Studies were undertaken to assess genetic relationships in seven species of Plantago and to evaluate the genetic variance within populations of P. ovata (Forsk.), P. indica (L.), P. arenaria (Waldst.), P. psyllium (Linn.), P. lanceolata (Linn.), P. serraria (Linn.) and P. coronopus (Linn.) by using random amplified polymorphic DNA (RAPD) markers. A total of 629 distinct DNA fragments ranging from 0.25 to >3.0 kbp were amplified using 75 selected random decamer primers. The cluster analysis indicated that the seven species of Plantago formed three major clusters: the first one consisted of three species and the second and third one represented by two species only. A maximum similarity of 85% was observed in P. arenaria and P. psyllium. Plantago indica shared up to 5% similarity with P. ovata. The wide genetic distance was observed within populations of different Plantago species. Thus, these RAPD markers have the potential for conservation of identified clones and evaluation of genetic relatedness among the species. This is also helpful in breeding programme and provides a major input into conservation biology.
The nitrobenzene oxidation method is widely used for structural analysis of lignin. However, the conventional nitrobenzene oxidation method has several drawbacks including the requirement of a sizeable amount of sample material and the rather slow completion of the reaction process. In this paper, we describe a microscale nitrobenzene oxidation method using deuterium-labeled vanillin and syringaldehyde as internal standards. Using this method, we show that the microscale nitrobenzene oxidation method realized high-throughput determination of lignin components using a small amount of sample, i.e. 10 mg per reaction, and high reproducibility. Due to the small sample sizes, the oxidation reaction and extraction steps of a number of samples can be completed in parallel, thus enabling us to process more than 40 samples per day.
A gram-negative bacterium, Mesorhizobium loti, contains a NADP+-malic enzyme (mlr5329) and a malate oxidoreductase (mlr0809) in the genome. We have screened transposon-induced mutants from the signature-tagged mutant library to survey their roles in nodule nitrogenase activity. The nodules induced by malate oxidoreductase mutants failed to fix N2, although NADP+-malic enzyme (NADP+-ME) mutants induced nodules exhibiting no change in nodule nitrogenase activity. When malate-degrading enzyme activities were compared between malate oxidoreductase mutants and wild-type M. loti, NAD+-malic enzyme (NAD+-ME) activity was decreased significantly in malate oxidoreductase mutants, suggesting it is an NAD+-ME mutant. We found that NADP+-ME was not required for N2 fixation. The fact that significant accumulations of sucrose, starch granules and malate were observed in the nodules induced by malate oxidoreductase mutant suggests that low nitrogenase activity of the nodules resulted in photosynthate accumulation. These data suggest that this malate oxidoreductase has a similar function to NAD+-ME in both Bradyrhizobium japonicum and Sinorhizobium meliloti.
Lignan is a large class of plant secondary metabolites, which has long attracted pharmacological interest because of its anti-tumor and estrogenic activities. Forsythia plants are known to produce a wide variety of lignans, such as (−)-matairesinol, (−)-secoisolariciresinol, (+)-pinoresinol, and (+)-phillygenin. The majority of such lignans are accumulated in glucoside forms. However, their glucosylation mechanisms largely remain to be elucidated. Here we describe the sequence, enzymatic activities, and gene expression profiles of UDP-sugar dependent-glycosyltransferases (UGT) from Forsythia koreana through a reverse-genetic approach. A Forsythia UGT, UGT71A18 protein, expressed in E. coli, preferentially glucosylated furofuran-class lignans, including (+)-pinoreisnol, (+)-epipinoreisnol, and (+)-phylligenin. Moroeover, the recombinant UGT71A18 exhibited specificity to UDP-glucose as a glycosyl donor. Gene expression analysis revealed that UGT71A18 is expressed predominantly in leaves and the suspension cell culture of F. koreana, and that the UGT71A18 transcript is upregulated in the transgenic cell culture expressing the RNAi construct of the pinoresinol lariciresinol reductase (PLR) gene, compared to non-transformants. These results are consistent with the remarkable elevation of pinoresinol glucosides in the PLR-RNAi lines. Collectively, the present data strongly suggests that UGT71A18, in part, is responsible for glucosylation of furofuran-class lignans, including (+)-pinoresinol and/or structurally related lignans in vivo.
