Carotenoid cleavage dioxygenases (CCDs) catalyze the cleavage of carotenoids in various organisms. In Arabidopsis, the CCD enzyme family consists of nine members, which are further divided into two subfamilies: five 9-cis epoxycarotenoid dioxygenases (NCEDs) and four CCDs. The NCEDs are involved in the biosynthesis of the plant hormone abscisic acid (ABA). CCDs are only distantly related to the NCEDs, and their substrate specificities and activities differ from those of the NCEDs. Recent evidence indicates that apocarotenoids produced by the action of CCDs play various roles in the growth and development of plants; these apocarotenoids include a novel plant hormone that controls shoot branching and pollinator attractants produced in reproductive organs. This review focuses on enzymes belonging to the CCD subfamily; their enzymatic activities and the functions of their apocarotenoid products are discussed.
Melon (Cucumis melo L.) is an excellent model species for the study of functional genomics in the Cucurbitaceae, a plant family that includes melon, cucumber, watermelon, and squash, because of its unique traits such as a lianous structure of the plant body and unusual mode of sex determination. Following the first workshop on Cucurbit genomics in Barcelona in 2005, the International Cucurbit Genomics Initiative (ICuGI: http://www.icugi.org/) was established at an academic level, and melon became the model species for cucurbit study as a result of existing genomic resources for this species. The ICuGI is currently overseeing three projects concerned with the development of research tools for functional genomics in melon: sequencing of ESTs, merging of existing melon genetic maps, and development of an ICuGI webpage containing specific genomic tools available to the cucurbit research community. Additional efforts are also underway to develop tools for the study of melon functional genomics. Availability of these resources will promote research activities in the Cucurbitaceae, especially melon. This review summarizes the current status of these tools and discusses future aspects of the study of melon functional genomics.
Establishment of the efficient method for isolating the female germ unit (FGU; egg, synergid and central cell) is useful for the studies on the characterization of each FGU as well as in vitro fertilization and gametosomatic hybridization. In this study, an easy one-step enzymatic procedure was successfully developed to isolate FGU from ovules of Petunia hybrida without releasing the somatic protoplasts from ovular tissues, which could not be achieved in the previous studies. Each FGU was separately liberated after treating the ovules, which were collected from ovaries of flowers one day after anthesis, with an appropriate enzyme solution comprised of 10 g l−1 Cellulase Onozuka R-10, 10 g l−1 Macerozyme R-10, 0.6 M mannitol, 5 mM 2-morpholinoethane-sulfonic acid and 5 g l−1 potassium dextran sulfate, pH 5.8 with 50-rpm shaking for 2 h at room temperature. Isolated FGUs were distinguished by their specific size and characteristics. Fluorescent staining with 4′, 6-diamidino-2-phenylindole could identify the polar nuclei of central cell and the nuclear polarity of egg apparatus cells. After transfer into washing solution supplemented with 0.6 M mannitol using a micropump-connected microcapillary, about 80% of the isolated FGUs were viable for up to 8 h after the isolation, as determining by fluorescein diacetate staining.
Gametosomatic hybridization has so far been achieved by the fusion between male-gametic (microspore tetrad or young-stage pollen) protoplasts and somatic protoplasts but no successful results have been reported on the use of female gamete so far. In this paper, we demonstrate for the first time the successful gametosomatic hybridization by using female gametophyte (egg cell) as the gametic-haploid partner instead of male gametophyte based on the method established for isolation of female germ units from ovules in Petunia in our previous report. Using P. hybrida strains, each single protoplast from egg cell and mesophyll cell were manually collected by a micropump-connected microcapillary and were then fused together as an individual pair by electrofusion. Each heterokaryon thus produced was transferred into Millicell culture plate placed in a Petri dish containing nurse cells, where cell division and microcolony formation took place. Among the fusion combinations tested, only fusants between mesophyll protoplast and egg cell protoplast from the same Petunia strain could regenerate and develop into the complete plants, whereas inter-varietal combinations failed to grow after developing into microcolonies or to regenerate plants. The flowers of a triploid hybrid line (2n=3x=21), confirmed by flow cytometric analysis and chromosome observation, were smaller and shorter than those of the parent with original color, and showed male sterility. These results indicate the intactness of isolated female gametophyte protoplasts, which might enable not only to produce triploid plants with various genomic combinations through gametosomatic hybridization but also to conduct fundamental studies on in vitro fertilization with isolated sperm cell protoplasts.
