Benito Pineda

Tomato Fruit Photosynthesis Is Seemingly Unimportant in Primary Metabolism and Ripening But Plays a Considerable Role in Seed Development

Fruit of tomato (Solanum lycopersicum), like those from many species, have been characterized to undergo a shift from partially photosynthetic to truly heterotrophic metabolism. While there is plentiful evidence for functional photosynthesis in young tomato fruit, the rates of carbon assimilation rarely exceed those of carbon dioxide release, raising the question of its role in this tissue. Here, we describe the generation and characterization of lines exhibiting a fruit-specific reduction in the expression of glutamate 1-semialdehyde aminotransferase (GSA). Despite the fact that these plants contained less GSA protein and lowered chlorophyll levels and photosynthetic activity, they were characterized by few other differences. Indeed, they displayed almost no differences in fruit size, weight, or ripening capacity and furthermore displayed few alterations in other primary or intermediary metabolites. Although GSA antisense lines were characterized by significant alterations in the expression of genes associated with photosynthesis, as well as with cell wall and amino acid metabolism, these changes were not manifested at the phenotypic level. One striking feature of the antisense plants was their seed phenotype: the transformants displayed a reduced seed set and altered morphology and metabolism at early stages of fruit development, although these differences did not affect the final seed number or fecundity. Taken together, these results suggest that fruit photosynthesis is, at least under ambient conditions, not necessary for fruit energy metabolism or development but is essential for properly timed seed development and therefore may confer an advantage under conditions of stress.

Overexpression of dehydrin tas14 gene improves the osmotic stress imposed by drought and salinity in tomato

One strategy to increase the level of drought and salinity tolerance is the transfer of genes codifying different types of proteins functionally related to macromolecules protection, such as group 2 of late embryogenesis abundant (LEA) proteins or dehydrins. The TAS14 dehydrin was isolated and characterized in tomato and its expression was induced by osmotic stress (NaCl and mannitol) and abscisic acid (ABA) [Godoy et al., Plant Mol Biol 1994;26:1921-1934], yet its function in drought and salinity tolerance of tomato remains elusive. In this study, transgenic tomato plants overexpressing tas14 gene under the control of the 35SCaMV promoter were generated to assess the function of tas14 gene in drought and salinity tolerance. The plants overexpressing tas14 gene achieved improved long-term drought and salinity tolerance without affecting plant growth under non-stress conditions. A mechanism of osmotic stress tolerance via osmotic potential reduction and solutes accumulation, such as sugars and K(+) is operating in tas14 overexpressing plants in drought conditions. A similar mechanism of osmotic stress tolerance was observed under salinity. Moreover, the overexpression of tas14 gene increased Na(+) accumulation only in adult leaves, whereas in young leaves, the accumulated solutes were K(+) and sugars, suggesting that plants overexpressing tas14 gene are able to distribute the Na(+) accumulation between young and adult leaves over a prolonged period in stressful conditions. Measurement of ABA showed that the action mechanism of tas14 gene is associated with an earlier and greater accumulation of ABA in leaves during short-term periods. A good feature for the application of this gene in improving drought and salt stress tolerance is the fact that its constitutive expression does not affect plant growth under non-stress conditions, and tolerance induced by overexpression of tas14 gene was observed at the different stress degrees applied to the long term.

Functional Analysis of the Arlequin Mutant Corroborates the Essential Role of the ARLEQUIN/TAGL1 Gene during Reproductive Development of Tomato

