Plants shed organs by abscission, removing leaves, flowers or fruits when the organs are senescent, damaged, diseased or mature. Abscission also affects agriculture; for example, abscission of fruits or cereal grains can significantly reduce crop yield. Abscission of organs typically occurs in a predetermined tissue region, the abscission zone (AZ). Organ abscission can be disturbed in two ways, inhibition of AZ differentiation in the organ or suppression of abscission processes in AZ cells. Recent studies, mainly in Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and tomato (Solanum lycopersicum), have identified many genes involved in regulation of AZ differentiation and activation of abscission of flowers or floral organs, seeds, and fruits. In this review, we discuss the functions of these genes, the developmental regulation of AZ tissues, and the signaling pathways that induce abscission. We also discuss the emerging concept that the regulation of abscission involves many of the same regulators that function in determination of shoot apical meristem cell fate.
Nicotine and tropane alkaloids are specialized metabolites produced in certain species of Solanaceae, and some of these alkaloids have been used as pharmacological agents. In tobacco plants, nicotine is a defensive toxin against herbivorous insects, and jasmonate (JA) signaling leads to the induction of nicotine biosynthesis. JA-responsive structural genes of the nicotine pathway have been identified as being down-regulated in a low-nicotine tobacco mutant, which possesses mutant alleles at two loci, NICOTINE1 and NICOTINE2 (NIC1 and NIC2). A group of JA-responsive genes that encode homologous ERF transcription factors are clustered at the NIC2 locus and deleted in the mutant. These NIC2-locus ERFs up-regulate the structural genes of the biosynthetic pathway by recognizing GCC-like boxes in their promoters, forming a regulon for nicotine biosynthesis with the downstream targeted genes. The three basic components in JA signaling, COI1, JAZ, and MYC2, are required for JA-induced nicotine formation in tobacco. The bHLH transcription factor MYC2 positively regulates the structural genes, both directly by recognizing G boxes in their promoters and indirectly by up-regulating NIC2-locus ERF genes. Molecular elucidation of nicotine regulation would lead us to better understand the JA-dependent regulation of a wide range of phytochemicals.
Tomato is currently the model plant for fleshy fruit development and for Solanaceae species. Recent genomic approaches including transcriptome, proteome and metabolome analyses and genetic mapping have produced a wealth of candidate genes whose function needs to be assessed. The recent development in model and crop plants of TILLING (Targeting Induced Local Lesions IN Genomes), which reveals allelic series corresponding to several independent point mutations, and the current availability of deep sequencing tools further increase the interest of generating artificially-induced genetic diversity in tomato. We describe here the generation and use of EMS (ethyl methanesulfonate) tomato mutants in the miniature cultivar Micro-Tom and provide as example the identification of new fruit size and morphology mutants. We further propose new deep sequencing-based strategies for the discovery of mutations underlying phenotypic variations observed in mutant collections that will considerably increase the interest of exploiting Micro-Tom mutant collections for gene discovery in tomato.
Gene silencing through transcriptional repression can be induced by double-stranded RNA (dsRNA) that targets a gene promoter. This phenomenon, termed RNA-mediated transcriptional gene silencing (TGS), was first discovered in plants using a transgene that transcribes an inverted repeat of promoter sequence. However, endogenous genes differ from transgenes in the feasibility of TGS induction, by being more resistant to silencing. Heritable, transgenerational silencing of an endogenous gene has been induced by targeting dsRNA to the promoter in petunia and tomato plants, using a vector based on Cucumber mosaic virus. Efficient TGS depends on the function of a viral protein, which can facilitate epigenetic modifications through the transport of short interfering RNA to the nucleus. The efficiency of the TGS also depends on the length and nucleotide composition of the promoter RNA segments. Such epigenetic changes induced by the viral vector results in a novel class of modified plant, a plant that does not carry a transgene but has altered traits. Thus, TGS to modify the epigenetic state of a plant is now a feasible tool to engineer novel traits. Here we review epigenetic changes induced in a particular gene through RNA-directed DNA methylation and those induced randomly on the genome in terms of their use for plant biotechnology.
The tomato (Solanum lycopersicum) draft genome, along with a draft of the wild relative, Solanum pimpinellifolium, were released in 2012, almost a decade after the International Tomato Genome project was initiated. Tomato is an important domesticated crop species, as well as a model organism for many aspects of plant biology such as fleshy fruit development, ripening, disease resistance, plant architecture, and compound leaf development. For these reasons, there has been a substantial effort for producing a high quality reference genome that will serve as an anchor for tomato species, and for closely related Solanaceae plants. The utility of this genome has already been demonstrated by a relatively large number of studies that have been published since the release of the sequence, covering a wide range of topics including gene expression, genetic diversity, phylogeny, comparative genomics, and epigenetics. With the availability of the potato genome, it is now possible to perform detailed comparative genomic analysis of gene families in the Solanaceae, facilitated by conservation and synteny between their genomes. A large number of ongoing efforts will result in the sequencing of hundreds of wild and domesticated tomato accessions from various populations, uncovering the breeding history of tomatoes and introducing new genomic technologies to accelerate breeding processes. In this review, we provide an overview of the origins of tomato and its position in the wider Solanaceae, and demonstrate the impact of the tomato genome sequence on Solanaceae research on the basis of recent literature that has made use of this new resource.
