Sesamin is a major lignan constituent of sesame seeds and beneficial to human health. We previously reported that sesamin is contained in leaves as well as seeds of sesame and proposed that sesame leaves could be a new sesamin source. Growth and constituents of plants are affected by light wavelength. In this study, growth and leaf sesamin content of sesame variety ‘Gomazou’ were investigated in plants grown under continuous white fluorescent and monochromatic red or blue light emitting diode (LED) light. Under red LED light, plants developed pale-green, epinastic leaves. Compared with white fluorescent light, red LED light promoted stem elongation 1–3 weeks after sowing but retarded it 3–5 weeks after sowing. Under blue LED light, plants exhibited interveinal necrosis in the leaf blades and excessive stem elongation occurred irrespective of plant age. Leaf yields were lower in plants grown under red and blue LED lights relative to those under white fluorescent light. Blue LED light increased leaf sesamin content by 2.0 and 4.5 times compared with white fluorescent and red LED lights, respectively. From these results, we concluded that blue (LED) light may be effective at producing sesamin-rich leaves if the unfavorable morphological changes and reduction in growth can be prevented.
Sphingolipids and their metabolites including long-chain bases (LCBs) and long-chain base 1-phosphates (LCBPs) have been shown to be involved in regulation of various aspects of biological processes in plants. However, little is known about the biological function of LCB kinases (LCBKs), which catalyze the phosphorylation of LCBs to form LCBPs in plant abiotic stress tolerance. In the present study, we performed a functional analysis in transgenic tobacco to explore the possible involvement of a rice LCBK gene OsLCBK1 in abiotic stress tolerance. Root elongation of the transgenic tobacco seedlings with constitutive overexpression of OsLCBK1 was less sensitive to exogenous abscisic acid as compared with the vector-transformed seedlings. The OsLCBK1-overexpressing transgenic tobacco plants showed increased tolerance against oxidative stress after treatment with methyl viologen or H2O2, and up-regulated expression of oxidative stress-related genes. However, the OsLCBK1-overexpressing transgenic tobacco plants showed similar phenotype as vector-transformed plants in response to salt stress and had no change in expression of salt stress-related genes. Our results suggest that OsLCBK1, an enzyme involved in synthesis of LCBPs, may be involved in ABA response and has functions in regulation of oxidative stress tolerance in plants.
Seedlings of the Japanese morning glory (Pharbitis nil, formerly Ipomoea nil) cv. Violet are induced to flower by a single short day treatment, and endogenous levels of 9,10-ketol-octadecadienoic acid (KODA) in cotyledons were found to correlate with this mode of short-day induction. When 100 µM KODA solution was sprayed on 7-day-old seedlings before and after a marginal short-day induction, the seedlings bore approximately 4 flower buds, a one-bud increase compared with control seedlings. In treated seedlings, the second node most commonly bore the first flower bud, one node lower than in the control seedlings. We then used RT-PCR to determine expression levels of 10 genes related to photoperiodic induction of flowering. In the cotyledons, no differences were observed in expression of any genes, including P. nil FLOWERING TIME LOCUS T (PnFT1 and PnFT2), between the KODA-treated and the control seedlings. In the apical buds, P. nil APETALA1 (PnAP1) was expressed earlier in the KODA-treated seedlings than in the control seedlings. A decrease in the expression of P. nil TERMINAL FLOWER1b (PnTFL1b) was also observed in the KODA-treated seedlings. These results suggest that KODA acts as a weak enhancer of flower bud formation.
Lignin is a major component of the secondary cell walls of vascular plants, and an obstacle in the conversion of plant cell wall polysaccharides into biofuels. Erianthus spp. are large gramineous plants of interest as potential energy sources. However, lignocelluloses of Erianthus spp. have not been chemically characterized. In this study, we analysed lignins, related compounds, enzymatic saccharification efficiencies, and minerals in the ash of the inner and outer parts of the internode, leaf blade and leaf sheath of Erianthus arundinaceus. Lignins in four organs consisted of guaiacyl, syringyl, and p-hydroxyphenyl units. The ratios of syringyl to guaiacyl lignins and lignin contents ranged from 0.43 to 0.79 and 20 to 28%, respectively, with values highest in the outer part of the internode. The amounts of ferulic acid were similar (7.3–11.8 mg g−1 dry weight of cell-wall material) in all four organs, while there was more p-coumaric acid in the inner part of the internode (44.7 mg g−1 dry weight of cell-wall material) than in other organs (25.7–28.8 mg g−1 dry weight of cell-wall material). The enzymatic saccharification efficiency (24 h reaction time) of the leaf blade was 21.6%, while those of the other organs ranged from 10.0 to 15.2%. The leaf blade had the highest ash content (17.1%); the main inorganic element was silicon. This paper provides the first fundamental knowledge of E. arundinaceus lignins.
