Perennial plants such as the long-lived trees of boreal forest cycle between periods of active growth and dormancy. Transition from active growth to dormancy is induced by short day (SD) signal. Once dormancy is established, prolonged exposure to low temperature is required for breaking dormancy before warm temperatures can induce growth. We have studied global changes in gene expression in the apex of model plant hybrid aspen during the distinct stages of activity-dormancy cycle. Our data shows that all stages of activity-dormancy cycle in the apex are associated with substantial modulation of the transcriptome. Detailed analysis of core cell cycle genes indicates that with the exception of plant specific B-type CDKs, all of the other CDKs are regulated post-transcriptionally during growth cessation. SD signal appears to target the expression of cyclin genes that are down regulated during growth arrest. Several of the cold hardiness related genes e. g. dehydrins are induced during transition to dormancy although temperature is not reduced and the up-regulation of the expression of these genes does not appear to rely on SD mediated induction of classical CBF transcription factors. Our results suggest that transcriptional control plays a key role in modulation of hormones such as ABA and GA that are known to play a central role in various processes during activity-dormancy cycle. Analysis of histone and DNA modification genes indicates that chromatin remodeling could be involved in coordinating global changes in gene expression during activity-dormancy cycle.
A metabolic profiling approach using high performance liquid chromatography-mass spectrometry (LC-MS) was applied to seedlings of transgenic rice plants (Oryza sativa) that over-express the OASA1D gene encoding a feedback-insensitive α-subunit of anthranilate synthase (AS, EC 188.8.131.52). Analysis revealed that the seedlings accumulated tryptophan (Trp) at a high concentration without marked effects on the amounts of other major metabolites. Some minor indole metabolites showed a certain degree of increase in the amounts in Trp-accumulating tissues, while no active catabolic conversion of Trp was indicated. Analysis also revealed that the distribution of Trp in the plant was uneven, with the highest level being observed in young developing tissues, despite tissue-independent expression of the OASA1D gene under the control of ubiquitin promoter. Differences in AS activity and anthranilate content among organs were small. A feeding experiment with radiolabeled Trp clearly demonstrated one-way Trp movement from old to young leaves; thus, the uneven distribution of Trp in OASA1D plants is the manifestation of Trp translocation. The negligible effects of Trp accumulation in other metabolic pathways, low metabolic activity of Trp, and efficient translocation of Trp in rice plants expressing OASA1D transgene are favorable characters from the aspect of metabolic engineering of Trp production.
The combination of homologous and site-specific recombination, if possible, could create mutants, such as tissue- or growth phase- specific gene knockout. In higher plants, there is no information on the site-specific recombination of the endogenous gene locus modified by gene targeting (GT) because of still limited success of reproducible GT for the endogenous gene. Our method of rice GT mediated by a strong positive–negative selection is appropriate for combined use with site-specific recombination because, in our method, it is possible to manipulate genome sequence variously without any unintentional random transformation, including ectopic targeting. We had previously created a targeted-waxy mutant by inserting the hygromycin phospho transferase (hpt) with ΔEn (a functional transcriptional stop sequence from En/Spm transposon), into the Waxy intron 1 region upstream of the translational initiation site. Since this inserted sequence was sandwiched by a pair of loxP in the same orientation, here, we tested Cre-loxP mediated elimination of this targeted insertion. The Cre was transiently expressed by a highly active promoter of CaMV 35S with the castor bean catalase intron 1 in the calli of the targeted-waxy homozygote. Cells derived from the treated calli were grown independently and applied to PCR screening as a bulk of calli clumps. Although the efficiency was quite low, Cre-loxP mediated hpt-ΔEn elimination and Waxy reactivation in the pollen of regenerated plants were detected as expected in some cells.
