Hairy roots were induced from leaf segments of Crotalaria spectabilis, which is used as a green manure crop antagonistic to nematodes, by infection with a mikimopine type wild strain of Agrobacterium rhizogenes A13 (MAFF02-10266). For initial proliferation of hairy roots after induction, addition of gibberellin A3 to the medium was indispensable. These roots exhibited vigorous growth and abundant lateral branching on half-strength Murashige and Skoog (1/2MS) medium without plant growth regulators. The adventitious shoots were induced from 24% of root segments 2 months after transfer onto B5 medium containing 5 mg l－1 6-benzyladenine. The axillary buds excised from these shoots produced roots 1 month after transfer onto 0.4% (wv－1) gellan gum-solidified 1/2MS medium containing 30 mM fructose without plant growth regulators. Regenerated plants were successfully grown under greenhouse conditions. Infection of hairy roots of C. spectabilis with root-knot nematodes Meloidogyne hapla and M. incognita led to the growth inhibition of these nematodes.
Seven sugarcane (Saccharum sp.) commercial cultivars, viz., UT-94-2-483, LK92-11, K84-200, K97-32, K95-84, K88-92 and K 92-80, originally derived from meristem cuttings were subjected to simulated osmotic stress (as 200 mM mannitol) under controlled environmental conditions. Proline content in the leaf tissues of all cultivars except K92-80, increased in plants subjected to mannitol-induced osmotic stress. Chlorophyll a (Chla), chlorophyll b (Chlb), total chlorophyll (TC), maximum quantum yield of PSII (Fv/Fm), and photon yield of PSII (ΦPSII) of all seven cultivars decreased under osmotic stress resulting in a reduction in net-photosynthetic rate (Pn). A positive correlation was found between Fv/Fm and ΦPSII, proline content and non-photochemical quenching (NPQ), ΦPSII and Pn, and Pn and plant dry weight in the sugarcane cultivars. Based upon Ward’s multivariate cluster analyses of data for proline content, photosynthetic capacity, chlorophyll fluorescence, and growth inhibition, three cultivars (K88-92, K92-80 and UT-94-2-483) were identified as water deficit sensitive, whereas four (K84-200, K95-84, K97-32 and LK92-11) as water deficit tolerant. These observations on different cultivar’s sensitivity/tolerance were confirmed by growth and yield attributes measured in a field trial. The plant dry weight (in vitro) correlated positively with total stalk weight of sugarcane cultivars
Heavy-ion beams are an effective mutagen for use in plant breeding and analyses of gene function. Heavy-ion beams have high linear energy transfer (LET) and thus they effectively induce DNA double-strand breaks. To rapidly evaluate effective LETs for inducing mutations, we investigated mutation frequencies in the M1 generation using the Arabidopsis heterozygous mutant of the ALBINO PALE GREEN 3 gene. Leaves of heterozygous plants in which intact alleles were mutated showed white sectors. We irradiated heterozygous plants with heavy-ion beams with LETs ranging from 22.5 to 61.5 keV µm－1 and doses ranging from 0 to 450 Gy. The results showed that the effect of LETs could be classified into three types: first, the LET of 22.5 keV µm－1 did not effectively induce mutations, regardless of the dose; second, LETs of 30.0 or 42.5 keV µm－1 were not effective at low doses (100–200 Gy) but were effective at 300 Gy; third, LETs of 50.0 or 61.5 keV µm－1 produced comparatively high mutation frequencies at all doses. Irradiation with 42.5–61.5 keV µm－1 reduced seed productivity at the dose providing the highest mutation frequencies (300 Gy). We concluded that an LET of 30.0 keV µm－1 was optimal for obtaining Arabidopsis thaliana mutants in the M2 generation.
Recent studies have revealed that β-amylases (EC 22.214.171.124) located in plastids participate in endogenous starch degradation. On the other hand, many plants possess a respectable amount of extraplastidic β-amylases. Because of their existence outside of the plastids, it has been believed that extraplastidic β-amylases do not contribute to starch degradation in plants. Here, we investigated the role of the major extraplastidic β-amylase of the radish (Raphanus sativus) (RsBAMY1) in starch metabolism. Amylase activity was elevated in the growing taproot of the radish. During the elevation of amylase activity, the starch content decreased, but the contents of soluble sugars such as maltose, glucose, fructose and sucrose increased. In the radish taproot, RsBAMY1 protein accumulated in the primary cambium and anomalous cambium of the storage parenchyma. The starch granules, however, were found just inside the cambium and adjacent to the anomalous cambium. When the RsBAMY1 gene was overexpressed in Arabidopsis, the plants contained lower starch contents than the wild-type plant. These results suggest that the extraplastidic β-amylase may affect the starch metabolism in some plants.
