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1.
Development ; 150(21)2023 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-37823342

RESUMO

Many developmental processes associated with fruit development occur at the floral meristem (FM). Age-regulated microRNA156 (miR156) and gibberellins (GAs) interact to control flowering time, but their interplay in subsequent stages of reproductive development is poorly understood. Here, in tomato (Solanum lycopersicum), we show that GA and miR156-targeted SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL or SBP) genes interact in the tomato FM and ovary patterning. High GA responses or overexpression of miR156 (156OE), which leads to low expression levels of miR156-silenced SBP genes, resulted in enlarged FMs, ovary indeterminacy and fruits with increased locule number. Conversely, low GA responses reduced indeterminacy and locule number, and overexpression of a S. lycopersicum (Sl)SBP15 allele that is miR156 resistant (rSBP15) reduced FM size and locule number. GA responses were partially required for the defects observed in 156OE and rSBP15 fruits. Transcriptome analysis and genetic interactions revealed shared and divergent functions of miR156-targeted SlSBP genes, PROCERA/DELLA and the classical WUSCHEL/CLAVATA pathway, which has been previously associated with meristem size and determinacy. Our findings reveal that the miR156/SlSBP/GA regulatory module is deployed differently depending on developmental stage and create novel opportunities to fine-tune aspects of fruit development that have been important for tomato domestication.


Assuntos
MicroRNAs , Solanum lycopersicum , Giberelinas/metabolismo , Solanum lycopersicum/genética , Flores , Meristema/metabolismo , Ovário/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Plantas/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo
2.
J Exp Bot ; 73(18): 6226-6240, 2022 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-35710302

RESUMO

Allelic variation in the CETS (CENTRORADIALIS, TERMINAL FLOWER 1, SELF PRUNING) gene family controls agronomically important traits in many crops. CETS genes encode phosphatidylethanolamine-binding proteins that have a central role in the timing of flowering as florigenic and anti-florigenic signals. The great expansion of CETS genes in many species suggests that the functions of this family go beyond flowering induction and repression. Here, we characterized the tomato SELF PRUNING 3C (SP3C) gene, and show that besides acting as a flowering repressor it also regulates seed germination and modulates root architecture. We show that loss of SP3C function in CRISPR/Cas9-generated mutant lines increases root length and reduces root side branching relative to the wild type. Higher SP3C expression in transgenic lines promotes the opposite effects in roots, represses seed germination, and also improves tolerance to water stress in seedlings. These discoveries provide new insights into the role of SP paralogs in agronomically relevant traits, and support future exploration of the involvement of CETS genes in abiotic stress responses.


Assuntos
Secas , Germinação , Germinação/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fosfatidiletanolaminas , Sementes/genética , Sementes/metabolismo
3.
Plants (Basel) ; 11(10)2022 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-35631734

RESUMO

The leaves of the wild tomato Solanumgalapagense harbor type-IV glandular trichomes (GT) that produce high levels of acylsugars (AS), conferring insect resistance. Conversely, domesticated tomatoes (S. lycopersicum) lack type-IV trichomes on the leaves of mature plants, preventing high AS production, thus rendering the plants more vulnerable to insect predation. We hypothesized that cultivated tomatoes engineered to harbor type-IV trichomes on the leaves of adult plants could be insect-resistant. We introgressed the genetic determinants controlling type-IV trichome development from S.galapagense into cv. Micro-Tom (MT) and created a line named "Galapagos-enhanced trichomes" (MT-Get). Mapping-by-sequencing revealed that five chromosomal regions of S. galapagense were present in MT-Get. Further genetic mapping showed that S. galapagense alleles in chromosomes 1, 2, and 3 were sufficient for the presence of type-IV trichomes on adult organs but at lower densities. Metabolic and gene expression analyses demonstrated that type-IV trichome density was not accompanied by the AS production and exudation in MT-Get. Although the plants produce a significant amount of acylsugars, those are still not enough to make them resistant to whiteflies. We demonstrate that type-IV glandular trichome development is insufficient for high AS accumulation. The results from our study provided additional insights into the steps necessary for breeding an insect-resistant tomato.