Plants absorb and metabolize formaldehyde, a C-1 compound that is one of the main indoor air pollutants. To elucidate the molecular mechanism of formaldehyde metabolism in plants, we isolated formaldehyde-responsive genes from golden pothos by means of GeneFishing PCR. We focused on the immediate-early response genes following formaldehyde treatment. Two full length cDNA sequences corresponding to a putative class II chitinase and a hypothetical novel protein, which we termed DEG2, were generated by rapid amplification of cDNA ends (RACE). The chitinase, which we designated EaCHI1, was up-regulated in the leaves and stems, whereas DEG2 was up-regulated in the leaves and roots of formaldehyde-treated golden pothos. Phylogenetic analysis showed that putative class II chitinases were split into two groups, monocot and dicot. EaCHI1 belonged to the former group, occupying the most basal level among the monocot chitinases analyzed. The identification of chitinase as a formaldehyde-responsive gene suggests a novel physiological role for this enzyme in plant carbon metabolism and environmental responses. The DEG2 sequence was not similar to any known protein sequence.
Gene dosage and genetic background are factors that influence transgene expression in transgenic plants. In our previous studies, we produced transgenic tomato plants that accumulate miraculin, a taste-modifying protein, in a genetically stable manner. To elucidate the effects of gene dosage and genetic background on miraculin accumulation in transgenic tomato fruits, we generated hybrid tomato lines between the homozygous transgenic line 56B (background cultivar ‘Moneymaker’) and the pure cultivars ‘Micro-Tom,’ ‘Moneymaker,’ ‘Ailsa Craig,’ ‘M82,’ ‘Rutgers’ and ‘Aichi-first’ and analyzed them for miraculin mRNA expression and miraculin protein accumulation. The hybrid lines exhibited variation in their fruit structures. Among the hybrid lines heterozygous for the miraculin gene, miraculin accumulation in the fruits varied from 111.0 μg g−1 fresh weight (FW) to 159.4 μg g−1 FW. Furthermore, the homozygous line 56B showed higher miraculin accumulation and miraculin mRNA expression than the heterozygous line 56B×‘Moneymaker.’ These results demonstrate the profound effects of gene dosage and genetic background on miraculin accumulation in transgenic tomato fruits.
Transgenic products and the creation of new organisms with innovative transgenic traits generally raise risk assessment concerns because of potential risks to nontarget organisms. Cartagena Protocol on Biosafety and Japanese government regulations require scientific environmental risk assessments of living modified organisms prior to release to avoid adverse effects on the environment. Soil microorganisms, such as the arbuscular mycorrhizal fungi, aid in plant nutrient acquisition and protection from environmental stresses such as salt stress. In this study, we used a salt-tolerant transgenic Eucalyptus camaldulensis transformed with the mangrin gene from a mangrove plant and evaluated the interactions between environmental stress-tolerant transgenic plants and arbuscular mycorrhizal fungi. Our results indicated that these transformants were substantially equivalent to nontransformants in terms of arbuscular mycorrhizal fungi colonization under both saline and nonsaline conditions, and that the arbuscular mycorrhizal fungi colonization could potentially enhance the salt tolerance of the transgenic plant.
Mutant lines covering all Arabidopsis genes allow us to pursue systematic functional genomics. A comprehensive phenotypic description, called phenome, is highly sought after in the profiling of -omics. We previously selected 4,000 transposon-insertional lines with transpson insertion in their gene-coding regions, systematically observed and recorded the visible phenotype of aerial portion of each line (ecotype Nossen) (Kuromori et al. 2006). In this paper, we tried to observe root phenotypes of seedling in these transposon-insertional lines with transpson insertion in their gene-coding regions. For example, we measured the length of main root and the length of the root-hair. We totally observed root-phenotypes of 1817 lines, and identified seven phenotypes in roots. We also ordered alleles from Salk-knock-out lines (ecotype Col-0) to make sure that one line showed the similar phenotype among them.
The floral dip protocol mediated by Agrobacterium tumefaciens is the most widely used transformation method for Arabidopsis thaliana. The “floral dip” process in which A. thaliana flower buds are dipped in an Agrobacterium cell suspension requires large volumes of bacterial cultures grown in liquid media, large shakers and centrifuges, and experimental space for them. These factors limit the number of transformations that can occur at once. We established that A. thaliana can be transformed by inoculating 5 μl of Agrobacterium cell suspension in flower buds, thus avoiding the use of large volumes of Agrobacterium culture. Using this modified protocol, we obtained 15–50 transgenic plants per transformation from each pot containing 3 A. thaliana plants. The protocol is satisfactory to be used for subsequent analyses. This simplified method, without floral dipping, which requires large volumes of Agrobacterim culture, offers as efficient a transformation as previously reported protocols. This method reduces the required workload, cost, time, and space. Furthermore, an important aspect of this modified protocol is that it allows many independent transformations to be performed at once.