The Arabidopsis ENHANCER OF SHOOT REGENERATION 1 (ESR1) is thought to be a key gene for commitment to in vitro shoot regeneration in tissue culture. ESR1 encodes a member of the ETHYLENE RESPONSIVE FACTOR (ERF) family of transcription factors. Here, we report identification of genes downstream of ESR1 during in vitro shoot regeneration. We previously demonstrated that the ESR1 protein functions as a transcriptional activator; in the present study, genes upregulated after induction of ESR1 overexpression were screened by microarray experiments. Seven genes, including CUP-SHAPED COTYLEDON 1 (CUC1), CLAVATA3/EMBRYO SURROUNDING REGION-RELATED PEPTIDE 2 (CLE2), and GCN5-related N-acetyltransferase 1 (GNAT1) were identified as ESR1-upregulated genes by screening. CUC1, CLE2, and GNAT1 were also upregulated by translocation of ESR1-ER (estrogen receptor) fusions to the nucleus in the presence of cycloheximide, suggesting that these genes are possibly the direct target of the ESR1 protein. Transcript levels of CUC1, CLE2, and GNAT1 as well as ESR1 increased during the early in vitro shoot regeneration process, although their time courses were not necessarily similar. Thus, these genes may function downstream of ESR1 and may be involved in the shoot differentiation process.
The Arabidopsis ENHANCER OF SHOOT REGENERATION 1 (ESR1) gene is thought to regulate the initiation of in vitro shoot regeneration. ESR1 encodes a transcriptional activator belonging to the ETHYLENE-RESPONSIVE FACTOR family. In this study, we investigated the functions of the C-terminal domain of the ESR1 protein. Effector-reporter assays using the GAL4 DNA-binding domain fused with portions of the C-terminal domain revealed that the ESR motif, which is a short amino acid sequence commonly seen in the C-terminal domain of ESR1 and ESR2, functions as a transactivation domain. We also examined the effects of partial deletions of the C-terminal domain on the in vitro shoot regeneration-enhancing effects of ESR1 overexpression. The results revealed that the ESR motif and a region between the AP2/ERF domain and the ESR motif were required for the shoot regeneration-enhancing effects of ESR1. The region between the AP2/ERF domain and the ESR motif appeared to have an essential function other than nuclear localization, DNA-binding, and transactivation. Substitution of the ESR motif by the VP16 transactivation domain abolished the ability of ESR1 to enhance shoot regeneration, suggesting that the ESR motif not only has transactivation activity but also has other functions.
A cDNA clone designated Sdrac (965 bp) was isolated from the seedlings of Scoparia dulcis. This gene contains an open reading frame encoding the protein of 196 amino acid residues with high homology to Rac/Rop small GTPases from various plant sources. In Southern hybridization analysis, the partial hydrolysates of genomic DNA of S. dulcis prepared by the digestion with XbaI, XhoI, or EcoRI showed one main band together with a few weakly hybridized signals. The change in the transcriptional activity of Sdrac was analyzed by RT-PCR under various conditions, and it showed a marked increase by the treatment of the leaf tissues of the plant with methyl jasmonate and 2-chloroethylphosphonic acid, an ethylene-generating reagent. However, no significant change in the expression activity was observed upon the treatment of the leaves with Ca2+-ionophore A23187. Treatment of the leaves with high concentration of NaCl also did not affect the expression level of the gene. These results suggest the possibility that Sdrac product plays roles in a certain cellular event in the signal transduction processes evoked by methyl jasmonate and ethylene accompanying the change in the transcriptional activity.
ATP-binding cassette (ABC) proteins are one of the largest protein families found in all organisms. More than 120 genes encoding ABC proteins have been identified in Arabidopsis and rice. Various functions of plant ABC proteins have been characterized so far, and most of these proteins contain transmembrane domains. However, at present there has been no report on one soluble ABC protein group, the ABCF subfamily. We analyzed expression of five members of the Arabidopsis ABCF subfamily using promoter-GUS fusion constructs, and identified that these members are expressed in various organs of Arabidopsis at different stages. We also isolated knockout lines of four members of the ABCF subfamily, and showed that AtABCF3 is involved in root growth and development. These results suggest that the Arabidopsis ABCF subfamily functions throughout the plant and that it has important roles in development.
