WRKY genes encode proteins which belong to a large family of transcription factors that are involved in various developmental and physiological processes, response to pathogen infections and wound stress in plants. The molecular characteristics of WRKY genes involved in these are becoming clear in model plants, like Arabidopsis. However, knowledge of WRKY genes in other plants, for instance vegetable, is still not enough. In the present studies, using a yeast one-hybrid system we isolated a cDNA from a spinach cDNA library and characterized its function. The cDNA, designated SoWRKY1, encodes a putative polypeptide of 362 amino acids which is highly homologous to Arabidopsis WRKY40 (AtWRKY40). The putative primary structure of SoWRKY1 contains a single WRKY domain of Cys2His2 zinc finger motif and a potential nuclear localization signal (NLS), whose structure is characteristic of group IIa type WRKY protein. SoWRKY1-sGFP fusion protein was localized to the nucleus when the protein was expressed in onion epidermal cells. SoWRKY1 showed high binding affinity to DNA molecules containing TTGAC(C/T) W-box sequences and transcriptional activation activity in yeast. SoWRKY1 transcripts in spinach leaves were transiently induced by wounding treatment and salicylic acid (SA). The transcripts accumulated following treatment with cycloheximide (CHX), a protein biosynthesis inhibitor. Functional analysis of SoWRKY1 in vivo was performed by overexpression in Arabidopsis, and pathogenesis-related PR1 and PR2 gene expression level increased in the transgenic plants. These results suggest that SoWRKY1 might be involved as a transcription factor in defense-related signaling transduction pathways of spinach.
In suspension-cultured carrot cells, a phenylalanine ammonia-lyase gene, DcPAL3, plays an important role in regulation of anthocyanin synthesis. In the DcPAL3 promoter region, a putative cis-element, box-L, which is committed to the upregulation of promoter activity, has been identified. Here, we isolated DcMYB5 cDNA using yeast one-hybrid screening with box-L as a bait from a cDNA library prepared from cells of a variant cultured cell line constitutively synthesizing anthocyanin. Although expression of DcMYB2, 3, and 4 was observed, all of which were previously isolated by plaque hybridization from a subtracted cDNA library of anthocyanin-synthesizing cells of a normal cultured cell line cultured in medium lacking 2,4-dichlorophenoxyacetic acid, DcMYB5 expression was not observed in cells of the normal cultured cell line; high expression of DcMYB5 was observed in cells of the variant cultured cell line. Although not only DcMYB5 but also DcMYB2, 3, and 4 could bind to the box-L sequence in yeast, DcMYB3 and 5 showed strong transcriptional activation activity for DcPAL3 promoter in carrot protoplasts. These results suggest that DcMYB3 and 5 might play an important role in the upregulation of DcPAL3 promoter activity in the different regulatory paths between the normal and variant cultured cell lines.
Dioscorea batatas tuber lectin 1 (DB1) is a storage protein isolated from yam tuber, and is shown to be a mannose-binding lectin. It has 58% amino-acid identity to insecticidal snowdrop bulb lectin GNA. In this study, we demonstrated that ≥1 mg ml−1 DB1 in an artificial diet significantly decreased the survival and fecundity of green peach aphid, Myzus persicaeca. We produced transgenic tobacco plants expressing cDNA of DB1 under the control of Cauliflower mosaic virus 35S promoter (35S-DB1) or phloem-specific promoter of rice sucrose synthase-1 gene (RSs1-DB1), and evaluate the degree of aphid resistance in whole plant bioassays. The number of survival aphids was reduced to 60% in transgenic lines with 35S-DB1 and RSs1-DB1, which accumulated DB1 at a level of 1.8% and 0.25%, respectively, of total soluble protein. Our results indicate that DB1 can be used to enhance resistance to sap-sucking insects in transgenic crops.
