Involvement of the tomato BBX16 and BBX17 microProteins in reproductive development.

BBXs are B-Box zinc finger proteins that can act as transcription factors and regulators of protein complexes. Several BBX proteins play important roles in plant development. Two Arabidopsis thaliana microProteins belonging to the BBX family, named miP1a and miP1b, homotypically interact with and modulate the activity of other BBX proteins, including CONSTANS, which transcriptionally activates the florigen, FLOWERING LOCUS T. Arabidopsis plants overexpressing miP1a and miP1b showed delayed flowering. In tomato, the closest homologs of miP1a and miP1b are the microProteins SlBBX16 and SlBBX17. This study was aimed at investigating whether the constitutive expression of SlBBX16/17 in Arabidopsis and tomato impacted reproductive development. The heterologous expression of the two tomato microProteins in Arabidopsis caused a delay in the flowering transition; however, the effect was weaker than that observed when the native miP1a/b were overexpressed. In tomato, overexpression of SlBBX17 prolonged the flowering period; this effect was accompanied by downregulation of the flowering inhibitors Self Pruning (SP) and SP5G. SlBBX16 and SlBBX17 can hetero-oligomerize with TCMP-2, a cystine-knot peptide involved in flowering pattern regulation and early fruit development in tomato. The increased expression of both microProteins also caused alterations in tomato fruit development: we observed in the case of SlBBX17 a decrease in the number and size of ripe fruits as compared to WT plants, while for SlBBX16, a delay in fruit production up to the breaker stage. These effects were associated with changes in the expression of GA-responsive genes.

Approaching the genetic dissection of indirect adventitious organogenesis process in tomato explants.

The screening of 862 T-DNA lines was carried out to approach the genetic dissection of indirect adventitious organogenesis in tomato. Several mutants defective in different phases of adventitious organogenesis, namely callus growth (tdc-1), bud differentiation (tdb-1, -2, -3) and shoot-bud development (tds-1) were identified and characterized. The alteration of the TDC-1 gene blocked callus proliferation depending on the composition of growth regulators in the culture medium. Calli from tds-1 explants differentiated buds but did not develop normal shoots. Histological analysis showed that their abnormal development is due to failure in the organization of normal adventitious shoot meristems. Interestingly, tdc-1 and tds-1 mutant plants were indistinguishable from WT ones, indicating that the respective altered genes play specific roles in cell proliferation from explant cut zones (TDC-1 gene) or in the organization of adventitious shoot meristems (TDS-1 gene). Unlike the previous, plants of the three mutants defective in the differentiation of adventitious shoot-buds (tdb-1, -2, -3) showed multiple changes in vegetative and reproductive traits. Cosegregation analyses revealed the existence of an association between the phenotype of the tdb-3 mutant and a T-DNA insert, which led to the discovery that the SlMAPKKK17 gene is involved in the shoot-bud differentiation process.

The Tomato SlVIPP1 Gene Is Required for Plant Survival Through the Proper Development of Chloroplast Thylakoid Membrane.

Since membranes play essential roles in all living beings, all cells have developed mechanisms for efficient and fast repair of membrane damage. In Escherichia coli, the Phage shock stress A (PspA) protein is involved in the maintenance of the integrity of its inner membrane in response to the damage produced by exposure to stress conditions. A role in thylakoid membrane maintenance and reorganization has been proposed for Vesicle Inducing Protein in Plastid 1 (VIPP1), the putative PspA ortholog in Arabidopsis thaliana. While some membranes of plant cells have been extensively studied, the biosynthesis and maintenance of chloroplast thylakoid membrane remains poorly known. Here, we report the cloning and functional characterization of the tomato (Solanum lycopersicum L.) ortholog of Escherichia coli PspA and Arabidopsis thaliana VIPP1, which we dubbed SlVIPP1. Our genetic and molecular characterization of slvipp1, an insertional mutant, allowed us to conclude that the tomato SlVIPP1 gene is needed for development, as Arabidopsis VIPP1, but not Escherichia coli PspA. Homozygous slvipp1 tomato plants are albino and exhibit early lethality and highly aberrant chloroplast development with almost complete absence of thylakoids. The phenotype of tomato RNAi lines and that of additional slvipp1 alleles generated by CRISPR/Cas9 gene editing technology confirmed that the morphological and histological aberrations shown by slvipp1 homozygotes are caused by VIPP1 lack of function. We also found that tomato SlVIPP1 overexpression does not cause any visible effect on plant morphology and viability. Our work with slvipp1 plants evidences that SlVIPP1 is an essential gene required for tomato survival, since its function is crucial for the proper formation and/or maintenance of thylakoid membranes.

