Fleshy Fruit Development Research Articles

Metabolomics-Inspired Insight into Developmental, Environmental and Genetic Aspects of Tomato Fruit Chemical Composition and Quality.

Tomato was one of the first plant species to be evaluated using metabolomics and remains one of the best characterized, with tomato fruit being both an important source of nutrition in the human diet and a valuable model system for the development of fleshy fruits. Additionally, given the broad habitat range of members of the tomato clade and the extensive use of exotic germplasm in tomato genetic research, it represents an excellent genetic model system for understanding both metabolism per se and the importance of various metabolites in conferring stress tolerance. This review summarizes technical approaches used to characterize the tomato metabolome to date and details insights into metabolic pathway structure and regulation that have been obtained via analysis of tissue samples taken under different developmental or environmental circumstance as well as following genetic perturbation. Particular attention is paid to compounds of importance for nutrition or the shelf-life of tomatoes. We propose furthermore how metabolomics information can be coupled to the burgeoning wealth of genome sequence data from the tomato clade to enhance further our understanding of (i) the shifts in metabolic regulation occurring during development and (ii) specialization of metabolism within the tomato clade as a consequence of either adaptive evolution or domestication.

Gene Expression and Function Indry and Fleshy Fruit Development in Solanaceae

Gene Expression and Function Indry and Fleshy Fruit Development in Solanaceae

MADS-Box Transcription Factor FRUITFULL Orthologs in Nicotiana Promote Transition to Flower, but Did Not Significantly Alter Capsule Development

Understanding the genetics mechanisms of flower and fruit development is very crucial for efficient breeding of many agronomical important crops. MADS-box group of transcription factors has been known for its significant contribution of controlling fruit development. FRUITFULL (FUL) is one of the genes that plays important roles in this regard. For more understanding of FUL gene function in capsule dry-fruit development, this study used virus Induced Gene Silencing method to knockdown FUL gene expression in the newly developed model plant, Nicotiana obtusifolia. Treated plants exhibited delayed flowering, shorter inflorescence stems and longer fused sepal when compared to wild type and control group. However, no altered fruit phenotypes were observed among the treated N. obtusifolia plants. Downregulation of FUL gene in Nicotiana obtusifolia, allows us to assess a better understanding of its function in dry fruit species of the family Solanaceae. It also suggests that FUL protein plays different roles in the development of dry versus fleshy fruits in Solanaceae.

Functional analysis of tomato calmodulin gene family during fruit development and ripening.

Calmodulin is a ubiquitous calcium sensor to recognize the different developmental and/or stimulus-triggered calcium changes and regulate plant growth and development. However, the function of calmodulin remains elusive for fleshy fruit development. We performed expression studies of a family of six calmodulin genes (SlCaMs) in tomato fruit. All calmodulins showed a double peak expression pattern. The first flat peak appeared at 10–30 days after anthesis, but their expression rapidly declined at mature green and breaker. Then a sharp and even higher peak came at turning/pink stages. Among six calmodulins, SlCaM1 had the highest expression during fruit enlargement, whereas SlCaM2 was the major calmodulin during fruit ripening. However, SlCaMs showed different patterns in three ripening mutants rin, Nor and Nr. In particular, at the stages corresponding to mature green and breaker, the expression levels of SlCaMs in those mutants were significantly higher than wild-type. Furthermore, SlCaMs, especially SlCaM2 were upregulated by ethylene. Transiently overexpressing SlCaM2 in mature green fruit delayed ripening, while reducing SlCaM2 expression accelerated ripening. Our results suggest that SlCaMs play double roles to regulate fruit ripening. Prior to the ethylene burst, the ethylene-independent repression of SlCaMs might be critical for fruit to initiate the ripening process. After the ethylene burst, SlCaMs could participate in the ethylene coordinated rapid ripening.

The auxin Sl-IAA17 transcriptional repressor controls fruit size via the regulation of endoreduplication-related cell expansion.