A multidrug resistance associated protein homolog gene, named FeABCC1, was isolated from a commercial brand of common buckwheat that has high lead tolerance and accumulation ability. The deduced amino acid sequence of FeABCC1 had 64% identity with AtMRP3 (AtABCC3) of Arabidopsis thaliana, and was predicted as integral membrane protein with 12 transmembrane domains. FeABCC1 expression was induced in the shoot of common buckwheat treated with lead. The yeast transformed with FeABCC1 showed a marked increasing in lead tolerance with the gene expression, and accumulated more lead compared to control. Not similar to AtMRP3, FeABCC1 expression did not effect to cadmium tolerance in yeast, and inhibition of glutathione synthesis by buthionine sulfoximine was not effect lead tolerance. These data suggests that FeABCC1 has some role for lead detoxyfication in common buckwheat independently to glutathione chelating.
Agrobacterium tumefaciens-mediated transformation is the preferred method for genetic transformation of rice. Here we provide a comprehensive and high-through-put protocol for Agrobacterium-mediated transformation of rice for generating the large numbers of transgenic plants that are required for functional analysis and for evaluating traits of agronomic importance. In a project designed to produce nitrogen use efficient rice plants, we refined/improved several factors including optimization of conditions for inducing vir genes prior to transformation, infection and co-cultivation medium for better interaction of target tissues with Agrobacterium. These optimizations supported not only the survival of co-cultured calli in high frequency, but also the production of multiple resistant cell lines per co-cultured embryogenic and nodular callus. In addition, improvements in plant tissue culture, selection and regeneration media has enabled us to produce large numbers of transgenic rice plants containing genes of agronomical importance. Partial desiccation and ABA treatment to hygromycin-resistant callus tissue significantly (P<0.05) enhanced both regeneration frequency and regeneration of transgenic plantlets per callus tissue. Regeneration frequency was further improved by optimizing the concentration of copper sulphate in the regeneration medium. The majority of the transgenic plants obtained using this improved protocol displayed a normal phenotype and the gene of interest was inherited by the progeny in a Mendelian fashion. Homozygous plants of selected lines showed stable phenotypes both in soil and hydroponic conditions even after five generations. Availability of such a high-throughput Agrobacterium-mediated transformation system will improve future opportunities for rice genetics and functional genomics study.
Dried roots and stolons of licorice plants (Glycyrrhiza uralensis) are among the most important drugs (Glycyrrhizae radix) in traditional oriental medicine; they are also important commercial products used worldwide in sweetening and flavoring. Here, we describe the establishment of an in vitro stolon culture system for G. uralensis. Stolon formation was induced in single node stems (with axillary buds) grown in Murashige and Skoog (MS) liquid culture medium, supplemented with 0.01 μM α-naphthaleneacetic acid (NAA). Stolons were cultured at 26°C in the dark on a rotary shaker, gyrating at 100 rpm. The same NAA concentration produced the highest rates of stolon proliferation (6.58-fold in 4 weeks). A 6% sucrose concentration also enhanced stolon proliferation (6.34-fold in 4 weeks). GC/MS analysis confirmed the accumulation of small amounts of glycyrrhizin (14 μg g−1 dry weight) in cultured stolons. Interestingly, betulinic acid and oleanoic acid production in vitro were higher than in field-grown stolons. Adventitious root and shoot regeneration from cultured stolons were readily achieved under illuminated conditions in MS medium containing 0.01 μM of NAA and 0.2% gerlite. Regenerated plants produced glycyrrhizin (7,600 μg g−1 dry weight) in their roots. Our in vitro stolon culture system is suitable for studying glycyrrhizin biosynthesis and for rapid propagation of elite licorice clones.