Reproductive development of higher plants comprises successive events of organ differentiation and growth which finally lead to the formation of a mature fruit. However, most of the genetic and molecular mechanisms which coordinate such developmental events are yet to be identified and characterized. Arlequin (Alq), a semi-dominant T-DNA tomato mutant showed developmental changes affecting flower and fruit ripening. Sepals were converted into fleshy organs which ripened as normal fruit organs and fruits displayed altered ripening features. Molecular characterization of the tagged gene demonstrated that it corresponded to the previously reported TOMATO AGAMOUS-LIKE 1 (TAGL1) gene, the tomato ortholog of SHATTERPROOF MADS-box genes of Arabidopsis thaliana, and that the Alq mutation promoted a gain-of-function phenotype caused by the ectopic expression of TAGL1. Ectopic overexpression of TAGL1 resulted in homeotic alterations affecting floral organ identity that were similar to but stronger than those observed in Alq mutant plants. Interestingly, TAGL1 RNAi plants yielded tomato fruits which were unable to ripen. They displayed a yellow-orange color and stiffness appearance which are in accordance with reduced lycopene and ethylene levels, respectively. Moreover, pericarp cells of TAGL1 RNAi fruits showed altered cellular and structural properties which correlated to both decreased expression of genes regulating cell division and lignin biosynthesis. Over-expression of TAGL1 is able to rescue the non-ripening phenotype of rin and nor mutants, which is mediated by the transcriptional activation of several ripening genes. Our results demonstrated that TAGL1 participates in the genetic control of flower and fruit development of tomato plants. Furthermore, gene silencing and over-expression experiments demonstrated that the fruit ripening process requires the regulatory activity of TAGL1. Therefore, TAGL1 could act as a linking factor connecting successive stages of reproductive development, from flower development to fruit maturation, allowing this complex process to be carried out successfully.

A multisite gateway‐based toolkit for targeted gene expression and hairpin RNA silencing in tomato fruits

A collection of fruit promoters, reporter genes and protein tags has been constructed in a triple-gateway format, a recombination-based cloning system that facilitates the tandem assembly of three DNA fragments into plant expression vectors. The new pENFRUIT collection includes, among others, the classical tomato-ripening promoters E8 and 2A11 and a set of six new tomato promoters. The new promoter activities were characterized in both transient assays and stable transgenic plants. The range of expression of the new promoters comprises strong (PNH, PLI), medium (PLE, PFF, PHD) and weak (PSN) promoters driving gene expression preferentially in the fruit, and covering a wide range of tissues and developmental stages. Together, a total of 78 possible combinations for the expression of a gene of interest in the fruit, plus a set of five reporters for new promoter analysis, was made available in the current collection. Moreover, the pENFRUIT promoter collection is adaptable to hairpin RNA strategies aimed at tissue/organ-specific gene silencing with only an additional cloning step. The pENFRUIT toolkit broadens the spectrum of promoter activities available for fruit biotechnology and fundamental research, and bypasses technical difficulties of current ligase-dependent cloning techniques in the construction of fruit expression cassettes. The pENFRUIT vector collection is available for the research community in a plasmid repository, facilitating its accessibility.

Neutralizing antibodies against rotavirus produced in transgenically labelled purple tomatoes

Edible fruits are inexpensive biofactories for human health-promoting molecules that can be ingested as crude extracts or partially purified formulations. We show here the production of a model human antibody for passive protection against the enteric pathogen rotavirus in transgenically labelled tomato fruits. Transgenic tomato plants expressing a recombinant human immunoglobulin A (hIgA_2A1) selected against the VP8* peptide of rotavirus SA11 strain were obtained. The amount of hIgA_2A1 protein reached 3.6 ± 0.8% of the total soluble protein in the fruit of the transformed plants. Minimally processed fruit-derived products suitable for oral intake showed anti-VP8* binding activity and strongly inhibited virus infection in an in vitro virus neutralization assay. In order to make tomatoes expressing hIgA_2A1 easily distinguishable from wild-type tomatoes, lines expressing hIgA_2A1 transgenes were sexually crossed with a transgenic tomato line expressing the genes encoding Antirrhinum majus Rosea1 and Delila transcription factors, which confer purple colour to the fruit. Consequently, transgenically labelled purple tomato fruits expressing hIgA_2A1 have been developed. The resulting purple-coloured extracts from these fruits contain high levels of recombinant anti-rotavirus neutralizing human IgA in combination with increased amounts of health-promoting anthocyanins.

