An extra copy of a rice pistil chitinase gene, termed chip, which is expressed predominantly in floral organs just before anthesis, was introduced into an economically important cultivar of rice (Oryza sativa L. cv. Koshihikari) together with the hygromycin-resistance gene (hph) as a marker gene by co-transformation. Over 100 regenerated rice plants (R0) and their self-pollinated first and second generations (R1 and R2) were genotypically characterized by PCR-based genomic DNA analysis of the two transgenes to obtain stable transgenic lines, in which chip was inserted into a single locus separately from hph. We obtained homozygous chip-transgenic R1 lines free of hph that stably expressed the chip mRNA and protein in rice leaf tissue. Chitin-hydrolytic activity in the leaves of these transgenic lines was higher than in those of non-transgenic controls. The resistance of the transgenic lines to rice blast disease fungus (Magnaporthe grisea) was examined by smearing water agar mixed with overcrowded conidia (race 007.0) on mechanically injured leaves, showing tendency to blast-disease resistance. Thus, the genotype-based selection was demonstrated to be effective at obtaining genetically stable transgenic plant lines without marker genes.
We identified a 23 kDa protein expressed abundantly in young barley seedlings; this protein was named P23k. P23k was shown to have evolved in barley and its allied species such as wheat, oat and rye, suggesting that P23k might be closely related to a specific biological trait of winter cereals. To determine the function of P23k, we examined the expression and localization of P23k and its mRNA in barley seedlings. P23k mRNA was markedly expressed in the scutellum of imbibed barley seeds, and was synchronously down-regulated with consumption of the starchy endosperm. In addition, ABA and sugar treatment of the scutellum showed that expression of P23k is highly dependent on the supply of glucose or sucrose, both of which are starch catabolites. Furthermore, immunocytochemical analysis showed that P23k is primarily localized around the membranes, where sugar translocation is active. These results indicate that P23k plays a role in sugar translocation in young barley seedlings.
Fragments of different length (4 kb and 1 kb) of the 5′ upstream region of the Wiv-1 (Lin6) gene encoding a wound-inducible cell wall-bound acid invertase in tomato were fused to the beta-glucuronidase (GUS) gene and expressed in tobacco and tomato plants. The GUS activity detected was similar in all transformed lines, suggesting that the 1 kb region contains most of the regulatory motifs responsible for the characteristics of the Wiv-1 promoter. GUS activity was observed in vascular tissues of transgenic tomato and tobacco. In stem sections of transgenic tobaccos, the strongest activity was observed in the internal phloem of the nodes adjacent to the bases of petioles. The GUS activity in leaves of transgenic tobacco was elevated by addition of soluble sugars, especially sucrose. Previous studies suggested that the Wiv-1 enzyme participated in the regulation of carbohydrate contents in tomato leaves (Ohyama and Hirai 1999). The results obtained in the present study suggest that the Wiv-1 enzyme controls the loading of sucrose at the phloem of nodes and Wiv-1 regulation depends on the concentration of sugars in source leaves.
The 4′-O-glucosides of 2′,4,4′,6′-tetrahydroxychalcone (THC) and 2′,3,4,4′,6′-pentahydroxychalcone (PHC) are present in the yellow snapdragon (Antirrhinum majus) flowers and serve as direct precursors for the synthesis of aurones, the yellow pigments of the flowers. Despite the metabolic significance of these chalcones in aurone biosynthesis, the biochemical properties of chalcone synthase (CHS), the enzyme responsible for their synthesis, remains to be clarified. In this study, one known CHS cDNA (niv, also termed AmCHS1) and one CHS-related cDNA, AmCHS2, were isolated from yellow snapdragon buds. AmCHS1 mRNA specifically accumulated in the petals in a coloration-dependent manner, whereas AmCHS2 mRNA accumulated only negligibly in petals and other organs, corroborating the importance of AmCHS1 in chalcone synthesis in the yellow flower. Recombinant AmCHS1 that was heterologously expressed in Escherichia coli cells efficiently utilized both p-coumaroyl-CoA and caffeoyl-CoA (relative activity, 50% of the activity for p-coumaroyl-CoA) as a starting ester to produce THC and PHC, respectively. Previous studies predicted that a single Thr197Ala or the double Val196Met/Thr197Ala substitution in AmCHS1 may provide a wider space for hydroxycinnamoyl-group binding and might enhance the ability of this enzyme to produce PHC [Austin MB, Noel JP (2003) Nat Prod Rep 20: 79–110]. However, these substitutions led to a decreased PHC-producing activity, implying that other factors may also be important for the efficient utilization of caffeoyl-CoA.