High-temperature stresses during seed development reduces rice grain yield and causes poor milling quality because of insufficient grain filling. We monitored the progress of seed enlargement and the fluctuation in ATP content during seed development. When the rice cultivars Nipponbare and Taichung-65 seeds were developed in a high-temperature condition, a shortage in the amount of ATP was detected in the early and middle stages of seed development. This observation suggested that high temperature caused the insufficient accumulation of storage substances leading to the formation of small seeds along with chalky or white core endosperm. The flo2 mutant, which is thought to lack a regulatory factor for storage starch biosynthesis, produced chalky endosperm that resembled the typical phenotype of the high-temperature ripening-grain. The flo2 mutant exhibited a shortage of ATP content in immature seeds, suggesting that the gene responsible for the flo2 mutation is also involved in ATP production during seed development. In contrast, Kinmaze seeds maintained sufficient ATP content and their features did not differ significantly between mature grains developed in the normal- and high-temperature environments. These findings suggest that Kinmaze has some trait that is involved in the tolerance to high-temperature stress.
Previously, we showed that four mutated acetolactate synthase (ALS) genes derived from rice and Arabidopsis (W548L/S627IOsALS, S627IOsALS, W574L/S653IAtALS and S653IAtALS) confer high levels of resistance to pyrimidinylcarboxylate type ALS inhibitors (Kawai et al. 2008). Mutated ALS genes of rice were obtained from rice cells cultured in the presence of an ALS-inhibitor. The mutated ALS genes of Arabidopsis, which have the same amino acid substitutions as those of rice, have been generated artificially. Here, we demonstrate that these mutated genes function as effective selectable markers for transformation of Arabidopsis. Specifically, we studied expression of the mutated ALSs in Arabidopsis and their effect on the sensitivity of transgenic Arabidopsis plants to the ALS inhibitors. Our results show that the degree of resistance to the ALS inhibitors of transformants expressing Arabidopsis mutated ALSs was greater than those of transformants expressing rice mutated ALSs. The amino acid sequences of ALSs derived from monocotyledonous plants and those derived from dicotyledonous plants were clearly divided into two clusters in a phylogenetic tree. Based on these results, it would be preferable to use rice and Arabidopsis mutated ALS genes for generating monocotyledonous and dicotyledonous transgenic plants, respectively. Moreover, our findings are particularly useful when generating transgenic plants with a known ALS nucleotide sequence. In such cases, their own ALS gene carrying these mutations could be used as a selectable marker because amino acid residues at the point of mutation are conserved among plant species.
In rice, the developmental process in leaf formation can be divided into 7 stages (stages P0 to P6). We investigated chloroplast biogenesis and physiological changes in the developing leaves at the stage P4, during which leaf blade elongation and establishment of basic leaf blade structure occur. Chlorophyll content was negligible in the leaves early in the P4 stage and increased rapidly as they enters the late P4 phase. Chlorophyll fluorescence ratio (Fv/Fm) also increased markedly and the final value was comparable with that of mature leaves. Gene expression analysis showed that during the P4 stage, chloroplasts in the leaf cell undergo all three steps of differentiation: (i) plastid division and DNA replication (ii) establishment of plastid genetic system (iii) activation of photosynthetic apparatus. These observations suggest that the P4 is key in the development of a leaf, during which leaf rapidly differentiated both morphologically and physiologically, and that the P4 leaf is suitable for investigation of physiological relationships between chloroplast and leaf development.
In carrot suspension-cultured cells, the expression of phenylalanine ammonia-lyase 1 (DcPAL1) gene was crucially regulated by the transcription regulatory factor Daucus carota MYB1 (DcMYB1). To elucidate the regulatory mechanism of DcMYB1 expression, we isolated and identified the transcription regulatory factor D. carota ethylene-insensitive3 (EIN3)-like protein (DcEIL) from a cDNA library prepared from suspension-cultured carrot cells treated with the “dilution effect” using a yeast one-hybrid system. DcEIL bound to a region of the DcMYB1 promoter containing a putative cis-element, which suppressed DcMYB1 promoter expression. The amino acid sequence of DcEIL contained the predicted signal sequence for nuclear localization, and transportation to the nucleus was confirmed using a green fluorescent protein-DcEIL fusion protein expressed in suspension-cultured Arabidopsis thaliana cells. The DcEIL gene was constitutively expressed irrespective of the elicitor treatment or the dilution effect. These results suggest that the binding of DcEIL to a cis-element of the DcMYB1 promoter might be regulated at the posttranslational level as a consequence of the regulation of DcPAL1 gene expression via DcMYB1 expression.