4.
Plasmid ; 121: 102630, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35398459

RESUMO

Gateway system is one of the most known cloning systems, which makes it compatible with several expression vectors, including those used for Yeast Two-Hybrid (Y2H) and Bimolecular Fluorescence Complementation (BiFC) assays. However, this system is laborious and expensive due to its two-step cloning. In this research, we developed a new cloning strategy named Brick into the Gateway (BiG). This approach uses GoldenBraid/Gate assemblies to create a DNA fragment of interest flanked by attL sites, which can be directly recombined into Gateway destination vectors. BiG method showed a high recombination efficiency and ensured the correct reading frame, which was successfully tested in Y2H and BiFC assays. BiG has proven to be a rapid, low-cost, reusable, and directional cloning method which allows the merged use of systems.


Assuntos
Vetores Genéticos , Clonagem Molecular , Vetores Genéticos/genética , Plasmídeos/genética
5.
Bio Protoc ; 12(24)2022 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-36618087

RESUMO

Cloning systems like Gateway and Golden Gate/Braid are known because of their efficiency and accuracy. While the main drawback of Gateway is the expensive cost of the enzymes used in its two-step (LR and BP) reaction, Golden Gate requires non-reusable components due to their specific restriction sites. We present the Brick into the Gateway (BiG) protocol as a new cloning strategy, faster and more economic method that combines (i) reusable modules or bricks assembled by the GoldenBraid approach, and (ii) Gateway LR reactions [recombination of attachment sites: attL (L from left) and attR (R from right)] avoiding the BP reaction [recombination of attachment sites: attP (P from phage) and attB (B from bacteria)] usually necessary in the Gateway cloning. The starting point is to perform a PCR reaction to add type IIS restriction sites into DNA fragments generating specific fusion sites. Then, this PCR product is used to design GoldenBraid bricks, including the attL Gateway recombination sites. Using the Golden Gate method, these bricks are assembled to produce an attL1 -gene of interest- attL2 fragment, which is integrated into a compatible vector producing a Gateway entry vector. Finally, the fragment containing the target gene is recombined by LR reaction into the Gateway destination vector. This protocol was validated in: Plasmid (2022), DOI: 10.1016/j.plasmid.2022.102630 Graphical abstract.

6.
Mol Hortic ; 2(1): 12, 2022 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-37789497

RESUMO

Tomato production is influenced by shoot branching, which is controlled by different hormones. Here we produced tomato plants overexpressing the cytokinin-deactivating gene CYTOKININ OXYDASE 2 (CKX2). CKX2-overexpressing (CKX2-OE) plants showed an excessive growth of axillary shoots, the opposite phenotype expected for plants with reduced cytokinin content, as evidenced by LC-MS analysis and ARR5-GUS staining. The TCP transcription factor SlBRC1b was downregulated in the axillary buds of CKX2-OE and its excessive branching was dependent on a functional version of the GRAS-family gene LATERAL SUPPRESSOR (LS). Grafting experiments indicated that increased branching in CKX2-OE plants is unlikely to be mediated by root-derived signals. Crossing CKX2-OE plants with transgenic antisense plants for the strigolactone biosynthesis gene CAROTENOID CLEAVAGE DIOXYGENASE (CCD7-AS) produced an additive phenotype, indicating independent effects of cytokinin and strigolactones on increased branching. On the other hand, CKX2-OE plants showed reduced polar auxin transport and their bud outgrowth was reduced when combined with auxin mutants. Accordingly, CKX2-OE basal buds did not respond to auxin applied in the decapitated apex. Our results suggest that tomato shoot branching depends on a fine-tuning of different hormonal balances and that perturbations in the auxin status could compensate for the reduced cytokinin levels in CKX2-OE plants.

7.
Plant Cell Environ ; 43(1): 76-86, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31691316

RESUMO

A major issue in modern agriculture is water loss through stomata during photosynthetic carbon assimilation. In water-limited ecosystems, annual plants have strategies to synchronize their growth and reproduction to the availability of water. Some species or ecotypes of flowers are early to ensure that their life cycles are completed before the onset of late season terminal drought ("drought escape"). This accelerated flowering correlates with low water-use efficiency (WUE). The molecular players and physiological mechanisms involved in this coordination are not fully understood. We analyzed WUE using gravimetry, gas exchange, and carbon isotope discrimination in florigen deficient (sft mutant), wild-type (Micro-Tom), and florigen over-expressing (SFT-ox) tomato lines. Increased florigen expression led to accelerated flowering time and reduced WUE. The low WUE of SFT-ox was driven by higher stomatal conductance and thinner leaf blades. This florigen-driven effect on WUE appears be independent of abscisic acid (ABA). Our results open a new avenue to increase WUE in crops in an ABA-independent manner. Manipulation of florigen levels could allow us to produce crops with a life cycle synchronized to water availability.