Auxin is known to regulate cell division and cell elongation, thus controlling plant growth and development. Part of the auxin signaling pathway depends on the fine-tuned degradation of the auxin/indole acetic acid (Aux/IAA) transcriptional repressors. Recent evidence indicates that Aux/IAA proteins play a role in fruit development in tomato (Solanum lycopersicum Mill.), a model species for fleshy fruit development. We report here on the functional characterization of Sl-IAA17 during tomato fruit development. Silencing of Sl-IAA17 by an RNA interference (RNAi) strategy resulted in the production of larger fruit than the wild type. Histological analyses of the fruit organ and tissues demonstrated that this phenotype was associated with a thicker pericarp, rather than larger locules and/or a larger number of seeds. Microscopic analysis demonstrated that the higher pericarp thickness in Sl-IAA17 RNAi fruits was not due to a larger number of cells, but to the increase in cell size. Finally, we observed that the cell expansion in the transgenic fruits is tightly coupled with higher ploidy levels than in the wild type, suggesting a stimulation of the endoreduplication process. In conclusion, this work provides new insights into the function of the Aux/IAA pathway in fleshy fruit development, especially fruit size and cell size determination in tomato.

Transcriptional control of fleshy fruit development and ripening.

Fleshy fruits have evolved to be attractive to frugivores in order to enhance seed dispersal, and have become an indispensable part of the human diet. Here we review the recent advances in the understanding of transcriptional regulation of fleshy fruit development and ripening with a focus on tomato. While aspects of fruit development are probably conserved throughout the angiosperms, including the model plant Arabidopsis thaliana, it is shown that the likely orthologues of Arabidopsis genes have distinct functions in fleshy fruits. The model for the study of fleshy fruit development is tomato, because of the availability of single gene mutants and transgenic knock-down lines. In other species, our knowledge is often incomplete or absent. Tomato fruit size and shape are co-determined by transcription factors acting during formation of the ovary. Other transcription factors play a role in fruit chloroplast formation, and upon ripening impact quality aspects such as secondary metabolite content. In tomato, the transcription factors NON-RIPENING (NOR), COLORLESS NON-RIPENING (CNR), and RIPENING INHIBITOR (MADS-RIN) in concert with ethylene signalling regulate ripening, possibly in response to a developmental switch. Additional components include TOMATO AGAMOUS-LIKE1 (TAGL1), APETALA2a (AP2a), and FRUITFULL (FUL1 and FUL2). The links between this highly connected regulatory network and downstream effectors modulating colour, texture, and flavour are still relatively poorly understood. Intertwined with this network is post-transcriptional regulation by fruit-expressed microRNAs targeting several of these transcription factors. This important developmental process is also governed by changes in DNA methylation levels and possibly chromatin remodelling.

The sugar transporter inventory of tomato: genome-wide identification and expression analysis.

The mobility of sugars between source and sink tissues in plants depends on sugar transport proteins. Studying the corresponding genes allows the manipulation of the sink strength of developing fruits, thereby improving fruit quality for human consumption. Tomato (Solanum lycopersicum) is both a major horticultural crop and a model for the development of fleshy fruits. In this article we provide a comprehensive inventory of tomato sugar transporters, including the SUCROSE TRANSPORTER family, the SUGAR TRANSPORTER PROTEIN family, the SUGAR FACILITATOR PROTEIN family, the POLYOL/MONOSACCHARIDE TRANSPORTER family, the INOSITOL TRANSPORTER family, the PLASTIDIC GLUCOSE TRANSLOCATOR family, the TONOPLAST MONOSACCHARIDE TRANSPORTER family and the VACUOLAR GLUCOSE TRANSPORTER family. Expressed sequence tag (EST) sequencing and phylogenetic analyses established a nomenclature for all analyzed tomato sugar transporters. In total we identified 52 genes in tomato putatively encoding sugar transporters. The expression of 29 sugar transporter genes in vegetative tissues and during fruit development was analyzed. Several sugar transporter genes were expressed in a tissue- or developmental stage-specific manner. This information will be helpful to better understand source to sink movement of photoassimilates in tomato. Identification of fruit-specific sugar transporters might be a first step to find novel genes contributing to tomato fruit sugar accumulation.