We examined the effect of pretreatment on the frequency of flower-color mutants induced by ion beams. We found that petunia seedlings treated with 3% sucrose from 8 days after sowing accumulated significant amount of pigments within 4 days compared to non-treated control seedlings. The petunia seedlings treated with sucrose were exposed to 320-MeV carbon ions. The sucrose treatment did not affect the survival rate and seed fertility of the M1 plants. In the M2 lines obtained by self-pollination of individual M1 plants, chlorophyll mutants were obtained in both treated and non-treated groups with a similar frequency. Flower-color mutants that included magenta, purple, light pink and white were obtained from the original violet color. The frequency of flower-color mutants was significantly higher in the sucrose-treated group than in the non-treated group. These results suggest that sucrose pretreatment specifically increases the frequency of flower-color mutation following ion beam irradiation.
In order to compare the profiles of hydrophobic secondary metabolites between the calli and plants of Euphorbia tirucalli, we analyzed their free sterol and free triterpenoid contents by GC-MS. We obtained the calli by culturing E. tirucalli internodes successively on solidified B5 medium containing hormones. The aerial parts of the plants or calli were extracted with CHCl3-MeOH (2 : 1, v/v) and subjected to GC-MS analysis. In a plant extract, only two sterol peaks, for campesterol and β-sitosterol, and at least 11 peaks of the total ion chromatogram (TIC) with a [M+] of 426, including peaks for euphol, β-amyrin, and glutinol, were detected. The ratio of triterpenoid- and triterpenoid-like-peaks of TIC with a [M+] of 426 to the total of all peaks detected was ca. 86% (n=3) in the plant extract. In a callus extract, 6 sterols, campesterol, stigmasterol, β-sitosterol, isofucosterol, cycloartenol, and 24-methylenecycloartanol, and two triterpenoids with a molecular weight of 426 (euphol and β-amyrin) were detected. The ratio of triterpenoid- and triterpenoid-like peaks of TIC with a [M+] of 426 to the total of all peaks detected was only ca. 6.3% (n=3) in the callus extract. These results indicate that the ratio of free triterpenoids and free triterpenoid-like compounds (MW: 426) to free sterols is lower in the calli than in the plants of E. tirucalli.
Cytoplasmic male sterility (CMS) is a maternally inherited trait that results in the inability of plants to produce functional pollen. CMS is considered as a phenomenon caused by aberrant mitochondrial genomes. In BT-type CMS of rice (Oryza sativa L.), a CMS-associated mitochondrial gene has been reported to be an orf79 gene. To confirm the effect of ORF79 on cell fate, we introduced the orf79 gene with or without a mitochondrial targeting sequence into Taichung 65 carrying normal cytoplasm. It was revealed that ORF79 was toxic to plant regeneration when expressed as a fusion with mitochondrial targeting sequence of the ATPase γ subunit. This result implicates ORF79 as cytotoxic, and its toxicity depends on its combination with a mitochondrial targeting sequence.
Arabidopsis response regulators (ARRs) are involved in the His-to-Asp phosphotransfer signal transduction mechanism induced by cytokinins, a class of phytohormones in Arabidopsis thaliana. Previous studies have reported ARR mRNA accumulation in response to cytokinins. ARRs consist of 2 families (types A and B). The expression of type-A ARR genes can be induced by cytokinins, while that of type-B genes is constitutive. Recently, we have shown that plant growth induced by the endocrine disruptor bisphenol A (4,4′-isopropylidenediphenol) was similar to that induced by cytokinins. The present study is the first to show that ARR (type-A family; ARR15 and ARR16) mRNA accumulation is influenced by bisphenol A, indicating that bisphenol A is involved in inducing intracellular signal transduction.