Transgenic torenia plants with various pink petal tones were obtained from blue or violet cultivars by combining downregulation of endogenous flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′, 5′-hydroxylase (F3′5′H) genes with expression of a heterologous gene. Knockdown of the F3′H and F3′5′H genes in a blue torenia cultivar resulted in pale pink lines by decreasing the cyanidin and delphinidin levels and increasing the pelargonidin-based anthocyanin levels. Additional expression of rose dihydroflavonol 4-reductase (DFR) gene elevated the level of pelargonidin and yielded darker pink petals. Expression of pelargonidin DFR gene instead of the rose DFR gene increased the level of pelargonidin and darkened the petal color. Introducing the two genetic constructs containing a DFR gene into a violet torenia cultivar, which had more anthocyanins and a darker color than the blue one, further increased the level of pelargonidin and pink color intensity. These results reveal that selection of a suitable gene source and host greatly affects the phenotypes of the resultant transgenic plants.
Copaiba (Copaifera officinalis) is a tropical plant that is also known as the ‘diesel tree’, previously noted for production of diesel-like oleoresin. The advancements in molecular tools such as expressed sequence tag (EST) library construction provide a novel opportunity for insight into the physiology of this tree. We generated a small set of ESTs for a young copaiba, sequencing and annotating a total of 613 unigenes. Of these, 84% showed similarity to the National Center for Biotechnology Information (NCBI) database. Annotation showed 70% of unigenes had at least one associated Gene Ontology (GO) term. We found a majority of ESTs to be associated with heat response genes. Based on these data, this EST library of C. officinalis represents a small but important step in helping to understand the general physiology and heat-response expression patterns. Additionally, this small collection of EST offers a modest starting point in helping to understand this enigmatic tropical plant.
Stability of the variegation was examined in the plants obtained by tissue culture of three variegated cultivars of Farfugium japonicum, ‘Ukigumo-nishiki’, ‘Temboshi’ and ‘Kinkan’ by culturing shoot tips on plant growth regulator-free 1/2 Murashige and Skoog (MS) medium and rhizome and leaf blade segments on MS medium supplemented with 1 mg l−1 6-benzylaminopurine and 1 mg l−1 α-naphthaleneacetic acid, respectively. In ‘Ukigumo-nishiki’, shoots regenerated from rhizome and leaf blade segments had only green leaves in the former and either green or albino in the latter, respectively, whereas those developed from the culture of shoot tips exhibited the same variegation as the original plant. In this cultivar, however, shoots propagated secondary by transferring the variegated shoots obtained by shoot tip culture onto 1/2MS medium containing 0.2 mg l−1 6-furfurylaminopurine segregated into either green or albino shoots without maintaining the original variegation phenotype. These results indicate the chimeric nature of variegation in ‘Ukigumo-nishiki’. In contrast, shoots derived from all explants of ‘Temboshi’ and ‘Kinkan’ exhibited the same variegation as original plants during the multiplication process, indicating no chimeric nature of both cultivars. In these two non-chimeric variegated cultivars, approximately 3.2 times proliferation rate in average was obtained after ca. 40 days of culture on gellan gum-solidified medium containing 0.2 mg l−1 6-furfurylaminopurine. These shoots were successfully rooted on 1/2MS medium containing 0.01 mg l−1 α-naphthaleneacetic acid and established in the soil.
The Arabidopsis thaliana cfd (cytochrome f deficient) mutant was isolated by the sensitivity of its photosystem II to low temperature using a chlorophyll fluorescence imaging technique. The cfd mutant is defective in intersystem electron transport even at 23°C, secondarily leading to photodamage of PSII at 4°C. Map-based cloning revealed that the cfd phenotype is due to a mutation in At1g49380, which encodes a putative plastid-targeting protein with high similarity to Ccs1 in Chlamydomonas reinhardtii. Ccs1 is required for c-type cytochrome (Cyt) assembly in chloroplasts. Consistent with the high sequence similarity of At1g49380 and Ccs1, the levels of Cyt f heme and Cyt f were low in the cfd mutant. We conclude that CFD is an ortholog of Chlamydomonas Ccs1. In vitro ferredoxin-dependent plastoquinone reduction activity was not affected in cfd, suggesting that system II c-type Cyt biogenesis is required for the machinery of photosystem I cyclic electron transport.
