Integrated regulation of the photosynthetic gene family from Arabidopsis thaliana in transformed tobacco cells

Summary Expression of the three chlorophyll a/b binding protein (cab) genes of Arabidopsis thaliana was studied in transformed tobacco tissues. For each cab gene, approximately 1000 bp of the promoter region plus a portion of the structural gene was inserted into a promoter-expression vector such that a translational fusion between the cab gene and the promoter-less chloramphenicol acetyltransferase (cat) gene was formed. The constructed molecules were introduced into either cultured tobacco cells or tobacco leaves and the promoter activity was monitored as chloramphenicol acetyltransferase activity. The light-grown tissues exhibited 1.5- to 60-fold greater promoter activity than did dark-grown tissues. Expression of the cab promoters was tissue specific: activities were much stronger in green leaves than other tissues. The cab promoters were almost equally active in transformed calli or shoots derived from leaves. However, in cultured tobacco cells, one promoter was two to three times stronger than the other two. The chimeric gene fusion, cab-cat, segregated in the F1 generation as a dominant Mendelian trait.     

Three distinct regulatory elements comprise the upstream promoter region of the nopaline synthase gene

Summary Fine deletion mutants were generated in the upstream control region of the nopaline synthase (nos) promoter to define the position and role of upstream regulatory elements. The results indicated that the 8 bp sequence (CAGAAACC) at -106/-113 and its inverted repeat (GGTTTCTG) at -140/-147 are important for promoter function. The downstream element appears more important than the upstream element since deletion of the former reduced promoter activity more significantly than deletion of the latter. Deletion of the element alone, however, did not abolish promoter function, whereas, deletion of the 10 bp potential Z-DNA-forming (Z) element located between the repeat elements nullified promoter activity. Therefore, it appears that the Z element is an essential upstream regulator and the repeated elements are upstream modulators of the nos promoter. These elements are functionally distinct since alteration of stereospecificity or insertion of short oligonucleotides between the elements did not significantly influence promoter activity. These regulatory elements were unable to function from 200 bp upstream of the CCAAT-TATA box region.     

Pollen-specific expression of theArabidopsis thaliana α1-tubulin promoter assayed by β-glucuronidase,chloramphenicol acetyltransferase and diphtheria toxin reporter genespromoter assayed by β-glucuronidase,chloramphenicol acetyltransferase and diphtheria toxin reporter genes

We have characterized the promoter specificity of theArabidopsis thaliana α₁-tubulin (α ₁-tub) gene by studying expression patterns of gene fusions between the 2.2 kbp 5′ upstream region of theα ₁-tub gene and each of three different reporters: chloramphenical acetyltransferase, β-glucuronidase or the diphtheria toxin chain A gene. Analysis of transgenic tobacco andArabidopsis plants carrying the transgene showed that the chloramphenicol acetyltransferase and β-glucuronidase activities were not detected in any vegetative or reproductive organs except mature pollen. Transgenic tobacco plants carrying the diphtheria toxin chain A gene under the control of theα ₁-tub promoter were of normal phenotype but seed fertility was drastically reduced. Furthermore, the transgene could not be transmitted to the next generation through pollen, supporting the observation that theα ₁-tub promoter is active only in pollen. It was observed that the promoter activity was most active in mature pollen and decreased significantly duringin vitro pollen germination, indicating that the promoter is inactive or subdued in germinating pollen. The promoter activity was not affected by various plant growth hormones during pollen maturation.     

Fertile Transgenic Indica Rice Produced by Expression of Maize Ubiquitin Promoter-bar Chimaeric Gene in the Protoplasts

We report production of fertile transgenic Indica rice plants by transferring a chimaeric construct consisting of promoter, first exon and intron of maize ubiquitin gene (Ubi-1) and the coding sequences of the bar gene from Streptomyces hygroscopicus to the rice protoplasts through electroporation. In total, 11 plants were regenerated. All of them were fertile and set seeds on maturity. These plants were resistant to high concentration of PPT (400 mg l^?1) which was otherwise toxic to the untransformed controls. The gene was inherited to the progenies of the five plants in Mendelian ratio.     

The stability of foreign protein production in genetically modified plant cells

Summary The stability of foreign protein production in genetically engineered plant cells was studied. A cultured tobacco cell line was transformed with a chimeric molecule carrying a bacterial gene, ß-glucuronidase (GUS), under plant regulatory sequences. The specific GUS activity was monitored for 294 days with ten independently transformed cell lines either in the presence or the absence of selectable antibiotics. Specific GUS activity was stably maintained in five lines. About a two-to four-fold increase in the GUS activity was observed from three cell lines. The remaining two cell lines lost the activity within the first 70 to 210 days. The presence of antibiotics did not significantly alter the stability of the foreign protein production in all cell lines examined.     

Utilization of T-DNA tagging lines in rice

We have previously developed more than 100,000 T-DNA insertion mutant populations in japonica rice. These include simple knockouts as well as those for activation tagging. T-DNA insertion sites have been determined from more than 50,000 lines. The database for insertion positions is now open to the public, and these tagging lines are widely distributed to members of the rice research community. To utilize these genetic resources more efficiently, we are summarizing the important features of these tagging vectors, rice varieties, and flanking sequences. We also provide methods for handling such materials.     

Chromatin interacting factor OsVIL2 increases biomass and rice grain yield

Grain number is an important agronomic trait. We investigated the roles of chromatin interacting factor Oryza sativa VIN3‐LIKE 2 (OsVIL2), which controls plant biomass and yield in rice. Mutations in OsVIL2 led to shorter plants and fewer grains whereas its overexpression (OX) enhanced biomass production and grain numbers when compared with the wild type. RNA‐sequencing analyses revealed that 1958 genes were up‐regulated and 2096 genes were down‐regulated in the region of active division within the first internodes of OX plants. Chromatin immunoprecipitation analysis showed that, among the downregulated genes, OsVIL2 was directly associated with chromatins in the promoter region of CYTOKININ OXIDASE/DEHYDROGENASE2 (OsCKX2), a gene responsible for cytokinin degradation. Likewise, active cytokinin levels were increased in the OX plants. We conclude that OsVIL2 improves the production of biomass and grain by suppressing OsCKX2 chromatin.     

Isolation and Characterization of a Rice Cysteine Protease Gene, OsCP1, Using T-DNA Gene-Trap System

The T-DNA gene-trap system has been efficiently used to elucidate gene functions in plants. We report here a functional analysis of a cysteine protease gene, OsCP1, isolated from a pool of T-DNA insertional rice. GUS assay with the T-DNA tagged line indicated that the OsCP1 promoter was highly active in the rice anther. Sequence analysis revealed that the deduced amino acid sequence of OsCP1 was homologous to those of papain family cysteine proteases containing the highly conserved interspersed amino acid motif, ERFNIN. This result suggested that the gene encodes a cysteine protease in rice. We also identified a suppressed mutant from T2 progeny of the T-DNA tagged line. The mutant showed a significant defect in pollen development. Taken together, the results demonstrated that OsCP1 is a cysteine protease gene that might play an important role in pollen development.