Multi-Auto-Transformation (MAT) vector system consists of positive selection, using the ipt or rol gene, with a site-specific recombination and DNA removal system, that generates morphologically normal marker-free transgenic plants without antibiotic selective-agent. This study describes rol-type MAT vector (pMAT101) containing lacZ gene as a model gene and the removable cassette with gus gene in the T-DNA region which was used to produce morphologically normal transgenic Kalanchoe blossfeldiana Poelln. employing rol gene as the selectable marker gene and gus gene as a reporter gene. Leaf explants inoculated with pMAT101 produced hairy roots with GUS expression on agar-solidified, half-strength MS medium without both plant growth regulators and selective agent under dark condition. These hairy roots produced shoots with Ri syndrome such as dwarfism, wrinkled leaves, and an over abundance of roots as a consequence of the morphogenic action of rol gene. They eventually produced morphologically normal shoots without GUS expression on the same fresh MS medium under 16h photoperiod. Molecular analysis of DNA from the hairy roots, shoots with Ri syndrome and morphologically normal shoots revealed that the normal shoots had only lacZ gene, and the removable cassette consisting of rol, R (recombinase) and gus genes was excised. This study indicates that the rol-type MAT vector could be used for the production of morphologically normal marker-free transgenic K. blossfeldiana plants without using selective chemical agents.
In order to induce horticulturally valuable mutants in the Liliaceous ornamental Tricyrtis hirta, embryogenic calluses of this species were irradiated with 12C+6 ion beams. Morphological characterization was performed on 35, 37 and 15 plants regenerated from calluses irradiated with 5, 10 and 20 Gy ion beams, respectively, and on 10 plants from non-irradiated calluses after 3 years of cultivation in pots. No plants were regenerated from calluses irradiated with 50 Gy ion beams. There were no large differences in the mean values of leaf length, leaf width, soil and plant analyzer development (SPAD) value of leaves, flower length and flower diameter between the control (division-derived plants from the mother plant of the embryogenic calluses) and the irradiation treatments at different doses. On the other hand, the mean number of shoots per plant increased, and the mean shoot length and the mean number of nodes per shoot decreased in the irradiation treatments. The mean number of flowers per plant was increased in the 20 Gy irradiation treatment. For most morphological characteristics investigated, the variation spectrum widened with increase in the irradiation dose. Several horticulturally attractive variations such as dwarfism, slender and deep green leaves, and large flowers were observed in regenerants from the irradiation treatments, and these variations were stable after additional 2 years of cultivation in pots or garden. Thus mutation induction by heavy ion beam irradiation of embryogenic calluses is a valuable tool for improving horticultural value of T. hirta.
Secretory and membrane proteins of eukaryotic cells must be properly folded and assembled in the endoplasmic reticulum (ER) before translocation to their final destination where they function. Perturbation of this process results in accumulation of unfolded proteins in the ER, so-called ER stress. The cells initiate a protective response to maintain cellular homeostasis, which is termed the ER stress response or the unfolded protein response (UPR). In the present study, we performed time-series transcriptome analysis of the ER stress response in Arabidopsis (Arabidopsis thaliana) with the N-linked glycosylation inhibitor tunicamycin, which causes misfolding of proteins in the ER, and therefore, triggers ER stress. A total of 259 genes were identified as tunicamycin-responsive genes, 175 of which were upregulated and 84 were downregulated. Hierarchical clustering and bioinformatic analysis demonstrated that 259 tunicamycin-responsive genes can be assigned to one of the six distinct expression classes and identified a potential novel cis-element, as well as known cis-elements, i.e., ER stress response element and UPR element. We also observed that a considerable number of tunicamycin-inducible genes, including those encoding the ER chaperone BiP and the membrane-bound transcription factor AtbZIP60, are coordinately upregulated at a late pollen development stage in Arabidopsis. This observation suggests that the ER stress response plays an important role in the development and function of pollens.