Transcription and enzymatic analysis of beta-glucosidase VvBG1 in grape berry ripening

Although beta-glucosidase was recently demonstrated to be involved at transcriptional level in fleshy fruit development, it remains unclear whether BG possesses biochemical activity. In the present study, real-time reverse transcription polymerase chain reaction analysis showed that the mRNA expression levels of β-glucosidase gene VvBG1 increased rapidly concomitant with berry red-colouring in Muscat Hamburg grapevine (Vitis vinifera), suggesting that the VvBG1 gene might be involved in the berry ripening. In order to explore VvBG1 biochemical activity, a 60-kD recombinant protein, VvBG1, was expressed in Escherichia coli BL21 (DE3) and purified. The results gained from a combination of VvBG1-pulp-homogeneity-incubation test and gas chromatography-mass spectroscopy analysis showed that VvBG1-treated berry samples contained more free ABA than the control, demonstrating that VvBG1 has high glycosyl hydrolase activity that can transform the ABA-glucosyl ester to free ABA. In conclusion, both the high expression and increased hydrolase activity of VvBG1 could suggest an important role of ABA in grape berry ripening.

MicroRNA156‐targeted SPL/SBP box transcription factors regulate tomato ovary and fruit development

Fruit ripening in tomato (Solanum lycopersicum L.) is well understood at the molecular level. However, information regarding genetic pathways associated with tomato ovary and early fruit development is still lacking. Here, we investigate the possible role(s) of the microRNA156/SQUAMOSA promoter-binding protein-like (SPL or SBP box) module (miR156 node) in tomato ovary development. miR156-targeted S.lycopersicum SBP genes were dynamically expressed in developing flowers and ovaries, and miR156 was mainly expressed in meristematic tissues of the ovary, including placenta and ovules. Transgenic tomato cv. Micro-Tom plants over-expressing the AtMIR156b precursor exhibited abnormal flower and fruit morphology, with fruits characterized by growth of extra carpels and ectopic structures. Scanning electron microscopy and histological analyses showed the presence of meristem-like structures inside the ovaries, which are probably responsible for the ectopic organs. Interestingly, expression of genes associated with meristem maintenance and formation of new organs, such as LeT6/TKN2 (a KNOX-like classI gene) and GOBLET (a NAM/CUC-like gene), was induced in developing ovaries of transgenic plants as well as in the ovaries of the natural mutant Mouse ear (Me), which also displays fruits with extra carpels. Conversely, expression of the MADS box genes MACROCALYX (MC) and FUL1/TDR4, and the LEAFY ortholog FALSIFLORA, was repressed in the developing ovaries of miR156 over-expressors, suggesting similarities with Arabidopsis at this point of the miR156/SPL pathway but with distinct functional consequences in reproductive development. Altogether, these observations suggest that the miR156 node is involved in maintenance of the meristematic state of ovary tissues, thereby controlling initial steps of fleshy fruit development and determinacy.

Genome-wide identification and expression analysis of aquaporins in tomato.

