Root-knot nematodes (RKN; Meloidogyne incognita) are phytoparasitic nematodes that cause significant damage to crop plants worldwide. Recent studies have revealed that RKNs disrupt various physiological processes in host plant cells to induce gall formation. However, little is known about the molecular mechanisms of gall formation induced by nematodes. We have previously found that RNA expression levels of some of genes related to micro-RNA, cell division, membrane traffic, vascular formation, and meristem maintenance system were modified by nematode infection. Here we evaluated these genes importance during nematode infection by using Arabidopsis mutants and/or β-glucronidase (GUS) marker genes, particularly after inoculation with nematodes, to identify the genes involved in successful nematode infection. Our results provide new insights not only for the basic biology of plant–nematode interactions but also to improve nematode control in an agricultural setting.
Bokashi fertilizer, an organic fertilizer made of plant residue, has been used in Japan not only to fertilize plants but to regulate their growth. Lactic acid bacteria have been found to play an important role in the fermentation process of Bokashi, but the relationship between these bacteria and plant growth activity has not been clarified. Using the adzuki rooting assay, this study identified 3-phenyllactic acid (PLA) produced by lactic acid bacteria as a root promoting compound in Bokashi. PLA showed synergistic effect with tryptophan, but no stem elongation activity. Lactic acid bacteria produced equal quantities of the L- and D-forms of PLA, which have similar root promoting activity. PLA did not significantly affect the amount of endogenous indole-3-acetic acid (IAA), although the chemical structure of PLA is highly similar to that of L-2-aminooxy-3-phenypropionic acid (L-AOPP), which inhibits IAA biosynthesis. These results indicate that the root promoting activity of PLA is not simply due to its increase in the amount of active auxin.
Tunisian pearl millet (Pennisetum glaucum L.) landraces are still growing in contrasting agro-ecological environments and are considered potentially useful for national and international breeders. Despite its genetic potential, the cropping areas of this species are still limited and scattered which increases the risk of genetic erosion. The chloroplast DNA polymorphism and maternal lineages classification of forty nine pearl millet landraces representing seven populations covering the main distribution area of this crop in Tunisia were undertaken based on informative cpSSR molecular markers. A total of 21 alleles combining to 9 haplotypes were detected with a mean value of 3.5 alleles per locus and a haplotype genetic diversity (Hd) of 0.82. The number of chloroplast haplotypes per population ranged from 1 to 4 with an average of 1.28. The haplotypes median-joining network and UPGMA analyses revealed two probable ancestral maternal lineages with a differential pearl millet seed-exchange rate between the investigated areas. Northern and Central populations presented unique genetic backgrounds while historical farmers’ practices in the South-East area resulted in the isolation of their own local landraces. The genetic evidences strongly support at least two introduction origins of pearl millet in Tunisia, one in the North and the other in the South followed by distinct local dispersal histories. Complementary in-situ and ex-situ conservation strategies taking into account the conservation of the maternal lineage cytoplasmic diversity are required. The investigated chloroplast SSRs provide useful molecular markers which could be used in further genetic studies and breeding surveys of pearl millet genetic resources.
Many abiotic stresses induce the generation of nitric oxide (NO) in plant tissues, where it functions as a signal molecule in stress responses. Plants modulate NO by oxidizing it to NO3− with plant hemoglobin (GLB), because excess NO is toxic to cells. At least eight genes encoding GLB have been identified in soybean, in three clades: GLB1, GLB2, and GLB3. However, it is still unclear which GLB genes are responsible for NO regulation under abiotic stress in soybean. We exposed soybean roots to flooding, salt, and two NO donors—sodium pentacyanonitrosylferrate (III) dihydrate (SNP) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP)—and analyzed expression of GLB genes. GmGLB1, one of two GLB1 genes of soybean, significantly responded to both SNP and SNAP, and its induction was almost completely repressed by a NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. GmGLB1 responded to flooding but not to salt, suggesting that it is responsible for NO regulation under NO-inducing abiotic stresses such as flooding. GmGLB3, one of two GLB3 genes of soybean, did not respond to NO donors at all but did respond to flooding, at a lower level than GmGLB1. These results suggest that flooding induces not only NO but also unknown factor(s) that induce GmGLB3 gene in soybean.