Agrobacterium-mediated transformation of Periploca sepium Bunge using proliferated clonal shoots was investigated to identify important factors affecting the transformation efficiency. Agrobacterium tumefaciens strains EHA105 and LBA4404 were used, both of which harbored a pKAFCR21 binary vector, which contained two reporter genes (GUS and sGFP, encoding β-glucuronidase and the synthetic green-fluorescent protein with S65T mutation) and two marker genes (encoding neomycin phosphotransferase II and hygromycin phosphotransferase). The factors evaluated were Agrobacterium strain, co-cultivation treatment, and antibiotic selection regime. The results revealed that the transformation efficiency could be synergistically increased to as high as 50–60% by infecting explants with Agrobacterium strain EHA105/pKAFCR21 and co-cultivating in the presence of 150 mg l−1 dithiothreitol, followed by selection at 100 mg l−1 kanamycin. Genomic DNA PCR, Southern hybridization, and quantitative real-time reverse transcription PCR analyses confirmed that the transgenes (GUS or sGFP) had presented, integrated, and expressed in all the tested transformant plants. The optimized protocol provides a basis for further genetic alteration of P. sepium for medicinal compounds and cis-polyisoprene production.
Three class-B MADS-box genes, the paleotype AP3 genes DMAP3A and DMAP3B, and the PI-like gene DMPI, were isolated from a native orchid in Japan named ‘Sekkoku’ (Dendrobium moniliforme) and ectopically expressed in Arabidopsis. DMAP3A and DMPI were expressed in all four floral whorls in the orchid, whereas DMAP3B was expressed only in whorls 2, 3, and 4. The ectopic expression of 35S::DMPI caused a partial sepal-to-petal conversion in whorl 1, increased the longevity of the flowers and delayed silique maturation in the transgenic plants, compared with wild-type plants. Transgenic Arabidopsis plants over-expressing 35S::DMAP3B/35S::DMPI exhibited a vegetative phenotype with leaf curling, a near-complete sepal-to-petal conversion in whorl 1, and the suppression of carpel development in whorl 4. Our results indicate that DMAP3B and DMPI are major class B MADS-box genes in D. moniliforme and may play an important role in the development of petals in the second floral whorl of the orchid. In addition, our results also suggest that these genes can be used for the genetic engineering of flower shape in plants.
Lipid peroxide-derived α,β-unsaturated aldehydes (2-alkenals) are potent toxins that inhibit enzymes in the Calvin cycle and in the mitochondrial photorespiratory pathway. Production of 2-alkenals in illuminated leaves and their participation in cellular damages have been suggested from the observation that the transgenic tobaccos overexpressing 2-alkenal reductase (AER), a 2-alkenal-detoxifying enzyme, showed tolerance to strong light. In order to identify the involved 2-alkenal species, we here analyzed the leaf aldehyde compositions in the AER-overproducing tobaccos and wild type (SR1), and compared their changes under photoinhibitory light. Aldehydes were extracted from the leaves at an early stage of photoinhibition, derivatized with 2,4-dinitrophenylhydrazine and analyzed on reversed-phase HPLC. In dark-adapted leaves of these tobacco lines, more than 40 carbonyl species were found, of which 17 were identified. In SR1 leaves, 2-alkenals such as (E)-2-pentenal, acrolein, and (E)-2-hexenal were increased by 70–290% after 30 min-illumination. In the leaves of AER-tobaccos, light-dependent increase of these 2-alkenals was apparently lower. Thus the production of highly reactive 2-alkenals in leaves was enhanced under photooxidative stress. The tolerance to strong light due to the overexpression of AER can be explained by the scavenging of these species.
