We report the isolation and characterization of three cDNAs encoding cinnamyl alcohol dehydrogenase (CAD) from Carthamus tinctorius (safflower). All three recombinant CADs were able to reduce coniferaldehyde and sinapaldehyde into coniferyl alcohol and sinapyl alcohol, respectively, and were designated as CtCAD1, CtCAD2, and CtCAD3. Phylogenetic analysis of CAD amino acid sequences and homology modeling revealed that CtCAD1 and CtCAD3 were closely related to the sinapaldehyde-specific aspen (Populus tremuloides) sinapyl alcohol dehydrogenase (PtreSAD). CtCAD2 was in a clade containing class I plant CADs. Gas chromatography-mass spectrometry-based kinetic analysis using two different substrates, coniferaldehyde and sinapaldehyde, indicated that the CtCADs showed no strong preference for either substrate. CtCAD2 has the highest catalytic efficiency (kcat/Km) (81.49 mM−1 min−1 and 95.3 mM−1 min−1 for coniferaldehyde and sinapaldehyde, respectively) compared with the other CtCADs. Inhibition kinetics showed that coniferaldehyde was a stronger inhibitor than sinapaldehyde for all CtCADs. Quantitative real-time PCR revealed that CtCAD2 was expressed at higher levels than CtCAD1 and CtCAD3 in all samples, except developing seeds at 3 days after flowering, where CtCAD1 had a higher expression level. In plant protein assays with coniferaldehyde and sinapaldehyde, plant protein extracted from seeds at 7 days after flowering, showed the highest specific activity. The product yields in plant protein assays were strongly correlated with gene expressions of CtCAD2 and CtCAD3 in the respective organs.
Oils and proteins represent two major carbon reserves within oil seeds. Attempts to increase seed oil production through the genetic suppression of seed protein, however, have generally been unsuccessful. In those experiments, the total protein content remains stable because of compensation by storage proteins from different gene families. Arabidopsis thaliana may offer a solution to this problem, as only a small number of 12S-globulin and 2S-albumin proteins, which are major seed storage proteins, are found in these seeds. We obtained T-DNA-tagged mutants for the major 12S-globulin genes, CRA1, CRB, and CRC, and found elevated levels of oil in crc seeds. This was associated with the transcriptional upregulation of genes encoding the LEC2 and WRI1 transcription factors, diacylglycerol acyltransferase (DGAT1), and plastidial pyruvate kinase β subunit 1 (PKp-β1), all of which are important for oil production. Furthermore, cra1, crb, and crc single-mutant plants developed substantially more branches, thereby producing more flowers and fruits than did wild-type plants. Thus, cra1, crb and crc mutations resulted in 19%, 22% and 41% increases in seeds and 24%, 25% and 62% increases in oil content per plant, respectively, as compared with wild-type plants. Our results suggest that the perturbation of storage-protein synthesis in developing seeds of Arabidopsis influences the capacity of whole plants for producing sink organs such as shoot branches, flowers and seeds.
With the first approval of a genetically modified (GM) soybean for uncontained use in Japan, information about hybrids of wild and GM or non-GM soybeans has become increasingly important. Therefore, we generated and characterized various hybrids between wild and GM or non-GM soybeans in a containment greenhouse, and investigated the inheritance of the cp4 epsps transgene and its effects on the hybrids. The hybrids inherited the cp4 epsps gene in a Mendelian fashion. The gene was stably expressed and produced functional protein, conferring glyphosate tolerance to the hybrids. We also examined the germination and survival rates of the second filial (F2) seeds and those of hybrids backcrossed twice (BC2F2) after a 3-month treatment at 4°C. Hybrids displayed similar germination characteristics as wild soybean after cold treatment. The majority of the BC2F2 seeds survived as dormant seeds. There was no effect of the cp4 epsps gene on the survival rate. Furthermore, we examined the morphology, anthesis, and fecundity of the hybrids. On the whole, the F1, F2, and F3 hybrids exhibited morphology, anthesis, and fecundity phenotypes that were intermediate between wild and cultivated soybeans; those of BC2F2 hybrids were similar to those of wild soybean. Comparisons between GM and the corresponding non-GM hybrids did not reveal significant differences in fecundity. We suggest that hybrids containing half of their genes from wild soybean and half from cultivated soybean display fitness that are intermediate between wild and cultivated soybeans, whereas BC2F2 hybrids have fitness similar to that of wild soybean. We also suggest that, despite being stably inherited, the cp4 epsps gene does not affect fitness in the absence of glyphosate treatment.
