A highly regenerating cotton (Gossypium hirsutum L.) cultivar, Coker 310FR, was used to generate transgenic plants expressing the herbicide resistance gene, bar, encoding phosphinothricin acetyltransferase (PAT), under the transcriptional control of the ribulose-1, 5-bisphosphate carboxylase (Rubisco) small subunit (rbcS) atslA gene promoter from Arabidopsis thaliana. Expression levels of the rbcS ats1A-bar transgenes were compared to bar transgenes under the control of the high level constitutive promoter from the Cauliflower Mosaic Virus 35S gene containing a dual enhancer region (2xE CaMV 35S). Significantly higher levels of bar mRNA, PAT protein and enzymatic activity, and enhanced levels of resistance to the herbicide Basta were observed in transgenic plants expressing bar under the rbcS ats1A promoter compared to the 2xE CaMV 35S promoter. Transgenic plants containing 2xE CaMV 35S-bar transgenes tolerated the maximum herbicide (Basta) application up to 200 mg l-1 PPT whereas rbcS ats1A-bar transgenic plants were capable of detoxifying Basta up to 400 mg l-1 PPT. These findings indicate that the rbcS ats1A promoter may be useful for higher expression of transgenes in developing tissues of cotton for improving it further through genetic engineering.
Directional growth of plant cells requires proper control of cortical microtubule organization, for which a novel Arabidopsis microtubule-localizing protein SPIRAL1 plays an important role. To better understand cellular functions of SPIRAL1, we here identified two highly homologous Arabidopsis membrane proteins (SPI1 and SPI2) that interact with SPIRAL1 in a yeast two-hybrid assay. Disruption of either SPI1 or SPI2 genes enhanced the right-handed helical growth phenotype of spiral1, whereas a double mutant of null spi1 and spi2 alleles appeared to be embryonic lethal. The SPI proteins potentially link SPIRAL1-dependent microtubule functions to endomembrane compartments.
Putrescine N-methyltransferase (PMT) catalyzes the first committed step of nicotine biosynthesis in tobacco. Tobacco PMT genes are activated in the root after wounding or by jasmonate treatment. In this study, a dual luciferase transient expression assay was used to show that the promoter of Nicotiana sylvestris NsPMT2 gene is induced by methyljasmonate in tobacco BY-2 protoplasts. An 80-bp TATA-proximal region was necessary and sufficient for the jasmonate response. We further demonstrated that a tetramer of the 24-bp sequence including a T/G-box within the region confers jasmonate responsiveness to a cauliflower mosaic virus 35S minimal promoter.
Camptothecin-derived compounds are widely used for clinical treatment of human cancer. They are synthesized from natural camptothecin, which is obtained by extraction from intact plants. For the feasible production of camptothecin, tissue cultures of Ophiorrhiza liukiuensis and O. kuroiwai, an interspecies hybrid of O. liukiuensis and O. pumila, have been investigated. The aseptic plants and hairy roots of O. liukiuensis and O. kuroiwai were established in addition to the previously-established O. pumila. The camptothecin production by O. kuroiwai was better than that by O. liukiuensis. 10-Methoxycamptothecin was accumulated in tissue cultures of O. liukiuensis and O. kuroiwai but not in O. pumila. Methyl jasmonic acid slightly enhanced the production of camptothecin in the O. liukiuensis hairy roots. These results indicate that the tissue cultures of O. liukiuensis and O. kuroiwai would be the feasible ways for production of camptothecin and related alkaloids.
Occurrence of endopolyploid cells in somatic tissues of spinach (Spinacea oleracea L.) was investigated by flow cytometry. Endopolyploidy was not present in embryos during imbibition of seeds. Rapid endopolyploidization occurred in seedlings during germination. Spinach appears to become endopolyploid by repeated rounds of replication of its entire genome. Spinach contained cells with six ploidy levels that correspond to 2C, 4C, 8C, 16C, 32C and 64C, where C is the haploid nuclear genome complement. The endopolyploid nuclei fall into clear ploidy series (2C, 4C, 8C, 16C...). Therefore, the process of endopolyploidy corresponds to endoreduplication.The patterns of endopolyploidy was characteristic of tissue type and developmental stage. However, endopolyploidy was not observed in apical meristematic tissues. Endopolyploidization may give rise to genetic plasticity in spinach.