Alq mutation increases fruit set rate and allows the maintenance of fruit yield under moderate saline conditions.

Arlequin (Alq) is a gain-of-function mutant whose most relevant feature is that sepals are able to become fruit-like organs due to the ectopic expression of the ALQ-TAGL1 gene. The role of this gene in tomato fruit ripening was previously demonstrated. To discover new functional roles for ALQ-TAGL1, and most particularly its involvement in the fruit set process, a detailed characterization of Alq yield-related traits was performed. Under standard conditions, the Alq mutant showed a much higher fruit set rate than the wild type. A significant percentage of Alq fruits were seedless. The results showed that pollination-independent fruit set in Alq is due to early transition from flower to fruit. Analysis of endogenous hormones in Alq suggests that increased content of cytokinins and decreased level of abscisic acid may account for precocious fruit set. Comparative expression analysis showed relevant changes of several genes involved in cell division, gibberellin metabolism, and the auxin signalling pathway. Since pollination-independent fruit set may be a very useful strategy for maintaining fruit production under adverse conditions, fruit set and yield in Alq plants under moderate salinity were assessed. Interestingly, Alq mutant plants showed a high yield under saline conditions, similar to that of Alq and the wild type under unstressed conditions.

Phenotypic and genetic characterization of tomato mutants provides new insights into leaf development and its relationship to agronomic traits.

BACKGROUND: Tomato mutants altered in leaf morphology are usually identified in the greenhouse, which demands considerable time and space and can only be performed in adequate periods. For a faster but equally reliable scrutiny method we addressed the screening in vitro of 971 T-DNA lines. Leaf development was evaluated in vitro in seedlings and shoot-derived axenic plants. New mutants were characterized in the greenhouse to establish the relationship between in vitro and in vivo leaf morphology, and to shed light on possible links between leaf development and agronomic traits, a promising field in which much remains to be discovered. RESULTS: Following the screening in vitro of tomato T-DNA lines, putative mutants altered in leaf morphology were evaluated in the greenhouse. The comparison of results in both conditions indicated a general phenotypic correspondence, showing that in vitro culture is a reliable system for finding mutants altered in leaf development. Apart from providing homogeneous conditions, the main advantage of screening in vitro lies in the enormous time and space saving. Studies on the association between phenotype and nptII gene expression showed co-segregation in two lines (P > 99%). The use of an enhancer trap also allowed identifying gain-of-function mutants through reporter expression analysis. These studies suggested that genes altered in three other mutants were T-DNA tagged. New mutants putatively altered in brassinosteroid synthesis or perception, mutations determining multiple pleiotropic effects, lines affected in organ curvature, and the first tomato mutant with helical growth were discovered. Results also revealed new possible links between leaf development and agronomic traits, such as axillary branching, flower abscission, fruit development and fruit cracking. Furthermore, we found that the gene tagged in mutant 2635-MM encodes a Sterol 3-beta-glucosyltransferase. Expression analysis suggested that abnormal leaf development might be due to the lack-off-function of this gene. CONCLUSION: In vitro culture is a quick, efficient and reliable tool for identifying tomato mutants altered in leaf morphology. The characterization of new mutants in vivo revealed new links between leaf development and some agronomic traits. Moreover, the possible implication of a gene encoding a Sterol 3-beta-glucosyltransferase in tomato leaf development is reported.

The SlCBL10 Calcineurin B-Like Protein Ensures Plant Growth under Salt Stress by Regulating Na+ and Ca2+ Homeostasis.