Transcriptional Activity of the MADS Box ARLEQUIN/TOMATO AGAMOUS-LIKE1 Gene Is Required for Cuticle Development of Tomato Fruit

A ripening-related transcription factor regulates the cuticle development of tomato fruit as part of the reproductive developmental program. Fruit development and ripening entail key biological and agronomic events, which ensure the appropriate formation and dispersal of seeds and determine productivity and yield quality traits. The MADS box gene ARLEQUIN/TOMATO AGAMOUS-LIKE1 (hereafter referred to as TAGL1) was reported as a key regulator of tomato (Solanum lycopersicum) reproductive development, mainly involved in flower development, early fruit development, and ripening. It is shown here that silencing of the TAGL1 gene (RNA interference lines) promotes significant changes affecting cuticle development, mainly a reduction of thickness and stiffness, as well as a significant decrease in the content of cuticle components (cutin, waxes, polysaccharides, and phenolic compounds). Accordingly, overexpression of TAGL1 significantly increased the amount of cuticle and most of its components while rendering a mechanically weak cuticle. Expression of the genes involved in cuticle biosynthesis agreed with the biochemical and biomechanical features of cuticles isolated from transgenic fruits; it also indicated that TAGL1 participates in the transcriptional control of cuticle development mediating the biosynthesis of cuticle components. Furthermore, cell morphology and the arrangement of epidermal cell layers, on whose activity cuticle formation depends, were altered when TAGL1 was either silenced or constitutively expressed, indicating that this transcription factor regulates cuticle development, probably through the biosynthetic activity of epidermal cells. Our results also support cuticle development as an integrated event in the fruit expansion and ripening processes that characterize fleshy-fruited species such as tomato.

The HAL1 function on Na+ homeostasis is maintained over time in salt-treated transgenic tomato plants, but the high reduction of Na+ in leaf is not associated with salt tolerance

To achieve a deeper knowledge on the function of HAL1 gene in tomato (Solanum lycopersicum) plants submitted to salt stress, in this study, we studied the growth and physiological responses to high salt stress of T3 transgenic plants (an azygous line without transgene and both homozygous and hemizygous lines for HAL1) proceeding from a primary transformant with a very high expression level of HAL1 gene. The homozygous plants for HAL1 gene did not increase their salt tolerance in spite of an earlier and higher reduction of the Na(+) accumulation in leaves, being moreover the Na(+) homeostasis maintained throughout the growth cycle. The greater ability of the homozygous line to regulate the Na(+) transport to the shoot to long term was even shown in low accumulation of Na(+) in fruits. By comparing the homozygous and hemizygous lines, a higher salt tolerance in the hemizygous line, with respect to the homozygous line, was observed on the basis of fruit yield. The Na(+) homeostasis and osmotic homeostasis were also different in homozygous and hemizygous lines. Indeed, the Na(+) accumulation rate in leaves was greater in hemizygous than in homozygous line after 35 days of 100 mM NaCl treatment and only at the end of growth cycle did the hemizygous line show leaf Na(+) levels similar to those found in the homozygous line. With respect to the osmotic homeostasis, the main difference between lines was the different contribution of inorganic and organic solutes to the leaf osmotic balance. Taken together, these results suggest that the greater Na(+) exclusion ability of the homozygous line overexpressing HAL1 induces a greater use of organic solutes for osmotic balance, which seems to have an energy cost and hence a growth penalty that reverts negatively on fruit yield.