The E2F transcription factors play important roles in the regulation of gene expression at the G1/S transition in plants. Here, we show that the rice proliferating cell nuclear antigen (PCNA) promoter is activated by transient expression of tobacco NtE2F and Arabidopsis AtDPa in tobacco cells. This transcriptional activation is repressed by co-transfection with a plasmid encoding the tobacco Rb-related protein (NtRBR1), whereas further co-expression of cyclin D overcomes this repression. Importantly, the rice PCNA promoter is activated when cells are transfected with cyclin D alone, and this activation is enhanced by co-transfection with plasmids encoding NtE2F and AtDPa. These results suggest that the effect of cyclin D expression is mediated not only by its associated kinase, which allows it to phosphorylate NtRBR1 thereby releasing the NtE2F/NtDP complex to activate transcription, but also by a mechanism which does not involve transfected NtRBR1.
Cabbage (Brassica oleracea L.) seedlings pursue two contrasting morphogenetic patterns, depending on the light environment. In the light, cabbage seedlings show short hypocotyls and open, unfolded cotyledons. In darkness, seedlings have markedly elongated hypocotyls and folded cotyledons. Durig hypocotyl growth, a majority of cells goes through endoreduplication. In light-grown hypocotyls, up to three cycles of endoreduplication occur, whereas in darkness about 10% of the total cells undergo the fourth cycles of endoreduplication. In both light and dark conditions, two cycles of endoreduplication take place prior to any significant hypocotyl growth. In darkness, the fourth cycle is completed very early during hypocotyl growth. These results support the view that endoreduplication may be a developmental programs in cabbage plants.
Four species of Curcuma, C. longa, C. amada, C. aromatica and C. zedoaria were collected from different parts of West Bengal, India and propagated in our experimental garden. In vitro regeneration of C. longa and C. aromatica was carried out from nodal explants and that of C. zedoaria from rhizome explants. Shoots were successfully regenerated from both nodes and rhizomes in C. amada. Plants regenerated in vitro produced rhizomes when planted in pots containing sterile soil. Curcumin contents in rhizomes of these plants were determined by spectrophotometric analysis. All accessions of C. longa uniformly showed a high curcumin content, while C. amada and C. zedoaria did not. A novel accession of C. aromatica (accession number v10) was found to contain curcumin even higher than that of C. longa, suggesting it to be useful as an alternative source of curcumin.
The bio-active beads system has been verified as a novel transformation method. To enhance its versatility, we applied this method to the transformation of a monocot plant, rice. The transient GFP expression was observed 24 h after transformation. This indicates that the method can also be applied to monocotyledons.
Bisphenol A (4, 4'-isopropylinediphenol), an intermediate in the production of polymers and polycarbonates and an ingredient used in plastic dental fillings, is known to be an endocrine disruptor. Bisphenol A has been shown to exert estrogenic effects in animals, but its effects in plants were not known. We thus examined whether it has phytohormone effects. A cytokinin bioassay system that assessed the growth of soybean (Glycine max cv. ‘Acme’) calli showed that bisphenol A stimulated growth, and had maximum activity at a concentration of 10-1μg ml-1. Bisphenol A also induced shoot differentiation in carrot (Daucus carota L. var. sativa DC) calli. Thus, bisphenol A showed cytokinin-like activity.
In carrot (Daucus carota L.) somatic embryos, desiccation tolerance is induced by treatment with abscisic acid (ABA). Six cDNA clones that showed ABA-enhanced expression were isolated from carrot by differential screening with ABA-treated and ABA-untreated somatic embryos, and they were named the CAISE (carrot ABA-induced in somatic embryos) genes. Five of the clones encode late embryogenesis abundant (LEA) proteins, and the other clone encodes a glucose and ribitol dehydrogenase. The expression of the CAISE genes was detected in maturing seeds, embryogenic cells, and ABA-treated somatic embryos in which exhibit desiccation tolerance induced by endogenous or exogenous ABA. These results indicate that ABA-induced desiccation tolerance in carrot somatic embryos may be induced by the LEA proteins and the glucose and ribitol dehydrogenase encoded by these ABA-inducible genes.