Japanese honewort (Cryptotaenia japonica) is consumed as a traditional vegetable and has medicinal applications. In Japan, C. japonica is mainly produced using hydroponic culture systems; however, damping-off is often caused by the adherence of pathogens to its seeds. Therefore, the use of sterile artificial seeds in hydroponic culture is likely to be effective for preventing disease. In this study, we established methods for stress-induced somatic embryogenesis and artificial seed production in Japanese honewort. Shoot apex explants from seedlings were treated with 0.7 M sucrose as a hyperosmotic stress for 3 or 6 weeks, and then transferred to stress-free conditions. Somatic embryos were formed after culture in stress-free conditions for 7 weeks. Stress-treated shoot apex explants that formed somatic embryos were cultured in Murashige and Skoog liquid medium with shaking. After 2 weeks of culture, approximately 800 somatic embryos were formed from each explant. Somatic embryos were formed continuously during 37 weeks under the same culture conditions. Thus, somatic embryogenesis was effectively induced in Japanese honewort via hyperosmotic stress, and embryogenic competence was maintained under stress- and phytohormone-free conditions. The somatic embryos produced by liquid culture were used to produce artificial seeds by enveloping the embryos in whipped alginate gel to avoid hypoxic conditions. The artificial seeds had a high germination rate (72%). This system is suitable for the sterile, highly productive hydroponic culture of Japanese honewort.
In vitro acclimatization has been validated as the successful key to harden the plantlets before transplanting to ex vitro conditions. In the present study, we investigated the potential of different sugar types (glucose, fructose, galactose, sucrose) in regulating morphological, physiological and biochemical strategies, survival percentage and growth performance, and rhizome traits of turmeric under iso-osmotic potential. Leaf greenness (SPAD value) in acclimatized plantlets (4% glucose; −1.355 MPa osmotic potential) of ‘ST018’ was retained and greater than in ‘PB009’ by 1.69-fold, leading to maintain high Fv/Fm (maximum quantum yield of PSII), ΦPSII (photon yield of PSII) and Pn (net photosynthetic rate) levels, and retained shoot height, leaf length, leaf width, shoot fresh weight and shoot dry weight after one month upon transplanting to ex vitro conditions. In addition, Pn, Ci (intracellular CO2), gs (stomatal conductance) and E (transpiration rate) in acclimatized plantlets (6% sucrose; −1.355 MPa osmotic potential) of ‘PB009’ were stabilized as physiological adapted strategies, regulating the shoot and root growth and fresh and dry weights of mini-rhizome. Interestingly, the accumulation of total curcuminoids in mini-rhizome derived from 6% sucrose acclimatized plantlets of ‘ST018’ was greater than in ‘PB009’ by 3.76-fold. The study concludes that in vitro acclimation of turmeric ‘PB009’ and ‘ST018’ using 6% sucrose and 4% glucose, respectively, promoted percent survival, physiological adaptations, and overall growth performances under greenhouse conditions.
Cuscuta campestris, a stem parasitic plant, commences its parasitic behavior by forming a specialized disk-like adhesive structure called a holdfast, which facilitates tight adhesion to the stem surface of the host plant. The morphology of epidermal cells in the holdfast is similar to that of the leaf trichome and root hairs of dicotyledonous plants. However, the regulatory network underlying the development of the holdfast has not been elucidated to date. In this study, we assessed the roles of epidermal cell-patterning genes in the development of a holdfast. Epidermal cell-patterning genes of C. campestris, including CcWER, CcGL3, CcTTG1, CcGL2, and CcJKD, were expressed slightly before the initiation of the outgrowth of stem epidermal cells. CcJKD-silencing repressed CcJKD, CcWER, CcGL3, CcTTG1, CcGL2; therefore, CcJKD is an upstream regulator of other epidermal cell-patterning genes. Unlike other genes, CcCPC was not upregulated after attachment to the host, and was not repressed by CcJKD-silencing. Protein interaction assays demonstrated that CcJKD interacted with CcTTG1 and CcCPC. Furthermore, CcJKD-silencing repressed the outgrowth of holdfast epidermal cells. Therefore, C. campestris invokes epidermal cell-patterning genes for the outgrowth of holdfast epidermal cells, and their regulatory mechanism is different from those for leaf trichome or root hairs.
