Lignans are a group of plant phenolic compounds with various biological activities, including antitumor and antioxidant properties. O-Methylation is a critical step in biosynthesis of these compounds. However, little is known about the O-methyltransferase (OMT) enzymes that catalyze lignan O-methylation. We discovered a highly regioselective OMT activity in safflower (Carthamus tinctorius) seeds that catalyzed the methylation of matairesinol, a dibenzylbutyrolactone lignan, into 4′-O-methylmatairesinol (arctigenin) but not 4-O-methylmatairesinol (isoarctigenin). By examining such OMT activity in correlation with OMT transcript abundances during seed development, we cloned a few putative OMT cDNAs and produced their recombinant proteins in Escherichia coli. Among them, one protein exhibited O-methylation activity for matairesinol with the regioselectivity identical to that of the plant protein, and was named C. tinctorius matairesinol OMT (CtMROMT). CtMROMT did not show any detectable OMT activities towards phenylpropanoid monomers under the reaction conditions tested, while it methylated flavonoid apigenin efficiently into 4′-O-methylapigenin (acacetin). However, quantitative real-time polymerase chain reaction analysis demonstrated that expression of the CtMROMT gene was synchronized with the CtMROMT activity profile and arctigenin accumulation in the plant. These results demonstrated that CtMROMT is a novel plant OMT for lignan methylation.
Mitogen-activated protein kinase (MAPK) cascades play important roles in plant responses to various environmental stimuli, including high salt or drought levels. Arabidopsis MAP3Kδ4 with PAS (period circadian protein, arylhydrocarbon receptor nuclear translocator protein and single-minded protein) domain is one of the Raf-type MAPKKKs whose function has not been identified to date. Previous studies have shown that the MAP3Kδ4 over-expressing transformant exhibits vigorous growth. In this study, RT-PCR analysis showed that MAP3Kδ4 transcripts are increased through stress treatments, such as high salt, osmosis, drought and cold and the plant hormone, abscisic acid (ABA). The precipitation of MAP3Kδ4 using its specific antibody showed that ABA treatment markedly induces the activity of this enzyme. Furthermore, the ABA-mediated inhibition of seed germination was relieved in transgenic Arabidopsis over-expressing MAP3Kδ4. These results suggested that MAP3Kδ4 plays an important role in ABA signalling. Transgenic Arabidopsis over-expressing MAP3Kδ4 also exhibited enhanced tolerance to salt stress. The results obtained in this study demonstrated that MAP3Kδ4 was active during ABA-related responses and is involved in both stress tolerance and increased biomass. Therefore, MAP3Kδ4 and its counterpart genes are important with respect to agricultural developments.
Many flowering plants exhibit self-incompatibility (SI) to prevent inbreeding and promote outcrossing. This self/non-self discrimination mechanism is controlled by the S locus, which contains separate genes for pistil and pollen specificities. In the gametophytic SI (GSI) of Rosaceae, Solanaceae and Plantaginaceae, the pistil S determinant, S-RNase, encodes extracellular ribonuclease which is thought to act as a cytotoxin to the self pollen tube, while the pollen S determinant is the F-box gene called SLF/SFB/SFBB. In Petunia (Solanaceae), SLF is reported to be a component of the noncanonical E3 ubiquitin ligase complex with S-RNase binding protein1 (SBP1) and Cullin1 (CUL1), and interact with non-self S-RNases to ubiquitinate them for degradation. Here, we isolated an apple (Malus×domestica) homolog of SBP1 (MdSBP1) from pollen RNA by RT-PCR. MdSBP1 included a RING-HC domain required for E3 ubiquitin ligase activity, and showed 64.0–68.2% amino acid identities with solanaceous SBP1 proteins. Expression analysis showed that MdSBP1 was expressed in all the organs analyzed. We detected an interaction between recombinant MdSBP1 protein and S-RNase of apple using a pull-down assay.
Heading Chinese cabbage (Brassica rapa ssp. pekinensis) has been recalcitrant to regeneration and transformation. In particular, there are few reports concerning Japanese cultivars. We evaluated the factors that inhibit Agrobacterium-mediated transformation in heading Chinese cabbage. Then, we investigated the effects of selectable markers using heading Chinese cabbage cv. Chihiri 70 and compared the effects with those in broccoli cv. Ryokurei (B. oleracea var. italica) as a control for an easily transformable Brassica species. To utilize a selectable marker derived from a host plant cisgene, we cloned a genomic DNA fragment containing regulatory and coding sequences of the acetolactate synthase (ALS) gene from Chinese cabbage and mutagenized it to a herbicide resistant form. After transformation of Chinese cabbage and broccoli with this construct, transgenic plants were efficiently selected with the herbicide bispyribac sodium salt and screened by DNA gel blot analysis. The average transformation frequency of Chinese cabbage was 1.2±0.2%, which was similar to those in reports using antibiotic selectable markers and was lower than for broccoli (13.9±2.0%). Furthermore, the escape rate was restricted at a low level (about 35–50% lower than hygromycin selection), which is an advantage on practical transformation. We confirmed transgene inheritance and herbicide resistance of potted plants in the T1 generation. This report is the first to describe a selection system for the transformation of a Brassica crop that uses a herbicide-tolerant selectable marker derived from a cisgene.