Hybrid lethality was expressed in seedlings from crosses between Nicotiana nudicaulis and N. tabacum at 28°C. To clarify the subgenome responsible for this phenotype, we crossed N. nudicaulis with the two progenitors of N. tabacum (SSTT), N. sylvestris (SS) and N. tomentosiformis (TT). Seedlings of N. nudicaulis×N. tomentosiformis did not show any lethal symptoms, and these hybrid seedlings subsequently grew to maturity. On the other hand, in the cross N. sylvestris×N. nudicaulis, all seedlings cultured at 28°C died immediately after germination, and others cultured at 34°C and 36°C were nonviable or abnormal; only one hybrid plant survived. These findings suggest that the S subgenome in N. tabacum is responsible for the lethality occurring in hybrids between N. nudicaulis and N. tabacum. Furthermore, to determine whether the Q chromosome induces this hybrid lethality, we crossed a monosomic line of N. tabacum lacking the Q chromosome with N. nudicaulis. Subsequently, we determined the presence or absence of the Q-chromosome-specific DNA marker in hybrid seedlings. Hybrid seedlings both possessing and lacking the Q chromosome showed hybrid lethality when seedlings cultured at 34°C were transferred to 28°C. From these results, we concluded that the Q chromosome is not responsible for the hybrid lethality observed in crosses of N. nudicaulis×N. tabacum.
The first Japanese field trial testing the effects of genetically modified (GM) forest trees was conducted over a period of 4 years. Salt tolerance was conferred on Eucalyptus camaldulensis by introducing the soil microbe-derived choline oxidase (codA) gene into this plant. Three individual lines were planted in an isolated field owned by the Gene Research Center at the University of Tsukuba, following approval for type I use by the Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Japanese Ministry of the Environment. Soil populations of fungi, actinomycetes, and bacteria fluctuated during sampling, but any differences introduced were observed only transiently, and no long-term change in the population was observed during the trial. Both the soil-mix as well as the sandwich method was employed to evaluate allelopathic activity of codA-transgenic E. camaldulensis. By the soil mix method, both GM and non GM-derived plant samples were found to exert negative allelopathic effects on seed germination, with an annual fluctuation. Overall, no significant differences were observed between the transformant lines and their bulk seed-derived non-transformants. The sandwich method also revealed an annual fluctuation. A comparison of the non-transformants and transformants revealed no significant differences in their allelopathic activity. Considering the practical use of these transgenic E. camaldulensis, they will no doubt be planted in some salinized sites. Thus, evaluating their impact on the ecosystem in saline conditioned fields might be important in the future.
Several brown midrib (bm) mutants have so far been isolated from the C4 grasses, maize, sorghum and pearl millet, but have not been detected in C3 grasses including rice (Oryza sativa). In the present study we characterized the cad2 (cinnamyl alcohol dehydrogenase 2) null mutant isolated from retrotransposon Tos17 insertion lines of Oryza sativa L. ssp. japonica cv. Nipponbare. This mutant exhibited brown-colored midribs in addition to hulls and internodes, clearly indicating both bm and gold hull and internode (gh) phenotypes. The enzymatic saccharification efficiency in the culm of cad2 null mutant was increased by 16.1% than that of the control plants. The lignin content of the cad2 null mutant was 14.6% lower than that of the control plants. Thioacidolysis of the cad2 null mutant indicated the presence of cinnamaldehyde structures in the lignin. Taken together, our results show that deficiency of OsCAD2 causes the bm phenotype in addition to gh, and that the coloration is probably due to the accumulation of cinnamaldehyde-related structures in the lignin. Additionally, this cad2 null mutant was useful to silage purposes and biofuel production.