A suspension cultured cell line was established from the cultivar of Rosa hybrida ‘Charleston’ as a study model to understand the response of the anthocyanin biosynthesis pathway to environmental cues. The major identified anthocyanin in cell cultures was cyanidin 3-glucoside (chrysanthemin). The anthocyanin yield was enhanced by culturing cells in the EM medium with added sucrose at high concentration under additional UV-B radiation to white light. Three cDNA fragments were cloned with degenerate primers by RT-PCR and the obtained sequences shared high homology with putative key enzymes (DFR, ANS, and UF3GT) of other species. The expression levels of these three genes were promoted under optimum conditions for anthocyanin accumulation. These results suggest that expression levels of these genes were closely correlated with a temporal buildup of anthocyanins in response to environmental factors.
Gene flow via pollen dispersal leading to the escape of transgenes is a potential concern associated with the introduction of transgenic plants. Therefore, it is necessary to clarify the relationship between pollen dispersal and outcrossing rate for strict biosafety management during risk assessment. Maize (Zea mays) is one of the crops most at risk for gene leakage via pollen flow into the environment. Here, we report the results of a cross-pollination field investigation using maize varieties showing a xenia effect with tricolor kernels, which allowed us to designate a pollen donor with natural outcrossing and one with reduced outcrossing (via a screened greenhouse) in the same experimental field at the same time. Although a previous study showed that a special screened greenhouse covered by 1-mm single fine mesh may be effective in reducing outcrossing in maize, we used 1-mm duplex fine mesh to reduce further the possibility of outcrossing. We report how a special screened greenhouse covered by 1-mm duplex fine mesh reduced pollen dispersal, and affected the outcrossing rate of non-genetically modified (GM) yellow maize in the greenhouse and white maize outside the greenhouse, compared to natural outcrossing.
In plants, salicylic acid (SA), a molecule important for resistance to pathogens, is synthesized from phenylalanine or isochorismate. Although SA is reportedly synthesized via the isochorismate pathway in pathogen-infected Arabidopsis, the predominant pathway in pathogen-infected tobacco has not been known. To determine the pathway in tobacco, we studied the gene expression and enzyme activity of phenylalanine ammonia-lyase (PAL) and isochorismate synthase (ICS) in tobacco leaves infected with Tobacco mosaic virus (TMV). Two days after TMV inoculation, necrotic lesions had appeared, and the levels of SA, PAL activity, and transcripts of PAL A and PAL B had increased substantially. In contrast, no ICS activity was detected, and the levels of ICS transcript did not increase, after the formation of necrotic lesions caused by TMV infection. These results suggest that SA is synthesized mainly by the phenylalanine pathway in TMV-infected and necrotic-lesion-bearing tobacco leaves.
The cellular-localization pattern of promoter activities of two phenylalanine ammonia-lyase (PAL; EC 22.214.171.124) genes, palg1 and palg2b, of a hybrid aspen, Populus kitakamiensis, was determined using the GUS reporter system. The strong activities of palg2b promoter were detected in lignified tissues such as xylem and phloem fiber cells of the aspen stem, suggesting the specific function of the PALg2b in lignin biosynthesis. Immunoelectron microscopy and sub-cellular fractionation of xylem cells showed that the PAL activity was detected in both the plastid and cytosol of the xylem ray-parenchyma cells and the cytosol in the developing xylem cells of the aspen mature stem. Our results of the biochemical characterization of xylem PAL protein suggested that the PAL localization was varied during cell differentiation of the aspen xylem to function in lignin biosynthesis.
The peroxidase (POD) reaction for the industrial synthesis of etoposide proceeds effectively when suspension-cultured plant cells are used as catalysts. Tea (Camellia sinensis) suspension cultures express higher POD activity and produce a higher yield of reaction products than those of other plant cells. We intend to investigate the conditions necessary to efficiently increase the POD activity in a suspension-cultured tea callus. Here we examined the influence of photoenvironmental conditions, phytohormones, and carbohydrates on POD activity in the callus grown on solidified medium. POD activity in the callus was increased 1) in the dark; 2) on medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) at 10 mg l−1; or 3) on medium containing 0.3 M sucrose and 0.1 M mannitol. The highest POD activity, achieved under the third set of conditions, was about 3.6-fold higher than that achieved on control medium (with 0.09 M sucrose).