Over the last 10 years, considerable efforts have been undertaken to develop genomic resources in tomato, including genomic clones, physical maps, DNA markers, mapping populations, and genetic linkage maps. Such resources facilitate the investigation of genome structure and gene functions, and the identification of genes of agronomic importance. In parallel, an international project with the participation of over 90 groups from 14 countries has been utilizing these resources to proceed with the deciphering of all of the genetic information carried by tomato. With the aid of new sequencing technologies and sophisticated bioinformatics, sequencing of the whole genome of tomato was successfully completed and the results were published in May 2012. The resulting large number of DNA markers, high-density linkage maps, and information on the structure and function of almost all of the gene components in the tomato genome are expected to contribute to a wide variety of biological fields by accelerating the processes of identification, isolation, and functional assignment of genes of interest, understanding of the evolutional process of Solanaceae and other plants, and breeding of new varieties with better agronomic traits.
Pseudoperonospora cubensis and Phytophthoracapsici are plant pathogenic oomycetes that are severe threats to cucurbit cultivation because of the their global distribution, their broad host range among the Cucurbitaceae family, and their ability to overcome susceptibilities to host, environment, and chemical management. Historically, these pathogens have been extensively studied in terms of their life cycles and infection strategies in order to determine appropriate methods to manage disease. In recent years, the genomes of both pathogens have been sequenced, which will lead to greater opportunities for pathogen detection and will help researchers to better understand the host-pathogen interaction. In Ps. cubensis, the transcriptomes of both Ps. cubensis and Cucumis sativus (cucumber) have been sequenced, and this data is being analyzed to determine the function of Ps. cubensis effectors and the role of alternative splicing in the regulation of pathogen gene expression. Previous and ongoing work is being done to determine cucumber genes involved in resistance. In P. capsici, effectors have been identified in the genome sequence, and the genome being used to identify variation in different P. capsici isolates. Future work is needed to give biological meaning to genomics data and to determine mechanisms of pathogenicity in oomycetes and resistance in cucurbits. Herein, we will present an overview of the current and future objectives of genome-based research in this area, describing the molecular mechanisms of pathogen virulence and host response to infection.
Like many crops, cultivated melons present a very large phenotypic polymorphism compared with the low phenotypic polymorphism of wild melons. Domestication has not been intensively studied and the genetic control of domestication traits is still poorly understood. The results of the subsequent diversification and selection processes are the present day types of melons. Genetic control of a majority of the diversification traits is under recessive genetic control: sex expression, fruit shape, sutures, number of placentas, gelatinous sheath around the seeds, white flesh colour and so on. Other phenotypic traits are dominant (orange flesh colour, netting, yellow colour of mature fruit in the Amarillo type and so on) as are most of the disease resistances. Presence of the same traits in very different botanical groups can be the result of parallel evolution but also of intercrossing between groups and selection of preferred alleles. New results (genome sequencing) and methods will allow a better understanding of the genetic control of domestication and diversification.
ADP-glucose pyrophosphorylase (AGPase) is a key regulatory enzyme in starch biosynthesis. In this research, 2,885 bp of the predicted promoter sequence for the AgpS1 gene encoding the AGPase small subunit was isolated from tomato. Sequence analyses revealed a number of known cis-elements related to responses to salt and dehydration stress and sugar repression; predicted TATA boxes are located at −88 to −94 bp and −114 to −120 bp. The spatial expression pattern and tissue/organ specificity of AgpS1 were analysed in during development using promoter-GUS transgenic tomato plants. Based on GUS staining, the obtained sequence was proven to be the functional promoter and directed broad expression in both sink and source tissues/organs, including seedling, stem, flower, fruit stalk, fruit and root. In source leaf and early developing fruit, GUS staining was observed in all tissues, except for epidermal tissue. In contrast, GUS staining tended to be confined to vascular tissues in seedling, stem, fruit stalk and ripening fruit. In particular, a patchy staining pattern was observed in the phloem of the stem and fruit stalk, suggesting that AgpS1 is expressed in the phloem companion cells in those organs. These results also suggest that AGPase mainly functions in the vascular tissue of those organs.