Protoplasts with a yield greater than 2.0×106 cells per 0.3 g fresh weight were isolated from leaves of four bamboo species: an herbaceous bamboo, Lithachne pauciflora, and three woody bamboos, Phyllostachys meyeri, Sasa jotanii, and Bambusa vulgaris. For aseptic protoplast isolation, folded leaves were sterilized in 70% ethanol, and the internal tissues were drawn out from the leaf sheath. The leaf material was incubated for 4 h in a modified White's medium solution containing 0.8 M mannitol (pH 5.8, 32°C) and an enzyme mixture containing 3% Macerozyme R-10, 2.5% Meicelase, and 2% Cellulase Onozuka for woody bamboos, and 2.5%, 2.5%, and 1% of these enzymes, respectively, for L. pauciflora. Protoplasts were filtered through a double-layered Miracloth funnel and then washed with 0.6 M to 0.4 M mannitol-containing White's medium (pH 5.8) prior to analysis. Protoplast viability averaged 83% as determined by FDA staining.
Somatic embryogenesis is a valuable tool for investigating the totipotency of plant cells. We have established a simple culture system for inducing somatic embryogenesis from germinating embryos of Arabidopsis thaliana. One day after sowing, germinating embryos of the Nossen accession were placed on agar-solidified Gamborg's B5 medium supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid. After 14 days, light-green somatic embryos had formed in the region around the shoot apical meristems. After transfer to phytohormone-free medium, the somatic embryos grew into seedlings with cotyledon-like organs and roots, and finally into mature plants. The expression of several embryo-specific genes was detected in somatic embryos, suggesting that the somatic embryos retain embryonic features. We examined the effect of genotype on the formation of somatic embryos from germinating embryos of 352 Arabidopsis accessions. The frequency of somatic embryo formation differed markedly between the accessions and ranged from 0 to 92%. This result indicates that somatic embryogenesis from Arabidopsis germinating embryos is strongly affected by genotype.
Rapid shoot regeneration system from callus cultures of a sugarcane (Saccharum officinarum L.) cultivar NiF8 was established. Apical meristematic tissues harvested from young sugarcane shoots were cultured on modified Murashige and Skoog medium containing 2 mg l−1 of 2,4-dichlorophenoxyacetic acid for callus induction. The sugarcane callus was then transferred onto media with different concentrations of thidiazuron (TDZ) (0.5, 1, 2 and 3 mg l−1) with or without 0.1 mg l−1 of 1-naphthaleneacetic acid (NAA) for shoot regeneration. The highest regeneration frequency (80.0%) was observed after three weeks of culture on medium containing 1 mg l−1 TDZ and 0.1 mg l−1 NAA. Histological observation showed that differentiation of proembryoid-like structure with pro-vascular strands were observed 3 days after transfer onto the optimum medium followed by formation of apical meristematic tissue and leaf-like structures after 5 to 7 days, suggesting that TDZ induced rapid shoot regeneration via a process similar to somatic embryogenesis. This study demonstrated a rapid, reproducible and efficient regeneration procedure of sugarcane, which is suitable for biotechnological application including genetic transformation.
The excessive amounts of nitrogen applied in current farming systems can cause environmental problems. There is therefore a need to improve the ability of crop plants to utilize nitrogenous fertilizers. We screened for nitrogen deficiency-tolerant lines among transgenic rice plants that overexpressed full-length cDNAs (FL-cDNAs) corresponding to low-nitrogen response genes, genes related to nitrogen metabolism, and genes related to carbon metabolism. We found that overexpression of OsCPK12 FL-cDNA, encoding a calcium-dependent protein kinase (CDPK), conferred tolerance to low-nitrogen stress in rice. After two weeks of low-nitrogen treatment, dry weights of shoots from OsCPK12-overexpressing plants were greater than those from control plants. Furthermore, total nitrogen contents of OsCPK12-overexpressing plants were higher than those of the control plants. Our findings suggest that OsCPK12 is involved in the signal transduction pathway(s) in the low-nitrogen stress response and may be useful in engineering crop plants with improved tolerance to low nitrogen levels.