Characterization of a new tomato (Solanum lycopersicum) T-DNA mutant allowed for the isolation of the CALCINEURIN B-LIKE PROTEIN 10 (SlCBL10) gene whose lack of function was responsible for the severe alterations observed in the shoot apex and reproductive organs under salinity conditions. Physiological studies proved that SlCBL10 gene is required to maintain a proper low Na+/Ca2+ ratio in growing tissues allowing tomato growth under salt stress. Expression analysis of the main responsible genes for Na+ compartmentalization (i.e. Na+/H+ EXCHANGERs, SALT OVERLY SENSITIVE, HIGH-AFFINITY K+ TRANSPORTER 1;2, H+-pyrophosphatase AVP1 [SlAVP1] and V-ATPase [SlVHA-A1]) supported a reduced capacity to accumulate Na+ in Slcbl10 mutant leaves, which resulted in a lower uploading of Na+ from xylem, allowing the toxic ion to reach apex and flowers. Likewise, the tomato CATION EXCHANGER 1 and TWO-PORE CHANNEL 1 (SlTPC1), key genes for Ca2+ fluxes to the vacuole, showed abnormal expression in Slcbl10 plants indicating an impaired Ca2+ release from vacuole. Additionally, complementation assay revealed that SlCBL10 is a true ortholog of the Arabidopsis (Arabidopsis thaliana) CBL10 gene, supporting that the essential function of CBL10 is conserved in Arabidopsis and tomato. Together, the findings obtained in this study provide new insights into the function of SlCBL10 in salt stress tolerance. Thus, it is proposed that SlCBL10 mediates salt tolerance by regulating Na+ and Ca2+ fluxes in the vacuole, cooperating with the vacuolar cation channel SlTPC1 and the two vacuolar H+-pumps, SlAVP1 and SlVHA-A1, which in turn are revealed as potential targets of SlCBL10.

A collection of enhancer trap insertional mutants for functional genomics in tomato.

With the completion of genome sequencing projects, the next challenge is to close the gap between gene annotation and gene functional assignment. Genomic tools to identify gene functions are based on the analysis of phenotypic variations between a wild type and its mutant; hence, mutant collections are a valuable resource. In this sense, T-DNA collections allow for an easy and straightforward identification of the tagged gene, serving as the basis of both forward and reverse genetic strategies. This study reports on the phenotypic and molecular characterization of an enhancer trap T-DNA collection in tomato (Solanum lycopersicum L.), which has been produced by Agrobacterium-mediated transformation using a binary vector bearing a minimal promoter fused to the uidA reporter gene. Two genes have been isolated from different T-DNA mutants, one of these genes codes for a UTP-glucose-1-phosphate uridylyltransferase involved in programmed cell death and leaf development, which means a novel gene function reported in tomato. Together, our results support that enhancer trapping is a powerful tool to identify novel genes and regulatory elements in tomato and that this T-DNA mutant collection represents a highly valuable resource for functional analyses in this fleshy-fruited model species.

The sodium transporter encoded by the HKT1;2 gene modulates sodium/potassium homeostasis in tomato shoots under salinity.

Excessive soil salinity diminishes crop yield and quality. In a previous study in tomato, we identified two closely linked genes encoding HKT1-like transporters, HKT1;1 and HKT1;2, as candidate genes for a major quantitative trait locus (kc7.1) related to shoot Na+ /K+ homeostasis - a major salt tolerance trait - using two populations of recombinant inbred lines (RILs). Here, we determine the effectiveness of these genes in conferring improved salt tolerance by using two near-isogenic lines (NILs) that were homozygous for either the Solanum lycopersicum allele (NIL17) or for the Solanum cheesmaniae allele (NIL14) at both HKT1 loci; transgenic lines derived from these NILs in which each HKT1;1 and HKT1;2 had been silenced by stable transformation were also used. Silencing of ScHKT1;2 and SlHKT1;2 altered the leaf Na+ /K+ ratio and caused hypersensitivity to salinity in plants cultivated under transpiring conditions, whereas silencing SlHKT1;1/ScHKT1;1 had a lesser effect. These results indicate that HKT1;2 has the more significant role in Na+ homeostasis and salinity tolerance in tomato.

Heterologous expression of the yeast HAL5 gene in tomato enhances salt tolerance by reducing shoot Na+ accumulation in the long term.