Licorice (Glycyrrhiza uralensis) is a medicinal plant that contains glycyrrhizin (GL), which has various pharmacological activities. Because licorice is a legume, it can establish a symbiotic relationship with nitrogen-fixing rhizobial bacteria. However, the effect of this symbiosis on GL production is unknown. Rhizobia were isolated from root nodules of Glycyrrhiza glabra, and a rhizobium that can form root nodules in G. uralensis was selected. Whole-genome analysis revealed a single circular chromosome of 6.7 Mbp. This rhizobium was classified as Mesorhizobium by phylogenetic analysis and was designated Mesorhizobium sp. J8. When G. uralensis plants grown from cuttings were inoculated with J8, root nodules formed. Shoot biomass and SPAD values of inoculated plants were significantly higher than those of uninoculated controls, and the GL content of the roots was 3.2 times that of controls. Because uninoculated plants from cuttings showed slight nodule formation, we grew plants from seeds in plant boxes filled with sterilized vermiculite, inoculated half of the seedlings with J8, and grew them with or without 100 µM KNO3. The SPAD values of inoculated plants were significantly higher than those of uninoculated plants. Furthermore, the expression level of the CYP88D6 gene, which is a marker of GL synthesis, was 2.5 times higher than in inoculated plants. These results indicate that rhizobial symbiosis promotes both biomass and GL production in G. uralensis.
Heterosis refers to the improved agronomic performance of F1 hybrids relative to their parents. Although this phenomenon is widely employed to increase biomass, yield, and stress tolerance of plants, the underlying molecular mechanisms remain unclear. To dissect the metabolic fluctuations derived from genomic and/or environmental differences contributing to the improved biomass of F1 hybrids relative to their parents, we optimized the growth condition for Arabidopsis thaliana F1 hybrids and their parents. Modest but statistically significant increase in the biomass of F1 hybrids was observed. Plant samples grown under the optimized condition were also utilized for integrated omics analysis to capture specific changes in the F1 hybrids. Metabolite profiling of F1 hybrids and parent plants was performed using gas chromatography-mass spectrometry. Among the detected 237 metabolites, 2-oxoglutarate (2-OG) and malate levels were lower and the level of aspartate was higher in the F1 hybrids than in each parent. In addition, microarray analysis revealed that there were 44 up-regulated and 12 down-regulated genes with more than 1.5-fold changes in expression levels in the F1 hybrid compared to each parent. Gene ontology (GO) analyses indicated that genes up-regulated in the F1 hybrids were largely related to organic nitrogen (N) process. Quantitative PCR verified that glutamine synthetase 2 (AtGLN2) was upregulated in the F1 hybrids, while other genes encoding enzymes in the GS-GOGAT cycle showed no significant differences between the hybrid and parent lines. These results suggested the existence of metabolic regulation that coordinates biomass and N metabolism involving AtGLN2 in F1 hybrids.
Pollination is the crucial initial step that brings together the male and female gametophytes, and occurs at the surface of the stigmatic papilla cell in Arabidopsis thaliana. After pollen recognition, pollen hydration is initiated as a second critical step to activate desiccated mature pollen grains for germination, and thus water transport from pistil to pollen is essential for this process. In this study, we report a novel aquaporin-mediated water transport process in the papilla cell as a control mechanism for pollen hydration. Coupled with a time-series imaging analysis of pollination and a reverse genetic analysis using T-DNA insertion Arabidopsis mutants, we found that two aquaporins, the ER-bound SIP1;1 and the plasma membrane-bound PIP1;2, are key players in water transport from papilla cell to pollen during pollination. In wild type plant, hydration speed reached its maximal value within 5 min after pollination, remained high until 10–15 min. In contrast, sip1;1 and pip1;2 mutants showed no rapid increase of hydration speed, but instead a moderate increase during ∼25 min after pollination. Pollen of sip1;1 and pip1;2 mutants had normal viability without any functional defects for pollination, indicating that decelerated pollen hydration is due to a functional defect on the female side in sip1;1 and pip1;2 mutants. In addition, sip1;1 pip1;2 double knockout mutant showed a similar impairment of pollen hydration to individual single mutants, suggesting that their coordinated regulation is critical for proper water transport, in terms of speed and amount, in the pistil to accomplish successful pollen hydration.