Chimeric REpressor gene-Silencing Technology (CRES-T) is a reverse genetic method that converts transcriptional factors (TFs) to chimeric repressors by fusion with an ethylene-responsive element-binding factor (ERF)-associated amphiphilic repression domain. The plant expressing chimeric repressor is expected to show loss-of-function phenotype of the original TF even in the presence of other functionally redundant TFs. We used the CRES-T system for modification of flower shape in transgenic Ipomoea nil (formally Pharbitis nil). I. nil is emerging as a model plant for ornamental flowers because it has produced a wide variety of historical mutants, and it has been chosen to be a part of the National BioResource Project in Japan. We used cDNAs of TFs of Arabidopsis thaliana with the CRES-T system because A. thaliana TFs are well characterized compared with I. nil TFs. For this study, we selected two TCP (TEOSINTE BRANCHED1, CYCLOIDEA, and PCF) TFs, TCP3 and TCP5, because overexpression of these chimeric repressors TCP3SRDX and TCP5SRDX causes severe morphological alterations in A. thaliana. We found that these chimeric repressors cause morphological alterations, an undeveloped corolla and wavy petals in I. nil. In addition, the sympetalous corolla was easily disrupted to form choripetalous corolla. Although several TCP3SRDX transgenic ornamental flowers were reported, typical sympetalous flowers like I. nil have not yet been presented.
High temperature stress during seed development can reduce the rice grain yield and lead to poor milling quality because of inadequate grain filling. We tested the high temperature stress tolerance of eight rice cultivars and fluctuations in the ATP content during seed development. The phenotype of Norin-22 was very sensitive to high temperature conditions, which decreased the grain yield and produced defective characteristics. A significant reduction in the ATP content was also detected in the developing seeds. Koshihikari and Nipponbare, which are progenies derived from Norin-22, are known to be sensitive to high temperature stress. It is suggested that the high temperature stress sensitivity of Norin-22 was inherited by these cultivars. Shinriki was tolerant of high temperature stress, and it produced a large proportion of normal-shaped grains under high temperature stress conditions, with only a moderate decrease in its ATP content. These results suggest that there was a relationship between ATP deficiency and defective endosperm characteristics. Therefore, it is expected that identifying a rice cultivar that produces an adequate amount of ATP during seed development will be useful for breeding a cultivar that tolerates high temperature stress.
(S)-Tetrahydroberberine oxidase is the enzyme in the last step of berberine biosynthesis. While a previous report described the isolation of cDNA of tetrahydroberberine oxidase (THBO) from cultured Coptis japonica cells, we later found that purified THBO was heavily contaminated by triosephosphate isomerase. Here, we report the re-isolation of THBO cDNA from cultured C. japonica cells and its functional characterization in transgenic California poppy cells. A cDNA clone for (S)-tetrahydroberberine oxidase was isolated from an EST library prepared from high berberine-producing cultured C. japonica cells based on the partial amino acid sequence of the purified enzyme. Analyses of the nucleotide sequences of the cloned cDNA inserts of 1728 base pairs revealed an open reading frame that encoded a 540-amino acid polypeptide with putative 28-amino acid signal peptides and a mature polypeptide with a molecular mass of 57,748. A protein blast search also shows that CjTHBO belongs to the FAD-containing berberine bridge enzyme oxidoreductase family. Since all attempts to produce active recombinant CjTHBO in Escherichia coli and Saccharomyces cerevisiae cells failed, we tried to express CjTHBO in California poppy (Eschscholzia californica) cells. When transgenic California poppy cells that ectopically expressed CjTHBO under the control of Cauliflower mosaic virus 35S promoter were established, the transgenic cells showed small but evident new alkaloid peaks, which were scarcely detected in control cells that did not express CjTHBO. LC-MS analyses showed that these peaks were coptisine and dehydrocheilanthifoline, which were expected to be generated by the reaction of CjTHBO from pathway intermediates, i.e., cheilanthifoline and stylopine. The usefulness of CjTHBO for metabolic engineering in transgenic California poppy cells is discussed.
In the plant cell cycle, a group of genes with mitotic functions is transcribed specifically during G2 and M phases. Such G2/M-specific genes are regulated by a common mechanism involving a cis-acting element called mitosis-specific activator (MSA) and R1R2R3-class Myb transcription factors that bind to the element. We have previously shown that a group of structurally related R1R2R3-Myb proteins acts as transcriptional activators, which include NtmybA1 and NtmybA2 from tobacco and MYB3R1 and MYB3R4 from Arabidopsis thaliana. Loss-of-function mutations in MYB3R1 and MYB3R4 showed reduced shoot elongation and significant impairment in cytokinesis during somatic cell division in A. thaliana. However, it remained unclear if this type of Myb transcription factors also have equivalent developmental roles in evolutionarily distant species. Here, we showed virus-induced silencing of both NtmybA1 and NtmybA2 causes similar defects in cytokinesis and severely dwarfed phenotype. Our results showed that a group of evolutionarily conserved R1R2R3-Myb transcriptional activators has common physiological functions in the promotion of cytokinesis and shoot elongation in different plant species.
Suspension-cultured plant cells are feasible bioreactors for the production of pharmaceutical proteins. Post-translational modification, such as glycosylation, is important for in vivo biological and physiological roles of proteins. However, little is known about the effect of suspension-culture conditions on protein N-glycosylation. In this study, we studied the N-glycan structures of soluble and endogenous glycoproteins at different growth stages of suspension-cultured Arabidopsis thaliana T87 cells. At the late growth stage, the amount of N-glycans with terminal N-acetylglucosamine (GlcNAc) decreased, whereas that of N-glycans with fucose and xylose increased. This indicates that N-glycans have more plant-specific sugar residues as the cultivation reaches the late or end stage of plant growth.