High frequency in vitro plant regeneration via multiple shoots is reported from petioles of leaf explants of mungbean (Vigna radiata L.) on B5 culture medium supplemented with 6-benzyladenine (BA). The explants comprised of petiole cut at the node bearing the basal half of the lamina. Regeneration frequency varied with age of explant source and concentration of BA. Explants produced shoot-buds and shoots within 2 weeks. The explants harvested from 10-day-old seedlings produced higher number of shoots (8–9) than those from 4-day-old seedlings. Explants consisting of the petiole bearing either the entire lamina or atleast the basal half were more responsive than the explants without lamina. Histological studies confirmed the adventitious nature of shoot-buds. Shoots were rooted on B5 medium supplemented with 0.5 mg 1−1 indolebutyric acid. The plantlets, hardened and transferred to soil with 90% survival developed normal flowers and viable seeds. This efficient plant regeneration protocol can be used for genetic transformation to produce transgenic mungbean.
To establish a transient gene expression system for investigating the molecular function of genes in mature wheat seeds, we bombarded embryos or aleurones of three hexaploid wheat (Triticum aestivum L.) varieties Chinese Spring, RL4137 and OS21-5, two diploid wheat (T. monococcum L.) varieties KT3-1 and KT3-5 and a barley (Hordeum vulgare, L.) variety Himalaya with fusion constructs containing the GUS reporter gene driven by the Actin1 promoter or α-amylase promoter using a particle gun, and compared the efficiency of gene expression for the transient assay. The efficiency of transient expression was high in aleurone tissues of OS21-5, KT3-1, KT3-5 and Himalaya. In the aleurone tissues of KT3-5, the α-amylase promoter was especially markedly activated by gibberellic acid (GA) and by a GA-inducible MYB transcription activator of T. monococcum, TmGAMYB. This assay system using the aleurone tissues of KT3-5 may be especially useful for investigating which genes are expressed and how they are regulated in germinating wheat seeds.
γ-Glutamyltransferase (GGT) catalyzes the hydrolysis and transpeptidation of the γ-glutamyl moiety of γ-glutamyl peptides. Based on the N-terminal amino acid sequences of purified radish heterodimeric GGTs, we cloned and characterized three radish full-length cDNAs (RsGGT1, RsGGT2 and RsGGT3) encoding putative heterodimeric GGT isoforms. RsGGT proteins contained conserved amino acid residues that are required for the catalytic activity and the post-translational processing of GGT proteins in E. coli and mammals. Expression analysis indicated that RsGGT showed different organ expression patterns. The overexpression of RsGGT1 and RsGGT2 cDNAs, but not that of RsGGT3 cDNA, resulted in an increase of NaCl-extractable bound GGT activity in transgenic tobacco plants. These results suggest that RsGGT1 and RsGGT2 cDNAs encode heterodimeric bound GGT isoforms.
Two cDNAs (MdPAO1 and MdPAO2a) encoding polyamine oxidases (PAOs) were isolated from apple. Some notable features related to substrate specificity, catalytic activity and localization of MdPAO were predicted by comparison with those of other dicots- and monocots-PAOs. MdPAO was expressed in suspension cells at any time points tested during the culture period, but only in the young fruits among several organs analyzed.
The effects of photoperiod, light intensity and gibberellic acid (GA3) on adventitious shoot regeneration from spinach cotyledons were tested. The Analysis of Variance F test showed that the effects of photoperiod and GA3 on shoot regeneration were significant at high light intensity of 90–100 μmol m−2 s−1. The combined effect of optimum shoot regeneration and highest shoot multiplication was observed in explants of short day (SD)-grown seedlings cultured under the SD condition and at high light intensity, with 0.5 mg l−1 GA3 in Murashige and Skoog medium supplemented with 1 mg l−1 6-benzyladenine and 0.4 mg l−1 α-naphthaleneacetic acid. Scanning electron microscopy observation revealed multiple shoots regenerating directly from the basal end of cotyledon preceding callus. The regenerated shoots developed into normal plantlets and grew well upon transfer to pots. Some regenerated shoots flowered in vitro under the non-inductive SD condition.