Luciferases have been widely utilized as sensitive reporters to monitor gene expression and protein-protein interactions. Compared to firefly luciferase (Fluc), a recently developed luciferase, Nanoluciferase (NanoLuc or Nluc), has several superior properties such as a smaller size and stronger luminescence activity. We compared the reporter properties of Nluc and Fluc in rice (Oryza sativa). In both plant-based two-hybrid and split luc complementation (SLC) assays, Nluc activity was detected with higher sensitivity and specificity than that with Fluc. To apply Nluc to research involving the photoperiodic regulation of flowering, we made a knock-in rice plant in which the Nluc coding region was inserted in-frame with the OsMADS15 gene, a target of the rice florigen Hd3a. Strong Nluc activity in response to Hd3a, and in response to change in day length, was detected in rice protoplasts and in a single shoot apical meristem, respectively. Our results indicate that Nluc assay systems will be powerful tools to monitor gene expression and protein-protein interaction in plant research.
Allelopathic activities of three carotenoids of a natural pigment group, neoxanthin, crocin and β-carotene, were assayed by the protoplast co-culture method with digital image analysis (DIA-PP method). Effects on three different growth stages of lettuce protoplasts, i.e., cell wall formation, cell division, and yellow pigment accumulation, were investigated using 96-well culture plates. Cell division was inhibited 65–95% by all three carotenoids at 33–100 µM. Inhibition of cell division stage was stronger than at the cell wall formation stage in neoxanthin, and the water-soluble carotenoid, crocin, whose yellow pigment was incorporated into the vacuole of lettuce protoplasts. Neoxanthin at 33 µM and crocin at higher than 100 µM inhibited more than 100% of the yellow pigment accumulation. By contrast, at low concentrations (0.01–1 µM) β-carotene stimulated growth at the cell division stage. At high concentrations of β-carotene (100–500 µM), inhibition was prominent at all three stages, and also in neighboring wells of zero control, which suggested emission of a volatile compound by β-carotene. They were compared with the report of the volatile compound, tulipalin A. Differences in patterns of inhibition of carotenoids on lettuce protoplast growth were compared with reports of another natural pigment, anthocyanin, and anthocyanin-containing red callus cultured in the light, and with that of neoxanthin-containing yellow callus cultured in the dark.
The exine acts as a protectant of the pollen from environmental stresses, and the pollen coat plays an important role in the attachment and recognition of the pollen to the stigma. The pollen coat is made of lipidic organelles in the tapetum. The pollen coat is necessary for fertility, as pollen coat-less mutants, such as those deficient in sterol biosynthesis, show severe male sterility. In contrast, the exine is made of sporopollenin precursors that are biosynthesized in the tapetum. Some mutants involved in sporopollenin biosynthesis lose the exine but show the fertile phenotype. One of these mutants, cyp704b1, was reported to lose not only the exine but also the pollen coat. To identify the cause of the fertile phenotype of the cyp704b1 mutant, the detailed structures of the tapetum tissue and pollen surface in the mutant were analyzed. As a result, the cyp704b1 mutant completely lost the normal exine but had high-electron-density granules localized where the exine should be present. Furthermore, normal lipidic organelles in the tapetum and pollen coat embedded between high-electron-density granules on the pollen surface were observed, unlike in a previous report, and the pollen coat was attached to the stigma. Therefore, the pollen coat is necessary for fertility, and the structure that functions like the exine, such as high-electron-density granules, on the pollen surface may play important roles in retaining the pollen coat in the cyp704b1 mutant.