A high-throughput system for the evaluation of anti-herbivory genes in plant is proposed, in which genes are transiently expressed in a small area of leaf via Agrobacterium-infiltration, and the results can be obtained within 1 week. Transient expression of the cry1Ab gene, a well-known Bt toxin gene, in the leaves of tobacco (Nicotiana tabacum cv. Samsun NN) exhibited a lethal effect on both cotton cutworm (Spodoptera litura) and cabbage armyworm (Mamestra brassicae) larvae. In Arabidopsis leaves, this gene also provided potent inhibition of feeding of diamondback moth (Plutella xylostella) larvae. The expression of another anti-herbivory gene, MLX56, in tobacco and tomato (Solanum lycopersium cv. Micro-Tom) leaves resulted in enhanced mortality of M. brassicae and S. litura larvae. Significantly higher growth inhibition of S. litura larvae and feeding inhibition of P. xylostella larvae were also observed in tobacco and Arabidopsis leaves expressing the MLX56 gene. All results could be obtained within 5–7 days, indicating that this Agrobacterium-mediated transient gene expression system enables a high throughput evaluation of anti-herbivory genes.
Dioscorea batatas tuber lectin 1 (DB1) is a storage protein isolated from yam tuber and has been shown to be a mannose-binding lectin. Here we produced transgenic rice plants expressing cDNA of DB1 under the control of phloem-specific promoter of rice sucrose synthase-1 gene. DB1 accumulated at a level of 0.07% of total soluble protein. We then evaluated its efficacy for brown planthopper (BPH). After releasing the first instar of BPH on the transgenic rice plants, the number of survived BPH adults was reduced up to 30% compared to that of wild-type rice. The number of the next generation BPH was suppressed to 22% on average in the seven most-resistant plants compared to that of wild-type rice plants when female adult BPH was inoculated. These results demonstrated that DB1 is effective to confer BPH resistance in terms of decreased survival and fecundity.
Firefly luciferase (Fluc) is commonly used as a sensitive reporter for transient gene expression assays in plants. Although the protein level of Fluc can easily be quantified, it is difficult to quantify the mature mRNA level of the reporter gene in these assays. The mRNA level can be measured using quantitative reverse-transcription PCR (qRT-PCR), but the reaction often amplifies both the target mature mRNA and contaminating DNA or unspliced pre-mRNA, thus providing inaccurate results. To address this problem, we constructed a series of novel Fluc genes containing intronic sequences measuring 315 bp or longer. Here, we show that the contaminating DNA and unspliced mRNA were not PCR-amplified due to presence of these introns. Moreover, the intron-containing Fluc gene conferred superior Fluc activity to the original intron-less Fluc gene. We propose that the novel Fluc genes reported here can be used to quantitate both protein and functional mRNA levels in transient gene expression assays in plants.
To develop a model system for studying host tree–fungal pathogen interactions, we focused on Populus as a model tree and the fungus Colletotrichum gloeosporioides as a model pathogen. Although hybrid Populus is currently planted worldwide for biomass production and other purposes, the hybrid often lacks the tolerance of native species to cold, drought, and disease. Populus diseases have increased with the rapid development of Populus plantations worldwide. To investigate the susceptibility of hybrid aspen plants to the fungal pathogen Colletotrichum, which has a wide range of hosts, hybrid aspen plants were inoculated with Colletotrichum species such as C. dematium, C. destructivum, C. gloeosporioides, C. higginsianum, and C. orbiculare. C. gloeosporioides strains isolated from strawberry plants could infect hybrid aspen. These results indicate that monoculture of hybrid Populus may be potentially susceptible to local pathogens. The experimental system using hybrid aspen and C. gloeosporioides presented here will bring a lot of fruitful information, as first model system of host tree–fungal pathogen interactions.
Defense gene expression systems of higher plants responsible for protection against pathogen attack are predominantly regulated by salicylic acid (SA)- and jasmonic acid (JA)-mediated pathways, and control the expression of many downstream defense response genes. To monitor the regulated gene expression of SA-mediated signaling pathways, we previously described an assay system based on the bioluminescence of seedlings transformed with a promoter-luciferase fusion gene. Here, to develop a system suitable for JA-mediated gene expression monitoring, we compared the expression patterns of Arabidopsis gene promoters obtained from the plant defensin 1.2 (PDF1.2) and vegetative storage protein 1 (VSP1) genes in response to treatment with chemicals. Although both promoters responded well to treatment with JA in 3-week-old plants, only the VSP1 promoter exhibited marked and prolonged luciferase expression in response to JA treatment in 6-day-old seedlings. The use of transgenic Arabidopsis seedlings harboring the VSP1-luciferase reporter gene construct enables multiwell plates to be used for conducting high-throughput assays for the screening of chemicals that are involved in JA-mediated signaling pathways in Arabidopsis.