Arabidopsis thaliana is the most widely used model plant in the area of plant biosciences, and the cell line T87 is one of the most commonly used lines in Arabidopsis cell cultures. The characteristics of lignins in cultured cells often differ from those of lignins in intact plants. To date, nothing is known about the lignins of T87 cells. In this study, we characterized lignins of T87 cells using six analytical methods; phloroglucinol staining, thioacidolysis, nitrobenzene oxidation, alkaline hydrolysis, and Klason and acetyl bromide methods. These analyses clearly showed that lignins of T87 cells are composed of guaiacyl and p-hydroxyphenyl units, and are quite different from lignin found in the Arabidopsis inflorescence stem, which is composed of guaiacyl and syringyl units.
The Arabidopsis nsl2 (necrotic spotted lesion 2) mutant, which has been originally reported as the cad1 (constitutively activated cell death 1), shows a phenotype that mimics a hypersensitive response (HR)-like cell death. The NSL2 protein is suggested to negatively control the salicylic acid (SA)-mediated pathway of HR-like cell death in plant immunity. The induction of systemic acquired resistance (SAR) results in the induction of pathogenesis-related (PR) genes in systemic organs triggered by the local HR. In this report, we establish a NSL2 knockdown system in transgenic Arabidopsis based on constitutive or dexamethasone (DEX)-induced RNAi. The former showed a nsl2-like phenotype. In DEX-induced RNAi, localized knockdown resulted in the induction of PR1 gene expression and the restriction of bacterial growth in both DEX-treated and systemic leaves. These results indicate that NSL2 negatively controls SAR via HR.
An efficient Agrobacterium-mediated genetic transformation method was established for Brassica juncea by investigating several factors responsible for successful gene transfer. Four-day-old cotyledon explants from in vitro grown seedlings were co-cultivated with Agrobacterium strain GV3101 harboring the binary vector EnPCAMBIA1302-YCF1, which contained the hygromycin phosphotransferase (HPT) gene as a selectable marker and the yeast cadmium factor 1 (YCF1) gene. Two days co-cultivation period on shoot induction medium (MS medium supplemented with 0.1 mg l−1 α-naphthaleneacetic acid, 1.0 mg l−1 6-benzyladenine, and 2.0 mg l−1 silver nitrate) containing 20 mg l−1 acetosyringone and five days delaying exposure of explants to selective agent enhanced transformation efficiency significantly. A three-step selection strategy was developed to select hygromycin resistant shoots. Hygromycin-resistant shoots were subsequently rooted on root induction medium. Rooted plantlets were transferred to pot-soil, hardened, and grown in a greenhouse until maturity. Using the optimized transformation procedure, transformation efficiency reached at 16.2% in this study. Southern blot analysis was performed to confirm that transgenes (HPT and YCF1) were stably integrated into the plant genome. All transgenic plants showed single-copy of transgene integration in the host genome. Segregation analysis of T1 progeny showed that the transgenes were stably integrated and transmitted to the progeny in a Mendelian fashion.
Some cell wall enzymes of the xyloglucan endotransglucosylase/hydrolase (XTH) family catalyze molecular grafting between xyloglucan molecules. In tomato, 25 genes for XTH proteins have been identified. We studied the tomato gene SlXTH1, which has highest homology to the cDNA of the XTH first purified from hypocotyls of Vigna angularis. SlXTH1 mRNA was found to accumulate transiently at an early stage of fruit development, and the peak day of accumulation was about 12 days after pollination. The expression profile of SlXTH1 mRNA was roughly associated with that of the reported enzyme activity, suggesting that the SlXTH1 protein may be one of the main determinants of the enzyme activity. We then examined how alteration of expression of the SlXTH1 gene could influence the characteristics of fruits. Several lines of transgenic tomatoes with different levels of SlXTH1 transcript were produced by introducing a SlXTH1 transgene in either the sense or antisense orientation downstream of a constitutive promoter. In these transgenic tomatoes, fruit size was positively correlated with the level of SlXTH1 transcript, which was monitored at its peak stage. This is the first in vivo demonstration that SlXTH1 can control the morphological character of plants through changes in its level of expression.