The family of aquaporins, also called water channels or major intrinsic proteins, is characterized by six transmembrane domains that together facilitate the transport of water and a variety of low molecular weight solutes. They are found in all domains of life, but show their highest diversity in plants. Numerous studies identified aquaporins as important targets for improving plant performance under drought stress. The phylogeny of aquaporins is well established based on model species like Arabidopsis thaliana, which can be used as a template to investigate aquaporins in other species. In this study we comprehensively identified aquaporin encoding genes in tomato (Solanum lycopersicum), which is an important vegetable crop and also serves as a model for fleshy fruit development. We found 47 aquaporin genes in the tomato genome and analyzed their structural features. Based on a phylogenetic analysis of the deduced amino acid sequences the aquaporin genes were assigned to five subfamilies (PIPs, TIPs, NIPs, SIPs and XIPs) and their substrate specificity was assessed on the basis of key amino acid residues. As ESTs were available for 32 genes, expression of these genes was analyzed in 13 different tissues and developmental stages of tomato. We detected tissue-specific and development-specific expression of tomato aquaporin genes, which is a first step towards revealing the contribution of aquaporins to water and solute transport in leaves and during fruit development.

An extensive proteome map of tomato (Solanum lycopersicum ) fruit pericarp

Tomato (Solanum lycopersicum) is the model species for studying fleshy fruit development. An extensive proteome map of the fruit pericarp is described in light of the high-quality genome sequence. The proteomes of fruit pericarp from 12 tomato genotypes at two developmental stages (cell expansion and orange-red) were analyzed. The 2DE reference map included 506 spots identified by nano-LC/MS and the International Tomato Annotation Group Database searching. A total of 425 spots corresponded to unique proteins. Thirty-four spots resulted from the transcription of genes belonging to multigene families involving two to six genes. A total of 47 spots corresponded to a mixture of different proteins. The whole protein set was classified according to Gene Ontology annotation. The quantitative protein variation was analyzed in relation to genotype and developmental stage. This tomato fruit proteome dataset is currently the largest available and constitutes a valuable tool for comparative genetic studies of tomato genome expression at the protein level. All MS data have been deposited in the ProteomeXchange with identifier PXD000105.

Molecular programme of senescence in dry and fleshy fruits.

Fruits of angiosperms can be divided into dry and fleshy fruits, depending on their dispersal strategies. Despite their apparently different developmental programmes, researchers have attempted to compare dry and fleshy fruits to establish analogies of the distinct biochemical and physiological processes that occur. But what are the common and specific phenomena in both biological strategies? Is valve dehiscence and senescence of dry fruits comparable to final ripening of fleshy fruits, when seeds become mature and fruits are competent for seed dispersal, or to over-ripening when advanced senescence occurs? We briefly review current knowledge on dry and fleshy fruit development, which has been extensively reported recently, and is the topic of this special issue. We compare the processes taking place in Arabidopsis (dry) and tomato (fleshy) fruit during final development steps using transcriptome data to establish possible analogies. Interestingly, the transcriptomic programme of Arabidopsis silique shares little similarity in gene number to tomato fruit ripening or over-ripening. In contrast, the biological processes carried out by these common genes from ripening and over-ripening programmes are similar, as most biological processes are shared during both programmes. On the other hand, several biological terms are specific of Arabidopsis and tomato ripening, including senescence, but little or no specific processes occur during Arabidopsis and tomato over-ripening. These suggest a closer analogy between silique senescence and ripening than over-ripening, but a major common biological programme between Arabidopsis silique senescence and the last steps of tomato development, irrespective of its distinction between ripening and over-ripening.

Role of plant hormones and their interplay in development and ripening of fleshy fruits.

Plant hormones have been extensively studied for their roles in the regulation of various aspects of plant development. However, in the last decade important new insights have been made into their action during development and ripening, in both dry and fleshy fruits. Emerging evidence suggests that relative functions of plant hormones are not restricted to a particular stage, and a complex network of more than one plant hormone is involved in controlling various aspects of fruit development. Though some areas are extensively covered, considerable gaps in our knowledge and understanding still exist in the control of hormonal networks and crosstalk between different hormones during fruit expansion, maturation, and various other aspects of ripening. Here, we evaluate the new knowledge on their relative roles during tomato fruit development with a view to understand their mechanism of action in fleshy fruits. For a better understanding, pertinent evidences available on hormonal crosstalk during fruit development in other species are also discussed. We envisage that such detailed knowledge will help design new strategies for effective manipulation of fruit ripening.