Soybean somatic embryos have attracted attention both as a model of zygotic embryos and as explants for the generation of transgenic plants. β-Conglycinin, which is composed of three subunits (α, α′, and β) that are encoded by a multigene family, is a major seed component of soybean, and accumulates in somatic embryos with their maturation. We subjected the somatic embryos to transformation with vectors encoding double-stranded RNA fragments of various sizes that correspond to the gene for the α′ subunit of β-conglycinin. These DNA fragments were put under the control of the promoter region of the gene for another major seed protein, the A2B1a subunit of glycinin. Transgenic somatic embryos were obtained within 2 months of transformation, and the resulting mature embryos manifested down-regulation of the α′ subunit of β-conglycinin in a manner dependent on the size of the RNAi vector insert. Accumulation of small interfering RNA and depletion of mRNA corresponding to the α′ subunit were indicative of RNAi. The amounts of α and β subunits of β-conglycinin, which share high sequence similarity with the α′ subunit, were also reduced in the mature somatic embryos. Moreover, the abundance of all β-conglycinin subunits was greatly reduced in the seeds of regenerated transgenic plants. Our results indicate that the application of RNAi to somatic embryos is a feasible and rapid option for functional studies of soybean seed components.
Protocorm-like bodies (PLBs) of Cattleya orchid CM2450 cultured either under constant illumination with cool-white-fluorescent lamps or in the dark were cocultivated with Agrobacterium tumefaciens strain EHA105 carrying plasmid pSMAHdN627-ORSV harboring genes coding for Odontoglossum ringspot virus (ORSV) replicase and hygromycin phosphotransferase. PLBs were maintained in liquid New Dogashima (ND) medium and then added to a bacterial suspension culture (OD600≈0.6) yielding medium dilution ratio of 1 : 10 and incubated for either 30 minutes or 3 h. Hygromycin-resistant secondary PLBs were induced after 4 weeks of culture on 2.5 g l−1 gellan gum-solidified ND medium containing 1 mg l−1 naphthaleneacetic acid (NAA), 0.1 mg l−1 benzyladenine (BA), 10 mg l−1 hygromycin, 20 mg l−1 meropenem, 10 g l−1 sucrose in both light- and dark-cultured PLBs. The number of resistant PLBs generated using dark-cultured PLBs was higher than those cultured under constant illumination. Presence of acetosyringone (AS) in the pre-culture medium was also effective for the transformation. The highest frequency of transformation was obtained when dark-cultured PLBs were pre-cultured with 100 μM AS for 3 days and inoculated with Agrobacterium liquid culture for 3 h. Transformation of hygromycin-resistant plantlets regenerated from different sites of inoculated PLBs was confirmed by Southern blot hybridization. Transcription of ORSV replicase gene in transgenic lines was successfully confirmed by Northern blot hybridization.
Tetraploid plants of Artemisia annua L. exhibiting high-artemisinin-yield were successfully induced by treating excised leaves of in vitro plant with 0.1% colchicine. The chromosome number of original diploid plant was confirmed to be 2n=2x=18 whereas that of the tetraploid plant was 2n=4x=36. Morphological and anatomical characteristics of tetraploid plants were obviously different from the diploid counterpart. The tetraploid plants had larger sizes of root system, stomata and glandular secretory trichomes than diploid plant, whereas leaf size in tetraploid was smaller but thicker than diploid. The highest amount of artemisinin content was produced at flower blooming stage in both diploid (2.4% dry weight) and tetraploid (3.8% dry weight) lines. Based on the maximum yield of artemisinin, the optimum harvest time for the diploid lines was flower initiation stage but it was at full blooming stage for tetraploid lines. The highest artemisinin yield in tetraploid plants up to 3.0 mg plant−1 was detected at this stage which was 1.5 times greater than diploid plants. This is a first report that clearly showed the potential of chromosome doubling strategy to produce high-yield line of A. annua plant for artemisinin production.