The aim of this research is to study the effects of salt stress during different stages of development in transgenic tomato plants overexpressing the apple spermidine synthase gene (MdSPDS1) compared to wild type (WT) plants. Under salt treatment (100 and 150 mM NaCl), tomato plants clearly displayed several stress symptoms such as impaired seedling growth, decreased chlorophyll content, reduction in fruit yield and increased electrolyte leakage (EL) in leaves. These changes were more prominent in WT plants compared to MdSPDS1 transgenic plants which accumulate significantly more polyamines, namely spermine and spermidine. The response of ascorbate peroxidase (APX) isoenzymes in tomato leaves under saline conditions was also investigated. The transcript levels of SlApx genes were significantly up regulated under 100 mM NaCl either in wild type or in transgenic plants. Under 150 mM NaCl, only transgenic plants were capable to maintain high expression of SlApx genes, whereas in WT plants the expression declined after one month treatment. As a consequence, APX activity was significantly higher in MdSPDS1 transgenic plants compared to WT under saline conditions. These results suggest that elevated APX activity might contribute largely to the protection against oxidative stress generated by NaCl treatments and thus confer salinity tolerance in tomato overexpressing MdSPDS1.
We developed a particle bombardment-mediated transformation protocol in Phyllostachys bamboo by optimizing the growth efficiency of a target cell culture system. Under the optimal condition, i.e. Murashige and Skoog medium containing 680 mg l−1 KH2PO4 and 10 μM Picloram, bamboo suspension cells actively proliferated at ca. 80 ml sedimented cell volume per 100 ml medium in 2 weeks. Log phased cells, i.e. 8 to 13-day-old suspension cells, which showed synchronous cell divisions with uniform morphology, were selected for the bombardment. We found that a target distance, 6 cm was much better for the transient gene expression (222 GUS-positive cells/dish/shot in average) than that of 9 cm (38 GUS-positive cells/dish/shot in average) in the target cells. When the bombardment was carried out using lag phased cells, e.g. 5-d-old cells, no or less GUS-positive cells could be seen. A high generation of stable transgenic bamboo cells was achieved with constructs expressing hygromycin phosphotransferase gene and enhanced fluorescent protein genes namely AcGFP1 and mCherry.
Cultured plant cell lines that grew and produced phytochemicals in the absence of exogenous plant hormones were established and then examined for their culture characteristics and the features of their produced phytochemicals. Cell lines that produce the following phytochemicals were established by selection and passage: anthocyanin-producing cell lines of cherry, peach, and Asiatic dayflower (Commelina communis L.); a carotenoid-producing cell line of Asiatic dayflower; a betacyanin-producing cell line of an Amaranthus species; and an isoflavone-producing cell line of soybean. The strains of cherry, peach, and Asiatic dayflower were established by passage in hormone-free media, whereas those of the Amaranthus sp. and soybean required passage with concentrations of auxins and cytokinins that were progressively halved. The anthocyanin-producing cell line of pear required low levels of phytohormones (0.156 μg l−1 of 2,4-dichlorophenoxyacetic acid and 0.0156 μg l−1 of 6-benzylaminopurine), and no cell line that grew in the absence of exogenous phytohormones could be obtained from pear. The anthocyanins malonylawobanin and flavocommelin of blue Asiatic dayflower petal were not found in extracts from the blue-violet anthocyanin-producing cultured cells. The anthocyanin-producing cherry cell line that requires no phytohormone is amenable to long-term culture over 300 passages. In suspension culture, the strain produces anthocyanin at a higher concentration in modified B5 medium than in Murashige and Skoog medium.
Production of transgenic lettuce seeds via in vitro flowering and fruit setting is reported here. Six days old cotyledons were co-cultivated with Agrobacterium tumefaciens strain EHA105 harbouring the binary vector pCAMBIA2301 carrying the reporter gene α-glucuronidase intron (GUS-INT) and the marker gene neomycin phosphotransferase (NPTII). Transgenic calluses and shoot buds were induced on MS medium augmented with 0.1 mg l−1 benzyladenine (BA), 0.1 mg l−1 α-naphtaleneacetic acid (NAA), 100 mg l−1 kanamycin and 500 mg l−1 timentin (ticarcillin clavulanate). After transferring the cultures onto MS basal medium augmented or not with kanamycin, in vitro flower induction was observed within 90 days and eventually matured and produced seed pods. Pollen grains obtained from flowers induced on basal medium without kanamycin were heterogeneous containing both GUS positive and negative pollen grains. In the corresponding seed population, a Mendelian ratio (3 : 1) of gusA transgene segregation was observed. On the other hand, flowers that were induced on basal medium under kanamycin selection, all the pollen grains were GUS positives.