A VIN1 GUS::GFP fusion reveals activated sucrose metabolism programming occurring in interspersed cells during tomato fruit ripening

The tomato is a model for fleshy fruit development and ripening. Here we report on the identification of a novel unique cell autonomous/cellular pattern of expression that was detected in fruits of transgenic tomato lines carrying a GFP GUS driven by the fruit specific vacuolar invertase promoter VIN1. The VIN1 promoter sequence faithfully reproduced the global endogenous VIN expression by conferring a biphasic pattern of expression with a second phase clearly associated to fruit ripening. A closer view revealed a salt and pepper pattern of expression characterized by individual cells exhibiting a range of expression levels (from high to low) surrounded by cells with no expression. This type of pattern was detected across different fruit tissues and cell types with some preferences for vascular, sub-epidermal layer and the inner part of the fruit. Cell ability to show promoter activity was neither directly associated with overall ripening – as we find VIN+ and – VIN− cells at all stages of ripening, nor with cell size. Nevertheless the number of cells with active VIN-driven expression increased with ripening and the activity of the VIN promoter seems to be inversely correlated with cell size in VIN+ cells. Gene expression analysis of FACS-sorted VIN+ cells revealed a transcriptionally distinct subpopulation of cells defined by increased expression of genes related to sucrose metabolism, and decreased activity in protein synthesis and chromatin remodeling. This finding suggests that local micro heterogeneity may underlie some aspects (i.e. the futile cycles involving sucrose metabolism) of an otherwise more uniform looking ripening program.

Identification of microRNA targets in tomato fruit development using high-throughput sequencing and degradome analysis

MicroRNAs (miRNAs) play important roles in plant development through regulation of gene expression by mRNA degradation or translational inhibition. Despite the fact that tomato (Solanum lycopersicum) is the model system for studying fleshy fruit development and ripening, only a few experimentally proven miRNA targets are known, and the role of miRNA action in these processes remains largely unknown. Here, by using parallel analysis of RNA ends (PARE) for global identification of miRNA targets and comparing four different stages of tomato fruit development, a total of 119 target genes of miRNAs were identified. Of these, 106 appeared to be new targets. A large part of the identified targets (56) coded for transcription factors. Auxin response factors, as well as two known ripening regulators, COLORLESS NON-RIPENING (CNR) and APETALA2a (SlAP2a), with developmentally regulated degradation patterns were identified. The levels of the intact messenger of both CNR and AP2a are actively modulated during ripening, by miR156/157 and miR172, respectively. Additionally, two TAS3-mRNA loci were identified as targets of miR390. Other targets such as ARGONAUTE 1 (AGO1), shown to be involved in miRNA biogenesis in other plant species, were identified, which suggests a feedback loop regulation of this process. In this study, it is shown that miRNA-guided cleavage of mRNAs is likely to play an important role in tomato fruit development and ripening.

Apple SEPALLATA1/2‐like genes control fruit flesh development and ripening

Flowering plants utilize different floral structures to develop flesh tissue in fruits. Here we show that suppression of the homeologous SEPALLATA1/2-like genes MADS8 and MADS9 in the fleshy fruit apple (Malus x domestica) leads to sepaloid petals and greatly reduced fruit flesh. Immunolabelling of cell-wall epitopes and differential staining showed that the developing hypanthium (from which the apple flesh develops) of MADS8/9-suppressed apple flowers lacks a tissue layer, and the remaining flesh tissue of fully developed apples has considerably smaller cells. From these observations, it is proposed that MADS8 and MADS9 control the development of discrete zones within the hypanthium tissue, and therefore fruit flesh, and also act as foundations for development of different floral organs. At fruit maturity, the MADS8/9-suppressed apples do not ripen in terms of both developmentally controlled ripening characters, such as starch degradation, and ethylene-modulated ripening traits. Transient assays suggest that, like the RIN gene in tomato, the MADS9 gene acts as a transcriptional activator of the ethylene biosynthesis enzyme, 1-aminocyclopropane-1-carboxylate (ACC) synthase 1. The existence of a single class of genes that regulate both flesh formation and ripening provides an evolutionary tool for controlling two critical aspects of fleshy fruit development.