Yatein is an important biosynthetic precursor of the lignan podophyllotoxin. In Anthriscus sylvestris, yatein biosynthesis is preceded by two regioselective methylations involving the intermediates: thujaplicatin and 5-O-methylthujaplicatin. The two methylation steps are most likely catalyzed by plant O-methyltransferases (OMTs). In this paper, we report the isolation and characterization of a cDNA encoding an OMT involved in the first methylation step. The OMT cDNA was isolated from an A. sylvestris cDNA library prepared from roots and young shoots. The OMT was expressed as a recombinant protein using the pET expression system. Of the substrates that were tested, the recombinant OMT exclusively catalyzed regioselective methylation of thujaplicatin to produce 5-O-methylthujaplicatin, and thus was designated as A. sylvestris thujaplicatin OMT (AsTJOMT). Kinetic analysis with its substrate (thujaplicatin) showed that AsTJOMT had a Km value of 3.8 µM and kcat value of 0.29 min−1. Quantitative real-time polymerase chain reaction showed that AsTJOMT had the highest expression level in roots compared with other organs. This was in accordance with plant protein assays in which specific activity for thujaplicatin was significantly higher in roots compared with other organs. To the best of our knowledge, this is the first report on the isolation and characterization of a thujaplicatin-specific plant OMT.
A novel gain-of-function mutant showing dwarfism and bushy inflorescences was isolated from Arabidopsis activation-tagging lines. Transformed plants, in which the gene responsible for the phenotype of the mutant was overexpressed, exhibited phenotypes similar to constitutive systematic acquired resistance (SAR)-like defense responses including simulation of lesions in leaves and a highly activated PATHOGENESIS-RELATED1 gene. We, therefore, designated the gene as DWARF AND LESION FORMATION 1 (DLE1) and showed it encodes a DUF 247 protein. Microarray and gene ontology analyses revealed that defense response genes, especially to biotic stimulus, were enriched in DLE1 overexpressing plants. Fusion proteins of the N-terminal fragment of DLE1 and GFP were likely to be localized in the endoplasmic reticulum (ER) in tobacco BY-2 cells. DLE1 may function in the plant defense system responding to environmental stimuli including biotic stresses in the ER.
Clubroot resistance (CR) is an important trait for Chinese cabbage (Brassca rapa L. ssp. pekinensis). Since no CR genes had been found throughout Chinese cabbage lines originally, they were introduced from CR European turnips. Distribution of CR genes in the commercial cultivar is important information for CR breeding, but it has been scarcely known at molecular level. In this work, CR of 47 Chinese cabbage cultivars were analyzed phenotypically and genotypically. More than 80% of them exhibited high resistance phenotype, and all of the cultivars with high resistance were found to have CRa, one of the genes controlling CR. Majority of these cultivars were heterozygote for resistance, which is reasonable because CRa controls the resistance single-dominantly. This is the first work reporting the distribution of CR gene in Chinese cabbage cultivars and provides important and practical information for breeders and farmers. Further applications of CR genes are also discussed.
A laticifer is an elongated tubular cell and its cytoplasmic content (latex) is thought to be involved in defense against herbivores and microbes. Previous studies investigated laticifer transcriptomes in the unlignified and lignified tissues of mulberry (Morus alba) but protein accumulation in laticifers in unlignified and lignified tissues is poorly understood, except the conclusion of a previous study that insecticidal chitinase-like proteins (LA-a and b) were abundant in the laticifers of unlignified tissues while antifungal class I chitinase (LA-c) was abundant in the laticifers of lignified tissues. In order to understand precisely the physiological roles of laticifers in these tissues, this study identified the major proteins in them using mass spectrometry and Edman sequencing after separation by ion-exchange chromatography and SDS-PAGE. In addition to LA-a, b and c, this study has shown that mulberry laticifers accumulate large amounts of biotic-stress-related defense proteins, e.g., pathogenesis-related protein-1, β-1,3-glucanase, class V chitinase, osmotin and lectins. The abundance of some proteins varied among the laticifers of unlignified and lignified tissues, which suggested that the laticifers may have adapted to different threats.