For salt tolerance to be achieved in the long-term plants must regulate Na(+)/K(+) homeostasis over time. In this study, we show that the salt tolerance induced by overexpression of the yeast HAL5 gene in tomato (Solanum lycopersicum) was related to a lower leaf Na(+) accumulation in the long term, by reducing Na(+) transport from root to shoot over time regardless of the severity of salt stress. Furthermore, maintaining Na(+)/K(+) homeostasis over time was associated with changes in the transcript levels of the Na(+) and K(+) transporters such as SlHKT1;2 and SlHAK5. The expression of SlHKT1;2 was upregulated in response to salinity in roots of transgenic plants but downregulated in the roots of wild-type (WT) plants, which seems to be related to the lower Na(+) transport rate from root to shoot in transgenic plants. The expression of the SlHAK5 increased in roots and leaves of both WT and transgenic plants under salinity. However, this increase was much higher in the leaves of transgenic plants than in those of WT plants, which may be associated with the ability of transgenic leaves to maintain Na(+)/K(+) homeostasis over time. Taken together, the results show that the salt tolerance mechanism induced by HAL5 overexpression in tomato is related to the appropriate regulation of ion transport from root to shoot and maintenance of the leaf Na(+)/K(+) homeostasis through modulation of SlHKT1 and SlHAK5 over time.

Production of engineered long-life and male sterile Pelargonium plants.

BACKGROUND: Pelargonium 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. RESULTS: The 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. CONCLUSION: The 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 the procera tomato mutant shows novel functions of the SlDELLA protein in the control of flower morphology, cell division and expansion, and the auxin-signaling pathway during fruit-set and development.

procera (pro) is a tall tomato (Solanum lycopersicum) mutant carrying a point mutation in the GRAS region of the gene encoding SlDELLA, a repressor in the gibberellin (GA) signaling pathway. Consistent with the SlDELLA loss of function, pro plants display a GA-constitutive response phenotype, mimicking wild-type plants treated with GA3. The ovaries from both nonemasculated and emasculated pro flowers had very strong parthenocarpic capacity, associated with enhanced growth of preanthesis ovaries due to more and larger cells. pro parthenocarpy is facultative because seeded fruits were obtained by manual pollination. Most pro pistils had exserted stigmas, thus preventing self-pollination, similar to wild-type pistils treated with GA3 or auxins. However, Style2.1, a gene responsible for long styles in noncultivated tomato, may not control the enhanced style elongation of pro pistils, because its expression was not higher in pro styles and did not increase upon GA3 application. Interestingly, a high percentage of pro flowers had meristic alterations, with one additional petal, sepal, stamen, and carpel at each of the four whorls, respectively, thus unveiling a role of SlDELLA in flower organ development. Microarray analysis showed significant changes in the transcriptome of preanthesis pro ovaries compared with the wild type, indicating that the molecular mechanism underlying the parthenocarpic capacity of pro is complex and that it is mainly associated with changes in the expression of genes involved in GA and auxin pathways. Interestingly, it was found that GA activity modulates the expression of cell division and expansion genes and an auxin signaling gene (tomato AUXIN RESPONSE FACTOR7) during fruit-set.

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.

An insertional mutagenesis programme with an enhancer trap for the identification and tagging of genes involved in abiotic stress tolerance in the tomato wild-related species Solanum pennellii.

Salinity and drought have a huge impact on agriculture since there are few areas free of these abiotic stresses and the problem continues to increase. In tomato, the most important horticultural crop worldwide, there are accessions of wild-related species with a high degree of tolerance to salinity and drought. Thus, the finding of insertional mutants with other tolerance levels could lead to the identification and tagging of key genes responsible for abiotic stress tolerance. To this end, we are performing an insertional mutagenesis programme with an enhancer trap in the tomato wild-related species Solanum pennellii. First, we developed an efficient transformation method which has allowed us to generate more than 2,000 T-DNA lines. Next, the collection of S. pennelli T(0) lines has been screened in saline or drought conditions and several presumptive mutants have been selected for their salt and drought sensitivity. Moreover, T-DNA lines with expression of the reporter uidA gene in specific organs, such as vascular bundles, trichomes and stomata, which may play key roles in processes related to abiotic stress tolerance, have been identified. Finally, the growth of T-DNA lines in control conditions allowed us the identification of different development mutants. Taking into account that progenies from the lines are being obtained and that the collection of T-DNA lines is going to enlarge progressively due to the high transformation efficiency achieved, there are great possibilities for identifying key genes involved in different tolerance mechanisms to salinity and drought.

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.

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.

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.