Mutations that reduce the expression of ribosomal proteins (RPs) or limit the activity of ribosome biogenesis-related factors frequently cause physiological and morphological changes in Arabidopsis. Arabidopsis OLI2/NOP2A, a homolog of yeast Nop2, encodes a nucleolar methyltransferase that is required for the maturation of the 25S ribosomal RNA of the 60S large ribosomal subunit. Mutant oli2 plants exhibit pointed leaves and shortened primary roots. In this study, detailed phenotypic analysis of oli2 mutant and OLI2 overexpressor lines revealed a range of phenotypes. Seeds produced by oli2 mutant and OLI2 overexpressor plants were lighter and heavier than wild-type seeds, respectively. Seeds of the oli2 mutant also showed delayed germination, whereas seeds from the OLI2 overexpressor lines germinated earlier than the wild type. The oli2 mutant also had fewer and shorter lateral roots than the wild type. The lateral root development phenotype in the oli2 mutant was similar to that of auxin-related mutants, but was not enhanced by exogenously supplied auxin. Furthermore, the oli2 mutant and OLI2 overexpressor lines were hypersensitive and less sensitive to high concentrations of sugar, respectively. Split-GFP-based bimolecular fluorescence complementation analysis revealed that OLI2 interacted with a nucleolar protein, BRX1-2, which is involved in rRNA processing for the large ribosomal subunit. Moreover, overexpression of OLI2 and BRX1-2 caused similar morphological changes, including extension of plant lifespans. These results suggest that the functions of OLI2 and its interactor BRX1-2 are intimately associated with a range of developmental events in Arabidopsis.
Glycyrrhiza plants are important resources for sweeteners and medicines, because underground parts of them contain glycyrrhizic acid (GL), which has sweet taste and various pharmacological activities (ex. anti-inflammatory, antiallergy, antiviral activity, etc.). Although such importance of them, their supply still depends principally on the collection of wild plants. Therefore, it is an important issue to develop stable and efficient production system of Glycyrrhiza plants. To overcome this problem, we established the hydroponic cultivation system of Glycyrrhiza uralensis and selected superior G. uralensis clones with high-GL contents in the containment greenhouse. In this study, we aimed to develop a method of selecting these superior G. uralensis clones by DNA sequence polymorphisms in biosynthetic genes. Among the DNA sequences of GL biosynthetic key enzyme gene (CYP88D6), we found Glycyrrhiza species and clone-specific polymorphisms in intronic regions. By using these polymorphisms, discrimination among Glycyrrhiza species and G. uralensis clones became possible. Furthermore, the appearance frequency of superior clone-specific alleles in cloned CYP88D6 sequences was correlated with GL contents in crude drugs collected from the Japanese market. We also observed the tendency that G. uralensis seedlings having superior clone-specific alleles of CYP88D6 gene showed higher secondary metabolite productivity than those without the alleles. These results indicated that superior clone-specific alleles of CYP88D6 gene could be applied as DNA markers for selecting G. uralensis clones accumulating high secondary metabolites.
In plant-pathogen interactions, pathogens employ secreted molecules, known as effectors to overcome physical barriers, modulate plant immunity, and facilitate colonization. Among these diverse effectors, some are found to mimic the plant peptides, to target host’s peptide receptors, and intervene in the peptide-regulated defense pathways and/or plant development. To better understand how pathogens have co-evolved with their plant hosts in order to improve disease management, we explored the presence of plant peptide mimics in microbes by bioinformatic analysis. In total, 36 novel peptide mimics belong to five plant peptide families were detected in bacterial and fungal kingdoms. Among them, phytosulfokine homologues were widely distributed in 22 phytopathogens and one bacterium, thereby constituted the largest proportion of the identified mimics. The putative functional peptide region is well conserved between plant and microbes, while the existence of a putative signal peptide varies between species. Our findings will increase understanding of plant-pathogen interactions, and provide new ideas for future studies of pathogenic mechanisms and disease management.