Our previous investigation demonstrated that ADP-glucose hydrolytic nucleotide pyrophosphatase/phosphodiesterase (NPP) 1 is transported from the endoplasmic reticulum (ER)-Golgi system to the plastids via a secretory pathway in rice cells [Nanjo et al. (2006) Plant Cell 18: 2582–2592]. In this study, we analyzed the enzymatic characteristics and subcellular localization of its isozyme, NPP3. Unlike NPP1, NPP3 exhibited no hydrolytic activity toward ADP-glucose and no plastid-targeting ability. Furthermore, there was a clear difference between their N-terminal proteolytic processing schemes to form mature enzyme proteins. NPP1 is matured to a 70-kDa protein by two-step proteolytic processing. We detected the 72 kDa form of NPP1 in the microsomes of rice cells in addition to the 70 kDa mature protein, strongly suggesting that proprotein processing occurs post-translationally in the ER-Golgi system. To clarify the existence of the plastid-targeting signal of NPP1, the plastid localization of a series of carboxy-terminal truncated NPP1 proteins fused with green fluorescence protein was tested in rice cells. The results showed that NPP1 cannot be delivered to the plastid by the N-terminal region, including the ER signal sequence and the proprotein processing site, and that the peptide region, from 308 to 478 amino acid residues, is probably important for the transport of NPP1 into plastids in rice cells.
We used Arabidopsis thaliana seedlings as biocatalysts for the reduction of ketones, and investigated the factors affecting the chemical yield and enantioselectivity of the reactions. One- to four-week-old Arabidopsis seedlings were incubated for 24 h in reaction mixture containing either aromatic or aliphatic ketone as a substrate. After the reaction, the ketones and corresponding alcohols were extracted and quantified. The results indicated that Arabidopsis seedlings can be used as a biocatalyst for asymmetric reduction of ketones such as trifluoroacetophenone, t-butyl acetoacetate, methyl benzoylformate, and 2-(trifluoroacetyl)thiophene. The highest chemical yields were observed in seedlings pre-incubated under light conditions and in leaves, suggesting that asymmetric ketone reduction might be related to photosynthesis. In contrast, the age and size of seedlings did not have a significant effect on chemical yield or enantioselectivity. The findings suggest that Arabidopsis, which is widely used as a model plant system, presents a new opportunity for biotransformation.
By previous proteomic analysis, the amount of proteins exhibiting similarity to 1-Cys peroxiredoxin (Prx), a thiol-dependent peroxidase, was shown to be higher in seedlings of soybean [Glycine max (L.) Merr.] suffering from flooding stress than in normally grown seedlings. In this study, we characterized soybean 1-Cys Prx to elucidate the relationship of the protein to flooding stress. In the soybean genome, two genes corresponding to 1-Cys Prx (designated as GmPer1a, GmPer1b) exist. GmPer1a encodes a polypeptide containing the putative catalytic site, and a recombinant GmPer1a protein exhibited peroxidase activity. On the other hand, the GmPer1b contains a stop codon inside the deduced polypeptide-coding region, indicating that GmPer1b might be a pseudogene. The GmPer1a was expressed in developing seeds and transiently in germinating seeds of soybean. It was no longer expressed in 2-day-old seedlings, and was not induced by flooding treatment. The GmPer1 protein was synthesized in developing seeds, and was degraded during germination and growth. In addition, two forms of GmPer1 protein existed in both submerged and normally grown seedlings, and the amount of both forms was higher in the submerged seedlings. These suggest that both normal and post-translationally modified forms of GmPer1 might remain in seedlings suffering from flooding stress as a result of growth retardation.
Quinolizidine alkaloids (QAs) are one of the representative groups of plant alkaloids. To isolate genes involved in QA biosynthesis, we performed a differential gene expression analysis by PCR-select subtraction between a QA-producing bitter cultivar and a nonproducing sweet cultivar of Lupinus angustifolius. We obtained 71 and 43 clones specific to the bitter and sweet cultivars, respectively. Among the genes specifically expressed in the bitter cultivar, an acyltransferase-like gene (LaAT: Lupinus angustifolius acyltransferase) showing homology to the BAHD protein family was isolated. LaAT showed the strongest homology to the Arabidopsis thaliana BAHD acyltransferases involved in the formation of conjugated polyamines. Semi-quantitative RT-PCR revealed that LaAT expression was highest in the young leaves but barely detectable in the other organs of the bitter cultivar plant, whereas LaAT expression was undetectable in the sweet cultivar.