Aliphatic glucosinolates (AGSLs) in Arabidopsis thaliana are synthesized from methionine derivatives with various side-chain length that are formed from methionine via chain elongation cycle involving MAM1 and MAM3. Biosynthesis of AGSLs is highly affected by defense signaling molecules, including jasmonic acid, salicylic acid and ethylene. In response to exogenously-applied these phytohormones, MAM genes exhibited different induction patterns in Wild-type of Arabidopsis. The changing patterns of AGSL contents were distinctive in single knockout mutants of MAM genes compared with those in Wild-type, suggesting that MAM1 and MAM3 play an important role in the diversity of AGSL profiles in response to hormonal changes.
Endoreduplication, a modified cell cycle process in which the cellular DNA level increases without subsequent cell division, is a key component of growth and development of plants. CCS52A1, a substrate-specific activator of the anaphase promoting complex/cyclosome (APC/C), is essential for the transition from mitosis to endoreduplication. Here, we show that DNA-binding with one finger (Dof) transcription factors directly regulate the transcription of the CCS52A1 gene. Dof proteins are plant-specific transcription factors involved in plant growth and development processes, such as phytohormone and light signaling, defense response, and seed germination. We performed yeast one-hybrid screening using an Arabidopsis transcription factor library and identified several Dof transcription factors as novel CCS52A1 promoter-binding proteins. Protoplast transient assay showed that the Dof proteins, such as CDF1, CDF2, CDF3, and COG1, function as transcriptional repressors of CCS52A1. All of these Dof transcription factors were localized to the nucleus. Our data imply that CDF1, CDF2, CDF3, and COG1 control plant growth and development through the regulation of CCS52A1 expression.
The establishment of adaxial–abaxial polarity in the early stage of leaf development is important for the expansion of lamina. In Arabidopsis thaliana, asymmetric leaves2 (as2) and as1 mutations cause defects in the leaf adaxial–abaxial polarity, which are exhibited as abaxialized filamentous leaves in the various modifier mutant backgrounds. Several modifier single mutants generate pointed leaves in common. Mutations in FASCIATA2 (FAS2) also cause pointed leaves. The FAS2 gene encodes a component of Chromatin Assembly Factor-1 (CAF-1), a histone chaperone, which affects mRNA levels of various genes through the regulation of chromatin states. In the present study, we demonstrate that as2 fas2 double mutants frequently generate abaxialized filamentous leaves and show increased mRNA levels of genes for leaf abaxialization, KANADI1 (KAN1), KAN2, YABBY5 (YAB5), ETTIN/AUXIN RESPONSE FACTOR3 (ETT/ARF3) and ARF4. In addition, the transcript levels of all four class 1 KNOTTED1-like homeobox genes that are involved in the maintenance of shoot apical meristem and the Kip-related Protein2 (KRP2) and KRP5 genes that are involved in the cell cycle progression are elevated in the as2 fas2. The mRNA levels of all genes other than YAB5, whose transcript levels rise in as2 fas2, were increased in the fas2 single mutants. Our data suggest that FAS2 participates in the establishment of leaf adaxial–abaxial polarity through the repression of the transcript levels of these genes on the as2 background.
Despite lacking Na+-ATPase as a sodium pump in vascular plants, a gene encoding KPA (K+ P-type ATPase), a putative animal type-Na+/K+-ATPase, has been isolated from the marine red alga Porphyra yezoensis and designated PyKPA1. To characterize the properties of PyKPA1 and also to confirm its ability to confer salinity tolerance in land plants, transgenic rice plants were produced that expressed the full-length PyKPA1 cDNA under the control of cauliflower mosaic virus 35S RNA promoter. We observed transcriptional activation of the transgene, plasma membrane-localization of the gene product fused with green fluorescent protein in onion epidermal cells, and Na+-ATPase activity in the plasma membrane fraction from transgenic rice plants, indicating that PyKPA1 was functionally expressed in rice plants. Transgenic lines were examined in terms of growth in salinity stress conditions, resulting in protection from a decrease in biomass, although growth of control rice plants was repressed. These results demonstrate the utility of a red algal animal type-sodium pump for conferring salinity tolerance to land plants.