Dof proteins are known as plant-specific transcription factors. We previously reported that Dof proteins might be involved in expression of the tobacco resistance gene N for Tobacco mosaic virus (TMV). In N-carrying tobacco cultivars such as Samsun NN, a rapid upregulation of N transcription is induced by TMV infection, which is followed by the defense response called the hypersensitive response (HR). In this study, the role of a tobacco Dof protein BBF1 in N transcription was investigated. We cloned a BBF1 ORF cDNA of Samsun NN and confirmed that a full-length recombinant BBF1 could bind in vitro to DNA with a Dof binding core motif. The transient overexpression of BBF1 alone did not induce any HR but activated the endogenous N gene expression in Samsun NN. The N promoter activation by BBF1 overexpression was also confirmed in the N-lacking Samsun nn plant by using the exogenously introduced N regulatory sequence connected to a reporter gene. Additional experiments suggested that BBF1 overexpression enhanced not only ROS production but also the transcription activity of certain defense signaling and HR marker genes even without HR induction. Regarding subcellular localization, BBF1 fused with a fluorescent protein was predominantly localized in the nucleus. Based on these data, we discuss potential roles of BBF1 and other Dof proteins as transcription factors for defense responses.
We characterized a rice monoculm mutant moc2, which showed significantly reduced tiller numbers, pale-green leaves, a reduced growth rate, and a consequent dwarf phenotype. The monoculm feature was attributed to a deficiency in the efficient outgrowth of tiller buds, although the moc2 mutant produced tiller buds. Inconsistent change was observed in the expression of genes involved in tiller bud outgrowth, suggesting that the moc2 mutant has a defective function necessary for the tiller bud outgrowth. The gene responsible for the moc2 mutant was mapped to a locus encoding cytosolic fructose-1,6-bisphosphatase 1 (FBP1), in which a Tos17 retrotransposon was inserted in exon 4. Reverse-transcription PCR for the FBP1 gene amplified a shorter transcript from the moc2 mutant than from the wild-type plant. The sequence of the shorter transcript revealed a deletion of exon 4 by abnormal splicing, and the resulting frameshift generated a new translation termination signal. The moc2 mutant showed a very low level of FBPase activity, suggesting that it involves a loss-of-function mutation of FBP1. Cytosolic FBPase is considered a key enzyme in the sucrose biosynthesis pathway. Defective FBPase activity is anticipated to lead a shortage of sucrose supply, which probably causes the inhibition of tiller bud outgrowth in the moc2 mutant. The monoculm phenotype of the moc2 mutant supports the idea that sucrose supply may be an important cue to outgrow tiller buds.
The pentatricopeptide repeat (PPR) family has been reported to be involved in many post-transcriptional processes, including RNA editing and RNA splicing in both chloroplasts and mitochondria. There are a large number of PPR proteins in plant genome, however, the biological functions of most PPR proteins have not yet been determined. We describe the involvement of a new PPR member (OsPPR4) for the splicing and RNA editing of chloroplast transcripts. The rice P-class PPR protein, OsPPR4, possesses an RNA recognition motif (RRM) and 15 PPR motifs. A retrotransposon (Tos17)-insertion mutant, osppr4, showed an albino phenotype with early seedling lethality. The same results were obtained from plants transformed with an OsPPR4 RNA interference (RNAi) construct. Disruption of OsPPR4 expression led to a strong defect in the splicing of atpF, ndhA, rpl2, and rps12-2 introns and influences the splicing of petB and rps16 introns. In addition, RNA editing of unspliced ndhA transcripts was decreased in the osppr4 line, whereas both spliced and unspliced ndhA were completely edited in WT plants. These results suggest that OsPPR4 is an essential factor for greening in plants and plays a non-redundant role in post-transcriptional regulation of chloroplast genes.