We examined the effects of five antimitotic agents using Antirrhinum majus L. ‘Maryland True Pink’ on the induction of adventitious shoots resulted in increase of frequencies of chromosome doubling without plant growth regulators. Seeds were treated in vitro with 0, 16.5, 32.9, 65.8, 131.6, or 263.2 µM oryzalin (ORY), amiprofos-methyl (APM), butamifos (BUT), or propham (IPC) or 800, 1,600, 3,200, 6,400, or 12,800 µM colchicine (COL) for 7 day. ORY, COL and APM promoted induction of adventitious shoots on the hypocotyls at maximum frequencies of 57.6% with 16.5 µM ORY, 5.6% with 800 µM COL and 88.8% with 131.6 µM APM. ORY and COL also induced adventitious shoots on the epicotyls adjacent to the cotyledons, particularly at high concentrations, with a maximum frequency of 26.0% at 12,800 µM COL. APM treatment increased frequencies of tetraploids from 0.0 to 93.1%, with a positive correlation between the frequency and concentration. By contrast, ORY and COL induced tetraploids at frequencies of 16.0 to 54.6% and 4.0 to 59.4%, respectively, with peaks at both low and high concentrations of each. Correlation analysis revealed that frequencies of adventitious shoot formation could be useful as an index for the induction of tetraploids. These results showed that three of the antimitotic agents tested induced both adventitious shoot and tetraploid without plant growth regulators, indicating that antimitotic action may play a common role in the induction of adventitious shoot.
Tomato transformation is conventionally performed using Agrobacterium tumefaciens-infected cotyledons. Here, we propose a simple procedure for tomato transformation, by which A. tumefaciens cells were smeared onto floral buds of a tomato plant using a paintbrush. Sufficient numbers of fruits were obtained from them, although the smearing of an excess number of A. tumefaciens cells led to an adverse effect on the plant growth. Progeny plants were screened by growth on a kanamycin-containing selection medium plate. The nptII gene was detected in 10 plants among 1,599 progenies. These transformants were derived from fruits other than those obtained from the smeared buds. This suggested that A. tumefaciens cells moved to the buds located near the smeared buds and caused the transformation event. Our findings suggest that this procedure can be used for the introduction of a foreign gene into plant cells.
Root-knot nematodes (RKNs, genus Meloidogyne) are a class of plant parasites that seek out and infect the roots of many plant species. The identification of RKN attractants can be used in agriculture in conjunction with nematode-trapping technology to redirect RKN movements and eventually reduce their prevalence in the field. Here, we discovered that some commercial silica gels can attract nematodes. Silica gels that attract nematodes contain calcium sulfate. Calcium sulfate and calcium carbonate showed strong nematode attraction properties. When plant seeds were surrounded by calcium sulfate or calcium carbonate, nematodes were not attracted to the plant seeds. We propose that calcium sulfate and calcium carbonate can be used in agriculture as a novel material to trap RKN.
The taste-modifying protein miraculin (MIR) has received increasing interest as a new low-calorie sweetener. In our previous study using the tomato variety ‘Micro-Tom,’ it was shown that in transgenic tomatoes in which MIR was expressed by using the cauliflower mosaic virus 35S promoter (p35S) and a heat shock protein terminator (tHSP) cassette (p35S-MIR-tHSP), higher levels of miraculin accumulated than when MIR was driven by the nopaline synthase terminator (tNOS) cassette (p35S-MIR-tNOS). ‘Micro-Tom’ is a dwarf tomato used for research and shows a low yield. To achieve high productivity of MIR, it is essential to improve the MIR accumulation potential by using high-yielding cultivars. In this study, we evaluate whether the high MIR accumulation trait mediated by the tHSP appears even when fruit size increases. A line in which the p35S-MIR-tHSP cassette was introduced into a high-yielding variety was bred by backcrossing. The line homozygous for MIR showed higher accumulation of MIR than the heterozygous line. Despite large differences in fruit size, the MIR level in the backcross line was similar to that in the p35S-MIR-tHSP line (background ‘Micro-Tom’). It was approximately 3.1 times and 4.0 times higher than those in miracle fruits and the p35S-MIR-tNOS tomato line 5B (‘Moneymaker’ background, which exhibits the highest miraculin productivity achieved thus far), respectively. These results demonstrate that the high MIR accumulation trait mediated by the tHSP appears even when fruit size is increased.