Protein-protein interactions are important for various biological and cellular processes. To analyze protein-protein interactions in living cells, the bimolecular fluorescence complementation (BiFC) assay, based on structural complementation of two non-fluorescent N- and C-terminal fragments from a fluorescent protein, has been developed and widely used in various research fields. Here I report a bright green-colored BiFC assay in living plant cells by using the N-terminal fragment (GN) of green fluorescent protein-S65T (GFP-S65T) and the C-terminal fragment (CC) of cyan fluorescent protein (CFP), but not GN and the C-terminal fragment (GC) of GFP. Fluorescence intensity of the GN/CC-based BiFC was 7-fold higher than that of the GN/GC-based BiFC. The emission spectrum of the GN/CC-based BiFC in planta was identical to that of full-length GFP-S65T. Ala163 residue within the CC fragment was found to be responsible for the improvement of the BiFC efficiency. These findings provide a BiFC method for in vivo protein-protein interaction studies to many GFP users in various research fields.
The mature seeds of 7 Cymbidium species, including those from different climatic regions (temperate or tropical), with different life-forms (terrestrial or epiphytic), and different nutritional modes (autotrophic or mycoheterotrophic) were cryopreserved by vitrification to demonstrate the effectiveness of this method for germplasm preservation. The period of exposure to vitrification solution was initially optimized for seeds of 3 species, and the optimum periods were found to be 60 min for C. goeringii and C. macrorhizon seeds and 30 min for C. finlaysonianum seeds. Cryopreservation under optimum conditions did not have any deleterious effect on the seeds, and the germination rates of the cryopreserved seeds were as follows (these rates were almost identical to those of untreated control seeds): 32% for C. goeringii, 82% for C. macrorhizon, and 76% for C. finlaysonianum. The cryopreservation method was also successfully applied to the seeds of other members of the genus Cymbidium, although the period of exposure to the vitrification solution varied with life-form. The periods were optimized to 60 and 30 min for the terrestrial and epiphytic species, respectively. These results suggest that cryopreservation by vitrification has wide applicability for the seeds of several orchid species with varying ecological and physiological adaptations.
The pollen allergy problem of Japanese cedar has been a nation-wide problem in spring time in Japan. It is said that about 20% of people suffer Japanese cedar pollinosis in urban areas. New projects substituting Japanese cedar forest stands with low-pollen or sterile male Japanese cedar trees started from 2008. The propagation of male sterile Japanese cedar genotypes is one of the most important steps in that project. We developed a micropropagation technique using an in vitro culture of male sterile Japanese cedar genotypes. Leafy shoot segments were used for explants. There was a genotypic difference in tissue culture response. Among the cytokinin tested, zeatin and 6-benzylaminopurine (BAP) were relatively effective for bud induction. More buds were induced in the medium containing zeatin than that containing BAP with the male sterile genotype Fukushima 5. Shoots developed from buds on the medium 1/2 LP containing 5 g l−1activated charcoal. Shoots produced roots on a medium containing 23.8 μM (5 mg l−1) 4-chloroindole-3-acetic acid and 0.044 μM (0.01 mg l−1) BAP. Rooted plantlets were successfully habituated and cultured in pots outside. The in vitro cultured materials will be used for the micropropagation of male sterile Japanese cedar in order to reduce the pollen in the air.
Visual selection enables transformation in plants that are sensitive to the stress of antibiotic selection. Furthermore, we proposed a method of isolating transformed cells in which the gene of interest is expressed at high level using visual selection. However, visual selection was inefficient and laborious because of the technical difficulties involved in clonal propagation of transformed calli selected using GFP (9.4–20.5% in the model japonica rice variety Nipponbare in our previous report). Very recently, we have discovered that an indica model cultivar, Kasalath, is very highly competent for Agrobacterium-mediated transformation compared to Nipponbare, suggesting that visual selection would be achieved more efficiently in Kasalath than in Nipponbare. We confirmed that the number of transformed cells emitting green fluorescence in Agrobacterium co-cultivated callus of Kasalath under no antibiotic pressure was >50-fold higher than the number obtained in Nipponbare. We succeeded in clonal propagation of GFP emitting cells at a frequency of 62.2–85% in Kasalath. Moreover, we showed that anthocyanin, that is an intrinsic pigmentation and thus might be more acceptable to consumers, can be used as an intrinsic marker in visual selection of rice. Thus, we propose that combination of the use of cultivar which has high transformation competency and visual selection system could enhance efficient production of transgenic rice expressing foreign gene product at higher level.