Prenylated aromatic compounds represent the chemical components in the glandular trichomes (lupulin glands) of hops (Humulus lupulus, Cannabinaceae), which give the characteristic flavor and taste of beer. To isolate cDNAs for prenyltransferase recognizing aromatic substrates in hops, we constructed a cDNA library from the lupulin glands and randomly sequenced 11,233 EST clones, to obtain 6,613 non-redundant EST sequence information. Among them, we found an aromatic prenyltransferase-like gene (HlPT-1), which possessed three features of the plant aromatic prenyltransferase family, i.e., a D-rich motif, membrane-spanning domains, and a transit peptide. The tissue-specific expression study of HlPT-1 in the intact plant revealed this gene to be highly expressed in hop corns (female flowers), especially in the lupulin glands. Subcellular localization analysis using GFP fusion proteins suggested that HlPT-1 was localized to plastids. Phylogenetic analysis predicted that the HlPT-1 gene evolved from homogentisate prenyltransferases involved with vitamin E and plastoquinone biosynthesis.
We analyzed metabolic changes after mechanical wounding in Arabidopsis leaves to reveal wound effects on metabolism. Arabidopsis thaliana leaves were wounded by rubbing the leaf surface with silicon carbide particles (carborundum), as for experimental viral infection, and left up to 48 hours. We analyzed the metabolites of five replicate samples at each time point after wounding by ultra performance liquid chromatography time-of-flight mass spectrometry. Jasmonic acid production was induced immediately after wounding, as reported previously. Interestingly, we found that the amount of salicylic acid started to increase significantly 6 hours after wounding, followed by transient increases in salicylic acid glucoside and salicylic acid glucose ester 24 hours after wounding. The expression patterns of genes in salicylic acid biosynthesis pathway, which are available at public transcriptome databases, also support the observation of metabolic changes in salicylic acid and its glucose conjugates. These results indicate activation of salicylic acid metabolism after wounding, suggesting a role of salicylic acid in wound healing. The metabolome data obtained from this study is available from the MassBase metabolome database (http://webs2.kazusa.or.jp/massbase/).
An efficient method for in vitro propagation has been developed for pecan [Carya illinoinensis (Wangenh) K. Koch], a highly recalcitrant but commercially important fruit species. Nodal explants from in vitro grown cultivars, ‘Desirable’ and ‘Cape Fear’, were cultured on modified liquid woody plant medium (WPM) supplemented with 2% glucose and different concentrations of 6-benzylaminopurine (BAP; 0.44–44.39 μM). At least nine multiple shoots per explants were induced on modified WPM containing 13.32 μM BAP after three weeks of culture. The efficiency of shoot induction was over 95% in both ‘Cape Fear’ and ‘Desirable’ cultivars. The multiple shoots were proliferated and/or elongated on plant growth regulator free liquid WPM. Subsequently, the multiple shoots were separated and successfully rooted in liquid WPM containing 49.20 μM indole-3-butyric acid. The efficiency of rooting was over 90% in both ‘Cape Fear’ and ‘Desirable’ cultivar. The pecan plantlets were initially transferred to peat pellets and subsequently to the greenhouse. This is a simple and efficient protocol that may be used to propagate pecan plants through tissue culture.
A Japanese soybean variety, Kariyutaka, known to form many adventitious shoots from sections of the cotyledonary node, had a transformation frequency (relative number of T0 transgenic plants, which stably transmitted transgenes to their T1 progenies, to number of explants subjected to Agrobacterium-infection) of about 1.0% in the conventional protocol. We modified this method to obtain the transformation frequency of 4.4%. Modified protocol included mechanical wounding of explants with a micro-brush and application of 0.02% of Silwet L-77 during the infection with Agrobacterium. We obtained T0 plants 8 to 12 weeks after co-cultivation with Agrobacterium. T1 seeds were rapidly obtained from 5 to 6 months after the co-cultivation, for T0 plants flowered precociously. Three T0 plants out of 4, i.e. 75% of all T0 plants, which were generated at 12 weeks after co-cultivation, possessed stable transgene(s) that were transmitted to their T1 progenies, although only 7 T0 plants out of 22, i.e. 32% of all T0 plants, which were generated at 8 weeks after the co-cultivation, stably transmitted the Basta-resistance to their T1 progenies.