to Cd than cv. Taichung Native 1 (TN 1). Following treatment with CdCl₂, protein content decreased with a progressive and substantial increase of protease activity and total amino acids in TN 1, but not in TNG 67. The patterns of individual amino acids in Cd-treated leaves of both cultivars were examined and, only in cv. TN 1 a substantial increase in the content of all amino acids analysed, except for methione, was recorded. The role of these changes in endogenous amino acids in Cd toxicity of TN 1 leaves is discussed.     

PFGE analysis of the rice genome: estimation of fragment sizes,organization of repetitive sequences and relationships between genetic and physical distances

Pulsed-field gel electrophoresis (PFGE) has been applied to analyze the rice nuclear genome. Probing 56 RFLP probes selected from the 12 rice chromosomes to PFGE blots of nine rare-cutting restriction enzymes revealed that there are relatively high numbers of rare-cutting restriction sites in the rice genome. The average sizes of restriction fragments detected by single-copy probes are smaller than 200 kb for all of the rare-cutting restriction enzymes examined. Sizes of fragments detected by repetitive probes are variable, depending on the probes analyzed. By using PFGE, a tandemly repeated sequence, Os48, was found to be tightly linked to telomeric tandem repeats but not physically linked to r5s genes with which sequence homology had been observed. Relationships between genetic and physical distances have been established for three different chromosomal segments. In these regions 1 cm corresponds to ca. 260 kb on average. Analysis of a cluster of RFLP markers on chromosome 3 revealed that genetically clustered RFLP markers are also physically closely linked, suggesting that clustering of genetic markers may result in part from uneven distribution of single-copy sequences.     

Foreign DNA introgression caused heritable cytosine demethylation in ribosomal RNA genes of rice

Significant cytosine demethylation in ribosomal RNA genes (18S or 25S) were detected in all four studied rice lines containing introgressed DNA from wild rice, Zizania latifolia Griseb. In each line, the changed RFLP (restriction fragment length polymorphism) patterns produced with the methylation-sensitive enzyme (HpaII) were identical between two randomly selected individual plants both within and between generations. This indicates that the methylation changes are non-random and stably inherited. Cytosine demethylation in ribosomal RNA genes could be a major cause for the drastically altered phenotypic variations observed in the introgression lines.     

琼脂糖凝胶电泳中DNA回收方法的比较

实验采用直接切取琼脂糖凝胶,进行Lambda DNA/EcoRI HindIII Marker的回收,将其与试剂盒回收进行比较,并对比切下的凝胶立即回收和在-20℃冰冻数小时回收的效果,结果表明实验采用的方法与试剂盒的比较产量相当,且重复性好,达到了分子生物学实验的要求.尤其实验方法在大片段的回收(21226bp)平均回收率是43.5956%与试剂盒的回收率:38.9761%相比有明显的优势,非常适合大多数实验室使用.     

Genetic analysis and gene mapping of a rice few-tillering mutant in early backcross populations ( Oryza sativa L.)

A rice mutant,G069, characteristic of few tiller numbers, was found in anther culture progeny from theF ₁ hybrid between anindica-japonica cross, Gui630×02428. The mutant has another two major features: delayed tillering development and yellowing apex and margin on the mature leaves. As a donor parent,G069 was further backcrossed with the recurrent parent,02428, for two turns to develop aBC ₂F₂ population. Genetic analysis in theBC ₂F₂ population showed that the traits of few-tillering and yellowing apex and margin on the mature leaves were controlled by one recessive gene. A pool of equally mixed genomic DNA, from few-tillering individual plants inBC ₂F₂, was constructed to screen polymorphism with simple sequence repeat (SSR) markers in comparison with the02428 genome. One SSR marker and three restriction fragment length polymorphism (RFLP) markers were found possibly linked with the recessive gene. By using these markers, the gene of few-tillering was mapped on chromosome 2 between RFLP marker C424 and S13984 with a genetic distance of 2.4 cM and 0.6 cM, respectively. The gene is designatedft1.     

Induction of a polyubiquitin gene promoter by dehydration stresses in transformed rice cells

The expression of the maize polyubiquitin gene promoter UBI1 in rice cells has been used to study the involvement of ubiquitin in cell protection responses to dehydration caused by osmotic, saline or freezing stress. The effect of these stresses on UBI1 activity was investigated by the use of stably transformed rice calli (UBI1:GUS), as well as by transient expression experiments performed with cell lines with high or low tolerance to each type of stress. The theoretical analysis of the UBI1 promoter shows several putative stress-regulated boxes that could account for the stress-related UBI1 induction pattern described in this work. We suggest that the study of the differential UBI1 promoter-driven expression in rice cell lines with different level of tolerance to stress might be useful to elucidate complex signal transduction pathways in response to dehydration stresses in monocots.     

Identification of specific proteins from seed embryos by two-dimensional gel electrophoresis for the discrimination between indica and japonica rice

Summary Proteins extracted from seed embryos of 29 different cultivated rice (Oryza sativa L.) and one wild rice (O. rufipogon Griff.) were compared by two-dimensional gel electrophoresis analysis. Among more than 300 protein spots on the gel we found some interesting variations in ten spots which were individually designated as proteins A-J. Protein E was observed in all indica cultivars but was not found in those of the subspecies japonica. In contrast, protein F was only detected in japonica cultivars. Protein A existed in all japonica cultivars but, with the exception of IR-36, could not be found in other indica cultivars. Therefore, proteins A, E and F can be used as markers for the identification of indica and japonica. Some so-called Javanica cultivars showed the characteristics of japonica subspecies with regard to proteins A and F, while one other cultivar of Javanica expressed a type intermediate between indica and japonica interms of proteins A and E. One feature discriminating between Javanica and japonica cultivars was found in the D, G, and J proteins which were expressed strongly in Javanica cultivars but were scarcely expressed in those of japonica. Expression of subspecies-specific proteins E and F in f₁ hybrids was also investigated.