In order to accelerate functional analysis of large transcription factor families, development of a simple and easy protocol to generate transgenic cells of Arabidopsis overexpressing the transgenes using collections of cDNAs for coding sequences (CDSs) of the family genes is promising. We conceived a batch processing protocol for construction of plant expression vector plasmids and Agrobacterium-mediated transformation of suspension-cultured cells using a Gateway system-compatible cDNA collection and demonstrated its feasibility using a cDNA collection of the DOF family. The present results suggested that the batch processing from the LR reaction to the transformation of Agrobacterium cells was properly performed. The specific overexpression of a single DOF transgene was observed in at least 6 of 8 lines, and a transgenic callus obviously overexpressing multiple DOF transgenes was not obtained. These results show the potential utility of the present protocol to gain a clue for detailed functional analyses of Arabidopsis genes.
Introduction of T-DNAs with foreign genes by Agrobacterium-mediated transformation is widely used in plants. Multiple-introduced complex T-DNA loci, however, are difficult to clarify by conventional DNA gel blot analysis. We performed fluorescence in situ hybridization on extended DNA fibers (fiber FISH) of transgenic tobacco plants harboring multiple 37-kb T-DNA constructs. Five and seven types of integrations were successfully visualized in two transgenic lines. Most of the loci suffered duplication, deletion and/or translocation, indicating the complex integration events of the medium-size T-DNA. We concluded that fiber-FISH analysis is a powerful tool to analyze organization of multiple T-DNA loci in detail.
This examination for simple and efficient RNA extraction methods was conducted in vegetative organs of persimmon, including leaves, which contain inhibitory substances. We applied the result to physiological genetic analysis. Among the four extraction kits and a traditional SDS+PVP method, the combination of FastPure and Fruit-mate was the most suitable, resulting in maximum yield of 3.96 μg with high quality from 20 mg of persimmon mature leaves. Extraction was also possible from several organs other than leaves. The standard curve of quantitative-PCR (Q-PCR) using extracted RNA presented a high correlation coefficient (r2=0.99). Using this method, the activity of photosynthesis, which is an important role of leaves, was analyzed using DkrbcL as a target. The seasonal expression pattern of DkrbcL mRNA in leaves which was clearly demonstrated, indicates that this RNA extraction method is very important for the genetic analysis of persimmon vegetative organs.
We previously reported that transgenic rice (Oryza sativa L.) lines overexpressing OsWRKY11 showed significant desiccation tolerance, as indicated by their slower water loss in detached leaves. Here we examined the contents of sucrose, glucose, fructose and raffinose. Raffinose was shown to accumulate at a significantly higher level in the transgenic plants overexpressing OsWRKY11. Microarray analysis of gene expression profile indicated that the gene expression of Os07g0209100 encoding raffinose synthase and that of Os07g0687900 encoding galactinol synthase were up-regulated. These results suggest that OsWRKY11 induced activation of these genes involved in raffinose synthesis and the accumulated raffinose played an important role in the desiccation tolerance of the OsWRKY11-overexpressed plants.
Tobacco BY-2 cells were transformed with the human UDP-galactose transporter 1 gene (hUGT1) under the control of a 35S promoter. Accumulation of mRNA and protein derived from hUGT1 was detected in the hUGT1-transformed BY-2 cells. To identify the cell surface carbohydrates, BY-2 protoplasts were treated with 21 kinds of biotinylated lectin, stained with fluorescein isothiocyanate (FITC)-conjugated streptavidin and observed by fluorescence microscopy. FITC-ConA and -RCA120 staining exhibited strong fluorescence on the surface of BY-2 protoplasts, whereas staining with other FITC-lectin conjugates showed faint or no fluorescence. Regarding FITC-ConA and -RCA120 staining, the fluorescence of hUGT-expressing BY-2 protoplasts was weaker than that of control cells. Decreased FITC fluorescence of hUGT-expressing BY-2 protoplasts was also detected by fluorescence-activated cell sorting (FACS) analysis, suggesting that the surface carbohydrate composition was modified.