Boron is an essential nutrient for plant growth and reproduction. To identify a novel genetic mechanism which contributes to plant tolerance to nutrient deficiency, we screened Arabidopsis thaliana mutants for those tolerant to nutrient deficiencies. One of the isolated lines was tolerant to boron deficiency. This line was designated as LoBT1. From morphological characteristics and ploidy analysis, LoBT1 was found to be tetraploid, although the original screening population was diploid. Because LoBT1 is most likely to be created by the spontaneous duplication of the same diploid genome, LoBT1 is ‘autotetraploid’. Independently isolated autotetraploid A. thaliana lines which were obtained from the ABRC stock center were also tolerant to boron deficiency, showing that autotetraploidization generally improves tolerance to boron deficiency. Our results represent the first demonstration that autopolyploidization can improve tolerance to environmental stress. Because the clear tolerance was observed under a model system, our observation offers considerable resources to analyze autotetraploid tolerance to environmental stress in more details.
The control of flowering by day length was the first photoperiodic response to be described and is also one of the most characterized in many plants. The circadian clock plays a pivotal role in the photoperiodic flowering pathway. In Arabidopsis, flowering time is controlled by the photoperiod as well as by the gibberellic acid and vernalization/autonomous pathways. FLOWERING LOCUS C (FLC), a MADS box protein, has been shown to be a key floral repressor in the vernalization/autonomous pathways. Although the roles and regulation of floral activators GIGANTEA, CONSTANS, and FLOWERING LOCUS T in the photoperiodic flowering pathway have been well characterized, those of the floral repressors are not well understood. Here, we demonstrate that the MADS AFFECTING FLOWERING 5 (MAF5) gene, one of the FLC family members, shows a diurnal expression pattern in light/dark cycles and that both gain- and loss-of-function mutations in the photoperiod pathway affect the gene expression of MAF5 and FLC. These results highlight the possible roles of MAF5 and FLC in crosstalk between the photoperiod and vernalization/autonomous pathways in Arabidopsis.
LHY and CCA1 play key roles in circadian clock functions and photoperiodic flowering in Arabidopsis. Double loss of function of LHY and CCA1 genes (lhy cca1) accelerated flowering under long days or short days, but the lhy cca1 delayed flowering time under constant-light (LL) conditions. FCA encodes an RNA binding protein that plays key roles in the autonomous pathway. Loss of function of FCA increases mRNA level of a major floral repressor gene, FLC. A mutation in FLC gene partially suppressed the late flowering phenotype of the lhy cca1 in LL. Based on this result, we have proposed that FLC may be involved in this process. Increased level of FLC mRNA in fca is responsible for the delay of flowering and the late flowering phenotype of the fca is suppressed by vernalization. In this paper, we isolated an enhancer of the late-flowering phenotype of the lhy cca1 in LL based on natural variation of two Arabidopsis accessions, Columbia and Landsberg erecata. The enhancer was named ELLCL and mapped near the FLC. This result suggested that the gene responsive to ELLCL might be FLC and was consistent with our previous results. The late-flowering phenotype of lhy cca1 was insensitive to vernalization. By contrast, flowering time of the fca was accelerated by vernalization as reported. These results suggested that posttranslational, but not the transcriptional, regulation of FLC might be involved in this process.
EARLY FLOWERING 3 (ELF3) is a circadian clock protein with a major role in maintaining circadian rhythms in plants. In this work, elf3-101 was mutagenized by EMS in plants of the Landsberg erecta (Ler) background to isolate suppressors of elf3 and to understand the molecular mechanisms of flowering time controlled by ELF3. Two suppressors, sel106 (Ler) obtained from this screen and sel5 (Col) from a previous study, were chosen for further analysis. Genetic mapping, gene expression analysis, and sequencing identified sel106 and sel5 as new alleles of gi and fca, respectively. Genetic interactions between elf3 and gi and between elf3 and fca in the control of the floral activator FLOWERING LOCUS T (FT) were also investigated. Six suppressors of elf3 were classified at least into four subgroups based on the expression of such floral regulators as GI, CO, FT, SVP, and FLC, and on their flowering times under LL, LD, and SD. This classification scheme is useful for the characterization of unidentified suppressor mutations.