Cucurbita species are refractory to transformation. Hence, the only 2 reports published regarding the transformation of Cucurbita species until our successful transformation of C. moschata (cv. Heiankogiku) in 2011. The efficiency was 2.7±1.3% using wounded explants vortexed with whisker suspension. To improve transformation efficiency, transformation experiments were carried out with various ages of cotyledonary explants. The highest efficiency was obtained with 1-day-old (3.2±0.9%) and 2-day-old (3.3±0.8%) explants after germination. Histochemical analysis of GUS activity revealed that wounding allowed Agrobacterium access to the deeper layer of explants. These results suggested that cells having a high ability of shoot organogenesis exist not on the surface but in the deeper layers of explants. We applied vacuum infiltration to wounded explants to enhance Agrobacterium access to the deeper layers, which resulted in improvement of transformation efficiency by 3-fold (9.2±2.9%). To verify the efficacy of our transformation procedure for other Cucurbita species, we attempted to obtain transformants with 3 Cucurbita species: C. maxima (cv. Ebisu), C. pepo (cv. Black Tosca), and C. ficifolia (cv. Kurotanenankin). The average regeneration efficiencies were 54.2±7.2%, 62.5±19.1%, and 72.2±13.4% under our regeneration system. Although the efficiency for production of tansgenic plants was very low (ca. 0.2–0.3%), a transgenic line was obtained from C. maxima and C. pepo, respectively. We discuss recent advances that may help in the development of beneficial applications for the transformation of Cucurbita species.
Fleshy fruits are important worldwide crops that are rich sources of useful and functional compounds in the human diet. Although fruit ripening has been extensively studied, early fruit development has not been paid much attention despite its contribution to the sensorial and nutritional quality of the fruit. This study aimed at identifying candidate genes involved in early fleshy fruit development that can contribute to the control of final fruit size and composition by comparative analysis of tomato and grape genes. By mining public sequences and microarray database, we identified 23 transcription factors belonging to 14 classes (AP2-EREBP, ARF, bHLH, bZIP, C2C2-GATA, FHA, GeBP, GRAS, HB, LIM, MYB, PBF-2-like, SBP and WRKY) as candidate regulatory genes for early fruit development. The function of these candidate genes will be confirmed by several reverse genetic approaches using the miniature tomato cv. Micro-Tom.
Endopolyploidy, i.e. the amplification of genomic DNA without mitosis, is a widespread process in plants. Cells from the tomato fruit pericarp are characterized by a wide range of ploidy levels (from 2C to 256C). Although various functional hypotheses have been attributed to endoreduplication according to the literature, evidence for a specific role of endoreduplication in transcription and metabolism control is still lacking. We have developed a new method based on bacterial artificial chromosome fluorescent in situ hybridization (BAC-FISH) that allows the in situ determination of DNA ploidy levels of individual nuclei. The advantage of this method is illustrated by the analysis of ploidy levels and cell sizes within the pericarp tissue from mature green tomato fruits. Using this cellular approach we established the ploidy map of the pericarp tissue. Based on this map, we performed a structural analysis of endoreduplicated nuclei at the level of chromatin organization, nuclear shape and relationship with mitochondria. We demonstrated a link between the ploidy level of nuclei, the complexity of their shape and the number of mitochondria at the vicinity of polyploid nuclei. The use of the DNA FISH method demonstrated that endopolyploidy leads to the formation of polytene chromosomes, whereas the use of a RNA FISH method demonstrated that the rDNA transcription was increased during polyploidization. Performing quantitative PCR (qPCR) and RT-qPCR on sorted nuclei respectively, we confirmed that endoreduplication did amplified exponentially loci for a set of specific genes allowing us to demonstrade that endoreduplication results in an increasing transcriptional activity.
Besides being a model for fleshy fruits and Solanaceae species, tomato represents one of the main sources of ascorbate (vitamin C) in human diet in many parts of the world. Ascorbate fulfills various roles in plants due to its antioxidant potential and to its connection with other metabolic pathways e.g. cell wall biosynthesis. Among the functional genomic tools recently developed in tomato, EMS (ethyl methanesulfonate) mutant collections provide an opportunity for identifying allelic series of mutations in target genes by TILLING (Targeting Induced Local Lesions IN Genomes). We describe here the use of tomato EMS mutant collections in the miniature cv. Micro-Tom for the discovery of allelic variants in three ascorbate biosynthetic genes encoding the GDP-D-mannose pyrophosphorylase (GMP), the GDP-D-mannose epimerase (GME) and the GDP-L-galactose phosphorylase (GGP) respectively. We report on the discovery of several missense, truncation and splice junction mutations in these genes affecting plant ascorbate content to various levels, and show that several tomato mutant lines with strongly reduced ascorbate content undergo severe bleaching upon exposure to high light intensity.