An efficient system for Agrobacterium-mediated transformation was established in dahlia (Dahlia×pinnata) ‘Yamatohime’. Mass of shoot primordia (MSP) induced on MS medium supplemented with 10 mg l−1 TDZ were inoculated with Agrobacterium tumefaciens strain EHA101 (pIG121-Hm) harboring both β-glucuronidase (GUS) and hygromycin resistant genes. After 2 days of co-cultivation, the MSP were transferred to a selection medium containing hygromycin with meropenem for bacterial elimination. Shoots were successfully regenerated from survived MSP on hormone-free medium without hygromycin and they rooted on hormone-free medium containing hygromycin. The hygromycin-resistant plants thus obtained showed histochemical blue staining for GUS. Transformation of plants was confirmed by PCR and Southern blot analyses. Transgenic ‘Kokucho’ were also produced by using the same transformation procedure, suggesting wide applicability of this Agrobacterium-mediated transformation procedure for other dahlia cultivars.
Protocol of plant regeneration from leaf and stem explants was established in dahlia (Dahlia×pinnata) ‘Yamatohime’ in in vitro culture. Nodular calli were successfully induced from leaf and stem explants on 30 g l−1 sucrose-containing MS medium supplemented singly with one of the cytokinins, 6-benzyladenine (BA), zeatin or thidiazuron (TDZ) and the highest frequency of nodular callus induction was obtained on medium containing 10 mg l−1 TDZ. The nodular callus induced on this medium subsequently produced a tissue with numerous shoot primordial structures called ‘mass of shoot primordia’ (MSP), which was maintained at least for more than 2 years on the same medium. Shoot induction occurred after transfer the MSP onto hormone-free MS medium, in which replacement of sucrose by maltose was necessary. The shoots excised from the MSP were rooted on hormone-free MS medium and successfully grown under greenhouse conditions. The regenerated plants showed the same morphological characteristics to those of the control plants and flowered normally.
During seed maturation, the water content of seeds decreases remarkably. Mature seeds can germinate after imbibition since the embryos are protected by mechanism of desiccation tolerance. To better understand the mechanism of desiccation tolerance in seeds, we analyzed the fluctuation of stress-related proteins in the desiccation phase of rice seeds by a real-time RT-PCR and gel-based proteomic approach. Based on the changes in water content of developing rice seeds, we defined stages from the beginning of dehydration (10 to 20 days after flowering) and the desiccation phase (20 to 40 days after flowering). The proteomic analysis revealed that late embryogenesis abundant proteins, small heat shock proteins and antioxidative proteins accumulate at the beginning of dehydration and remain at a high level in the desiccation phase, suggesting that these proteins are involved in acquisition of desiccation tolerance. The fluctuation in levels of mRNA encoding some stress-related proteins did not precisely reflect the change in levels of these proteins. Therefore, proteomic analysis, which provides an accurate assessment of changes in protein levels, is a more efficient technique than transcriptomics for inferring the role of stress-related proteins in rice seeds.
Little is known regarding syringyl lignin biosynthesis in rice (Oryza sativa L. cv. Nipponbare). In the present study, the role of rice caffeic acid O-methyltransferase (OsCOMT1, Q6ZD89), was examined. The recombinant OsCOMT1 catalyzed the 5-Omethylation of 5-hydroxyferulate (5-HFA) and 5-hydroxyconiferaldehyde (5-HCAld). 5-HCAld inhibited 5-HFA methylation by this O-methyltransferase (OMT), while 5-HFA mitigated self-inhibition in 5-HCAld methylation. A rice plant in which OsCOMT1 expression was downregulated exhibited weakened cell wall staining with Wiesner reagent in vascular bundle cells and sclerenchyma tissue, compared with wild-type plants. The lignin content of transgenic rice plants was decreased and the syringyl lignin content reduced largely compared with that of the wild type. Taken together, these data indicated that OsCOMT1 functioned as a 5-HCAld OMT (OsCAldOMT1) in the biosynthetic pathway to syringyl lignin.
Biosynthesis of plant secondary cell walls is controlled by several master transcription factors. Ubiquitin ligases, which mediate ubiquitination of proteins, including transcription factors in the protein degradation pathway, are also believed to regulate secondary wall biosynthesis; however, the exact ubiquitin ligases involved in secondary wall formation have not yet been identified. We conducted a gene co-expression network analysis and found that ATL54, annotated as a RING-finger protein, was highly co-expressed with several transcription factor and enzyme genes involved in secondary wall formation. A recombinant ATL54 protein showed ubiquitin ligase activity. The expression of several biosynthetic genes of cellulose, lignin, and xylan in apical portions of inflorescence stems was up-regulated by ATL54 knock-out. The expression of Xylem Cysteine Peptidase1 (XCP1), which participates in the programmed cell death process of xylem tracheary elements, was down-regulated in middle stem portions of both ATL54-knock-out and ATL54-overexpressed mutants. Alteration of ATL54 expression levels did not, however, affect lignin and polysaccharide content and composition in whole mature stems. Our results suggest that ATL54 is an E3 ubiquitin ligase involved in secondary wall biosynthesis and programmed cell death during xylogenesis.