Genetic and Physiological Characterization of the Arlequin Insertional Mutant Reveals a Key Regulator of Reproductive Development in Tomato

The genetic and phenotypic characterization of a new tomato (Solanum lycopersicum) insertional mutant, Arlequin (Alq) is reported. Alq mutant plants were affected in reproductive development and their sepals were homeotically converted into fleshy fruit-like organs. Molecular analysis demonstrated that a single copy of T-DNA was present in the mutant genome while genetic analysis confirmed that the mutant phenotype co-segregated with the T-DNA insertion and was inherited as a monogenic semi-dominant trait. The histological and scanning electron microscope analyses revealed cell identity changes in both external and internal tissues of Alq sepals. Flowers developed by Alq homozygous plants showed a severe mutant phenotype, since after fruit set, not only did the sepals become succulent but they also followed a ripening pattern similar to that of normal fruits. From a metabolic viewpoint, Alq sepals also behaved like a fruit, as they acquired the properties of a sink that acted alternatively and independently to the fruit. In fact, expression of regulatory genes controlling tomato fruit ripening was detected in Alq sepals at similar levels to those observed in mature fruits. Furthermore, the Alq mutation inhibited the development of the abscission zone in tomato flowers indicating that the JOINTLESS gene is regulated by ALQ. Results from the genetic and developmental characterization of the Alq tomato mutant suggest that the ALQ gene participates in the regulatory pathway controlling fruit ripening of tomato.

Production of engineered long-life and male sterile Pelargonium plants

BackgroundPelargonium is one of the most popular garden plants in the world. Moreover, it has a considerable economic importance in the ornamental plant market. Conventional cross-breeding strategies have generated a range of cultivars with excellent traits. However, gene transfer via Agrobacterium tumefaciens could be a helpful tool to further improve Pelargonium by enabling the introduction of new genes/traits. We report a simple and reliable protocol for the genetic transformation of Pelargonium spp. and the production of engineered long-life and male sterile Pelargonium zonale plants, using the pSAG12::ipt and PsEND1::barnase chimaeric genes respectively.ResultsThe pSAG12::ipt transgenic plants showed delayed leaf senescence, increased branching and reduced internodal length, as compared to control plants. Leaves and flowers of the pSAG12::ipt plants were reduced in size and displayed a more intense coloration. In the transgenic lines carrying the PsEND1::barnase construct no pollen grains were observed in the modified anther structures, which developed instead of normal anthers. The locules of sterile anthers collapsed 3–4 days prior to floral anthesis and, in most cases, the undeveloped anther tissues underwent necrosis.ConclusionThe chimaeric construct pSAG12::ipt can be useful in Pelargonium spp. to delay the senescence process and to modify plant architecture. In addition, the use of engineered male sterile plants would be especially useful to produce environmentally friendly transgenic plants carrying new traits by preventing gene flow between the genetically modified ornamentals and related plant species. These characteristics could be of interest, from a commercial point of view, both for pelargonium producers and consumers.

Characterization of vegetative inflorescence ( mc-vin ) mutant provides new insight into the role of MACROCALYX in regulating inflorescence development of tomato

Inflorescence development is a key factor of plant productivity, as it determines flower number. Therefore, understanding the mechanisms that regulate inflorescence architecture is critical for reproductive success and crop yield. In this study, a new mutant, vegetative inflorescence (mc-vin), was isolated from the screening of a tomato (Solanum lycopersicum L.) T-DNA mutant collection. The mc-vin mutant developed inflorescences that reverted to vegetative growth after forming two to three flowers, indicating that the mutated gene is essential for the maintenance of inflorescence meristem identity. The T-DNA was inserted into the promoter region of the MACROCALYX (MC) gene; this result together with complementation test and expression analyses proved that mc-vin is a new knock-out allele of MC. Double combinations between mc-vin and jointless (j) and single flower truss (sft) inflorescence mutants showed that MC has pleiotropic effects on the reproductive phase, and that it interacts with SFT and J to control floral transition and inflorescence fate in tomato. In addition, MC expression was mis-regulated in j and sft mutants whereas J and SFT were significantly up-regulated in the mc-vin mutant. Together, these results provide new evidences about MC function as part of the genetic network regulating the development of tomato inflorescence meristem.