Depository of low-molecular-weight compounds or metabolites detected in various organisms in a non-targeted manner is indispensable for metabolomics research. Due to the diverse chemical compounds, various mass spectrometry (MS) setups with state-of-the-art technologies have been used. Over the past two decades, we have analyzed various biological samples by using gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, or capillary electrophoresis-mass spectrometry, and archived the datasets in the depository MassBase (http://webs2.kazusa.or.jp/massbase/). As the format of MS datasets depends on the MS setup used, we converted each raw binary dataset of the mass chromatogram to text file format, and thereafter, information of the chromatograph peak was extracted in the text file from the converted file. In total, the depository comprises 46,493 datasets, of which 38,750 belong to the plant species and 7,743 are authentic or mixed chemicals as well as other sources (microorganisms, animals, and foods), as on August 1, 2020. All files in the depository can be downloaded in bulk from the website. Mass chromatograms of 90 plant species obtained by LC-Fourier transform ion cyclotron resonance MS or Orbitrap MS, which detect the ionized molecules with high accuracy allowing speculation of chemical compositions, were converted to text files by the software PowerGet, and the chemical annotation of each peak was added. The processed datasets were deposited in the annotation database KomicMarket2 (http://webs2.kazusa.or.jp/km2/). The archives provide fundamental resources for comparative metabolomics and functional genomics, which may result in deeper understanding of living organisms.
Epigenetic modifications, including DNA methylation, are involved in the regulatory mechanisms of gene expression in animals and plants. In this study, we investigated whether the action of 5-azacytidine (5-aza-Cd), which is a well-known DNA methylation inhibitor, in suspension-cultured tobacco cells is affected by treatment with nucleoside derivatives of 5-methylcytosine (5-mCs), namely 5-methylcytidine (5-mCd) and 5-methyl-2′-deoxycytidine (5-mdCd). In a tobacco cell line, 5-aza-Cd treatment reactivated an epigenetically silenced transgene containing the cauliflower mosaic virus 35S promoter fused to the β-glucuronidase coding region and the nopaline synthase polyadenylation signal. The reactivation was evident on the fifth day of treatment and was augmented during culture with application of 5-aza-Cd at every subcultivation. This treatment, provided only once in the initial culture, resulted in transient transgene reactivation, followed by attenuation of its activity. The reactivation induced by 5-aza-Cd was suppressed by concomitant treatment with either 5-mCd or 5-mdCd. These results suggest that the 5-mCs derivatives inhibit and/or reverse 5-aza-Cd-induced reactivation of a silent transgene in tobacco cells.
The Arabidopsis T87 cell line has been widely used in both basic and biotechnological plant sciences. Agrobacterium-mediated transformation of this cell line was reported to be highly efficient when precultured in Gamborg’s B5 medium for a few days. However, because we could not obtain the expected efficiency in our laboratory, we further examined the preculture conditions of Arabidopsis T87 cells in the Agrobacterium-mediated transformation. As a result, we found that preculture in an excess amount of Murashige and Skoog (MS) macronutrients before cultivation in the B5 medium enhanced the transformation efficiency up to 100-fold, based on the transformed callus number on selective gellan gum plates. In this study, transformants were labeled with green fluorescent protein (GFP), and we found multiple fluorescent spots on individual transgenic calli. Therefore, the actual number of transgenic clones seems much more than that of transgenic calli. In our MS macronutrient-rich culture condition, T87 cells tended to aggregate and formed bigger cell clumps, a change that might be related to the enhancement of transformation efficiency. Based on these results, we report an improved protocol of Agrobacterium-mediated transformation of Arabidopsis T87 cells with high efficiency.
A stable salt-tolerant cell-suspension culture of Alluaudiopsis marnieriana was established, and intracellular compounds that accumulated under salt-stress conditions were investigated. HPLC/MS, and NMR analyses indicated that enhanced accumulation of coniferin was found during the growth phase in medium containing 150 mM NaCl. Coniferin or its derivatives may play an important role in salt-tolerance mechanisms in this plant.