Plastid transformation methods have been developed for 20 plant species. However, only a few plant species, such as tobacco and lettuce, have been used in applied studies because transformation efficiencies are extremely low in other species. Plastid transformation has been mainly performed by particle bombardment using 0.6-µm gold particles as microcarriers of the transformation vector. Because the target materials in some plant species are undeveloped proplastids rather than fully developed chloroplasts, optimizing microcarrier size for the target size is a major consideration. In this study, we evaluated the availability of gold particles (0.07, 0.08, 0.1, 0.2, and 0.3 µm) that were smaller than those used for plastid transformation in previous studies. We obtained stable plastid transformants of tobacco with sufficient efficiency using all the tested small gold particles as the same level as 0.6-µm gold particles, even the smallest (0.07 µm). The average number of transformants obtained with 0.3-µm particles (9.3±4.6 per plate) was the highest among the tested gold particles. Because small gold particles were revealed to be sufficient for plastid transformation in a model tobacco plant, it is suggested that choosing appropriate small-sized gold particles which have never been used before will improve plastid transformation in many plant species.
Transgenic woody plants have been rapidly developed in recent years, and the commercial use of these transgenic plants has been recognized as an important approach to solving environmental and food problems. Concomitantly, the potential impact of transgenic woody plants on peripheral ecosystems should be considered before they are released for practical commercial use. In this study, we have used environmental biosafety assessments to evaluate three lines of transgenic Eucalyptus globulus that harbor the choline oxidase (codA) gene and have previously been proven to have different levels of salt tolerance. The assessments included two allelopathy bioassays and a survey on rhizosphere microbes, which have shown practicability in other transgenic plants. The two allelopathy bioassays were used for evaluating the potential impact of the transformants on the surrounding vegetation. The survey on rhizosphere microbes was performed to investigate the potential impact of transformants on the rhizosphere microbe community. The results indicated there was no significant difference between the transformants and non-transformants with respect to the impact on the surrounding vegetation and the rhizosphere microbe community. A combination of our biological evaluation of E. globulus was then used to successfully obtain approval for the plantation of transgenic E. globulus in a Type I field trial in Tsukuba.
β-fructofuranosidase (FFase) of Aspergillus niger ATCC 20611 can transfer fructosyl residues from one sucrose to another for the synthesis of glucose and fructooligosaccharides composed of 1-kestose (GF2), nystose (GF3), and β-fructofuranosylnystose (GF4). The FFase gene, under the control of the sporamin gene promoter from sweet potato, was introduced into tobacco plants. Sporamin promoter activity is induced by sugar and exhibits preferential expression in stem and root tissues. Thin-layer and high performance liquid chromatographic analyses showed that soluble extracts from the transgenic plants contained considerable amounts of fructooligosaccharides such as GF2 and GF3. The conversion of sucrose into fructooligosaccharides did not affect plant growth or development. Our results indicate that the transgenic plants could be utilized as bioreactors, and this opens up the possibility for efficient production of fructooligosaccharides in sucrose-producing plants such as sugar beet and sugarcane.
This study aimed to identify the proteins produced specifically in Betula platyphylla var. japonica plantlet No. 8 by infection with Inonotus obliquus strain IO-U1. Sterile Japanese birch plantlets were infected with the fungus, and the protein samples obtained at 2 days post-infection were subjected to two-dimensional electrophoresis to detect infection-specific proteins. The specific proteins were analyzed using MALDI-TOF-MS and identified by peptide mass fingerprinting with MASCOT software. Among the 735 protein spots detected in the infected plantlets, 169 spots were recognized as infection-specific proteins. Of these spots, 91 were analyzed using MALDI-TOF-MS, with two heat shock proteins (Hsp70 and Hsp60) being identified as infection-specific proteins. Hsp70 and Hsp60 may cooperate to refold proteins that were denatured by the infection of I. obliquus strain IO-U1 in the birch plantlets. It is suggested that these proteins are expressed in B. platyphylla plantlet No. 8 by infectious stress with I. obliquus strain IO-U1.
Botrytis cinerea is a ubiquitous necrotrophic fungal pathogen that infects over 200 different plant species. We have analyzed 17 Arabidopsis ecotypes for natural variations in their susceptibility to B. cinerea, and found compatible and incompatible Arabidopsis–Botrytis interactions. We determined that Arabidopsis ecotype Ler is resistant to 5 B. cinerea isolates used in this study. To further investigate the roles of the salicylic acid (SA)-dependent defense response pathways against B. cinerea, we inoculated various Arabidopsis mutants with the pathogen. Arabidopsis Ler plants expressing the nahG gene inoculated with B. cinerea showed as much resistance as the parental plants (Ler-wild type). The sgt1b-1 and rar1-10 mutants also showed resistance to the pathogen. In this study, we discuss the natural variations in the symptoms observed among various ecotypes upon inoculation with B. cinerea. In addition, SA plays only a minor role in preventing systemic infection with B. cinerea.