Mis‐expression of a PISTILLATA‐like MADS box gene prevents fruit development in grapevine

The FLESHLESS BERRY (Flb) somatic variant identified in the grapevine cultivar Ugni Blanc develops grape berries without flesh, suggesting a role for the altered gene in differentiation of flesh cells. Here we describe identification of the molecular defect responsible for this phenotype. Using a combination of genetic and transcriptomic approaches, we detected the insertion of a miniature inverted-repeat transposable element in the promoter region of the PISTILLATA-like (VvPI) gene, the grapevine homologue of Arabidopsis PISTILLATA. The transposon insertion causes specific ectopic expression of the corresponding VvPI allele during early fruit development, causing expression of genes specific for petal and stamen development within the fruit. A causal relationship between the insertion and the phenotype was demonstrated by phenotypic and molecular analyses of somatic revertants showing that ectopic expression and mutant phenotype were always linked to the presence of the transposon insertion. The various phenotypic effects of the flb mutation on ovary morphology, fruit set and fruit development, depending on the cell lineage affected, are presented for each phenotype, offering new insights into floral and fleshly fruit development. The results highlight the importance of VvPI repression after fertilization to achieve normal fleshy fruit development, and the complex genetic, genomic and cellular interactions required for the flower to fruit transition in grapevine.

The grape berry-specific basic helix–loop–helix transcription factor VvCEB1 affects cell size

The development of fleshy fruits involves complex physiological and biochemical changes. After fertilization, fruit growth usually begins with cell division, continues with both cell division and expansion, allowing fruit set to occur, and ends with cell expansion only. In spite of the economical importance of grapevine, the molecular mechanisms controlling berry growth are not fully understood. The present work identified and characterized Vitis vinifera cell elongation bHLH protein (VvCEB1), a basic helix–loop–helix (bHLH) transcription factor controlling cell expansion in grape. VvCEB1 was expressed specifically in berry-expanding tissues with a maximum around veraison. The study of VvCEB1 promoter activity in tomato confirmed its specific fruit expression during the expansion phase. Overexpression of VvCEB1 in grape embryos showed that this protein stimulates cell expansion and affects the expression of genes involved in cell expansion, including genes of auxin metabolism and signalling. Taken together, these data show that VvCEB1 is a fruit-specific bHLH transcription factor involved in grape berry development.

Advances in tomato research in the post-genome era

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.

Alteration of tomato microRNAs expression during fruit development upon Cucumber mosaic virus and Tomato aspermy virus infection

The economic importance of Solanaceae plant species is well documented, and tomato has become a model for fleshy fruit development and ripening studies. Plant microRNAs (miRNAs) are small endogenous RNAs that are involved in a variety of activities including plant development, signal transduction and protein degradation, as well as response to environment stress and pathogen invasion. Here in this study, we aimed at quantifying the expression alterations of nine miRNAs and target mRNAs in tomato flower and fruit development upon Cucumber mosaic virus (CMV) and Tomato aspermy virus infections. Three different CMV strains CMV-Fny, CMV-FnyΔ2b and CMV-Fny-satT1 were used in our investigation, and the miRNA/mRNA expression alterations were analyzed by real-time quantitative RT-PCR. The results shown the levels of several miRNA/mRNA pairs were increased upon virus infections. However, the increased level of individual miRNA differed for different virus strains, reflecting differences in severity of symptom phenotypes. The altered expression patterns of these miRNA/mRNA pairs and their predicted functions indicate the possible roles in flower and fruit development, and provide experimental data for understanding the miRNA-mediated phenotype alterations in tomato fruit.