Little is known about the mechanisms by which Ca2+-binding sensory proteins direct the plant heat shock (HS) response. Since two Ca2+-dependent protein kinases (CPK3 and CPK13) were recently shown to phosphorylate the heat shock transcription factor HsfB2a, we assessed in the current study whether these kinases are also involved in HS signal transduction, by monitoring the transcriptional profile of HS protein (Hsp) family genes in Arabidopsis Col-0 plants (WT) and the corresponding mutants. Both with and without HS, the gene transcript levels of Hsp70, Hsp101, Hsp17.4-CIII and Hsp15.7-CI were found to be lower in cpk3 and cpk13 mutants compared to WT, resulting in the impairment of basal thermotolerance in the mutants. To determine the in vivo function of CPKs, CPK3/13 and their substrate HsfB2a (heat shock transcription factor) were co-expressed as cofactors for the transient expression of a reporter (GUS) gene under the control of heat shock element (HSE) in Nicotiana benthamiana leaves. However, CPK3/13-phosphorylated HsfB2a did not function in the suppression/activation of HSE-promoted expression in the transient expression system. Implications for possible signal trafficking via CPKs and Hsfs are discussed.
It is well documented that metal-binding peptides, such as phytochelatins and metallothioneins, are involved in metal homeostasis and tolerance in plants. These peptides bind metals by means of the thiol groups of cysteine residues. Histidine is also known to be a metal-binding residue. It has been demonstrated that microorganisms and mammals possess histidine-rich metal-binding peptides for the storage and homeostasis of metals. In plants, however, only several examples which describe the characteristics of the histidine-rich metal binding peptides have been reported. We therefore searched for histidine-rich peptides in the Arabidopsis database. Here, we describe a candidate gene designated Arabidopsis thaliana histidine-rich peptide 1 (AtHIRP1). AtHIRP1, which belongs to a small auxin-up RNA (SAUR) family in Arabidopsis, shows the highest histidine content (19.7% of total amino acid residues) in the Arabidopsis genome. The recombinant AtHIRP1 apparently bound to Co2+, Ni2+, Cu2+, and Zn2+, but weakly to Cd2+. In the case of the AtHIRP1-Zn2+ binding, the dissociation constant was 0.58 μM and the maximum binding capacity was 12 mol Zn2+ per 1 mol AtHIRP1. The accumulation of AtHIRP1 transcripts increased by drought stresses. These results suggest that AtHIRP1 is a metal-binding peptide which may function in plants exposed to abiotic stresses.
The leaves of field-cultivated Gynura bicolor DC. are reddish purple on the abaxial side and green on the adaxial side. The leaves of cultured G. bicolor plantlets, however, appear almost completely green on both sides. Cultured plantlet leaves treated with sucrose accumulated anthocyanins on the abaxial side. In this study, to investigate the anthocyanin-accumulation mechanism in G. bicolor leaves, we isolated the MYB transcription regulatory gene from the leaves using a degenerate PCR method. The isolated gene, GbMYB2, was approximately 10-fold up-regulated in sucrose-treated leaves. In addition, co-expression of GbMYB2 and bHLH-type transcription factor, GbMYC1, activated GbDFR and GbANS promoters in tobacco leaf protoplasts. These results suggest that GbMYB2 might regulate anthocyanin biosynthesis genes in G. bicolor leaves.
The DnaK/Hsp70 family is a molecular chaperone that binds non-native states of other proteins, and affects various physiological processes in bacterial, plant and animal cells. In this study, a dnaK gene from a halotolerant cyanobacterium, Aphanothece halophytica, was introduced into Eustoma grandiflorum. The ApdnaK transformed plants could grow at a similar rate with the control plants expressing the vector alone. Although exposure to low temperature is required for the bolting of control Eustoma, it was not required for the ApdnaK transformant. Under normal growth conditions, the glutathione content in the ApdnaK transformants was higher than that of control plants. In the transformant, the pistil is not evident and consequently, no seed formation. The results suggest the involvement of molecular chaperone for the bolting of Eustoma.