Hybrid seedlings from reciprocal interspecific crosses between Nicotiana nudicaulis and N. tabacum were not viable when cultured at 28°C; this an example of hybrid lethality. Characteristically, shoot apices and root tips of these hybrids began to brown immediately after germination at 28°C. However, hybrid seedlings did not exhibit any symptoms of lethality at 34°C, and their growth was normal at this higher temperature. When hybrid seedlings were cultured at 34°C and then transferred to 28°C, lethal symptoms emerged rapidly after the transfer. Therefore, hybrid lethality associated with reciprocal interspecific crosses between N. nudicaulis and N. tabacum was due to the interaction of coexisting heterologous genomes and not to a cytoplasmic effect. Furthermore, as the N. nudicaulis×N. tabacum seedlings die, features of apoptotic cell death, including chromatin condensation, nuclear fragmentation, and fragmentation of DNA, were evident. These observations indicated that hybrid lethality of N. nudicaulis×N. tabacum seedlings is accompanied by apoptotic cell death. This report is the first to demonstrate that hybrid lethality with apoptotic cell death results from N. nudicaulis×N. tabacum hybridization.
Biosynthesis of indole-3-acetic acid (IAA) is crucial for the regulation of plant growth and morphological changes. However, the pathways of IAA biosynthesis have not been completely understood. Although indole derivatives have been proposed to be intermediates of the pathway, it has not been thoroughly elucidated whether the proposed intermediates are indeed converted into an active auxin, IAA, or intermediate metabolites other than IAA. In the present work, we examined 11 indole derivatives whether they are indeed IAA intermediates. The indole derivatives were examined by measuring their effects on recovery from auxin-deficiency conditions caused by L-aminooxyphenylpropionic acid (AOPP), which we previously identified as an IAA biosynthesis inhibitor. We estimated auxin activity of each indole derivative by analyzing auxin-inducible marker gene expression and root morphology in Arabidopsis. We found that indole-3-pyruvic acid most effectively recovered seedlings from auxin deficiency among the indole derivatives examined, followed by N-hydroxyl tryptamine and indole-3-acetonitrile, with effective recovery from the auxin deficiency caused by AOPP. We also found that tryptophol recovered the root growth defects. Among the indole derivatives examined, only indole-3-acrylic acid did not show effective recovery from auxin deficiency. These results provide information on which compounds are reliable intermediates of IAA biosynthesis pathways in Arabidopsis.
Promoter constructs with high levels of xylem specific expression are needed to obtain efficient expression of candidate genes, microRNAs (miRNAs) and artificial microRNAs (amiRNAs) for the genetic modification of wood properties. The gene for caffeic acid O-methytransferase (PtrCOMT2) has the second most abundant transcript level of all the genes in monolignol biosynthesis in Populus trichocarpa and a high level of specificity in differentiating xylem. To characterize the PtrCOMT2 promoter, we cloned a short (2.0 kb) and a long (3.3 kb) promoter segment and compared their expression using GUS as a reporter gene in the differentiating xylem of Nicotiana tabacum. Both the 2.0 kb and the 3.3 kb promoter segments showed high specificity for differentiating xylem in this heterologous system. GUS activity increased as much as 5 times when the 4×35S enhancer was inserted in front of the 2.0 kb promoter, but GUS activity was only increased 2 times when the enhancer was inserted behind the promoter. The enhancer inserted upstream reduced the expression of the 3.3 kb promoter. While expression of some of the enhancer-plus-promoter constructs increased expression, there was a loss of specificity.
This study investigated the time-course changes of the in situ peroxidase (POD) distribution and expression of POD isozymes in Betula platyphylla var. japonica plantlet No. 8 infected with a canker-rot fungus, Inonotus obliquus strain IO-U1. Intact (C1), wounded (C2), and infected (T) plantlets were collected at 2 h up to 30 d. In situ POD activity was detected in the C2 and T plantlets, and the POD activity in the T plantlets was more widely distributed compared to the C2 plantlets. In addition, the area of POD activity localization was almost the same as that of phenolic compounds, although a time lag was found between the appearance of POD activity and phenolic compounds. POD isozymes were clearly detected within the basic range (pI>8.5) in isoelectric focusing electropherograms. The activity of cationic POD isozymes in the C2 and T plantlets was induced strongly compared to the C1 plantlets. In addition, the pattern of time-course changes in the activities of in situ POD and POD isozymes was different between the C2 and T plantlets, suggesting that the responsive mechanisms against fungal infection are different from the responses to wounding. The obtained results suggest that cationic POD isozymes are related to the basal resistance in B. platyphylla var. japonica plantlet No.8 against infection with I. obliquus strain IO-U1.