Assuntos
Florígeno/metabolismo , Solanum lycopersicum/metabolismo , Água/fisiologia , Ácido Abscísico/metabolismo , Isótopos de Carbono/metabolismo , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , Secas , Ecótipo , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Solanum lycopersicum/genética , Fotossíntese , Desenvolvimento Vegetal , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estômatos de Plantas/genética , Estômatos de Plantas/metabolismo
8.
New Phytol ; 221(3): 1328-1344, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30238569

RESUMO

Age-regulated microRNA156 (miR156) and targets similarly control the competence to flower in diverse species. By contrast, the diterpene hormone gibberellin (GA) and the microRNA319-regulated TEOSINTE BRANCHED/CYCLOIDEA/PCF (TCP) transcription factors promote flowering in the facultative long-day Arabidopsis thaliana, but suppress it in the day-neutral tomato (Solanum lycopersicum). We combined genetic and molecular studies and described a new interplay between GA and two unrelated miRNA-associated pathways that modulates tomato transition to flowering. Tomato PROCERA/DELLA activity is required to promote flowering along with the miR156-targeted SQUAMOSA PROMOTER BINDING-LIKE (SPL/SBP) transcription factors by activating SINGLE FLOWER TRUSS (SFT) in the leaves and the MADS-Box gene APETALA1(AP1)/MC at the shoot apex. Conversely, miR319-targeted LANCEOLATE represses floral transition by increasing GA concentrations and inactivating SFT in the leaves and AP1/MC at the shoot apex. Importantly, the combination of high GA concentrations/responses with the loss of SPL/SPB function impaired canonical meristem maturation and flower initiation in tomato. Our results reveal a cooperative regulation of tomato floral induction and flower development, integrating age cues (miR156 module) with GA responses and miR319-controlled pathways. Importantly, this study contributes to elucidate the mechanisms underlying the effects of GA in controlling flowering time in a day-neutral species.


Assuntos
Flores/crescimento & desenvolvimento , Giberelinas/metabolismo , MicroRNAs/metabolismo , Solanum lycopersicum/crescimento & desenvolvimento , Solanum lycopersicum/genética , Inflorescência/crescimento & desenvolvimento , Meristema/crescimento & desenvolvimento , MicroRNAs/genética , Modelos Biológicos , Mutação/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
9.
Plant Physiol ; 176(4): 2904-2916, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29500181

RESUMO

The SELF PRUNING (SP) gene is a key regulator of growth habit in tomato (Solanum lycopersicum). It is an ortholog of TERMINAL FLOWER1, a phosphatidylethanolamine-binding protein with antiflorigenic activity in Arabidopsis (Arabidopsis thaliana). A spontaneous loss-of-function mutation (sp) has been bred into several industrial tomato cultivars, as it produces a suite of pleiotropic effects that are favorable for mechanical harvesting, including determinate growth habit, short plant stature, and simultaneous fruit ripening. However, the physiological basis for these phenotypic differences has not been thoroughly explained. Here, we show that the sp mutation alters polar auxin transport as well as auxin responses, such as gravitropic curvature and elongation of excised hypocotyl segments. We also demonstrate that free auxin levels and auxin-regulated gene expression patterns are altered in sp mutants. Furthermore, diageotropica, a mutation in a gene encoding a cyclophilin A protein, appears to confer epistatic effects with sp Our results indicate that SP affects the tomato growth habit at least in part by influencing auxin transport and responsiveness. These findings suggest potential novel targets that could be manipulated for controlling plant growth habit and improving productivity.


Assuntos
Ciclofilina A/metabolismo , Frutas/metabolismo , Ácidos Indolacéticos/metabolismo , Proteínas de Plantas/metabolismo , Solanum lycopersicum/metabolismo , Transporte Biológico , Ciclofilina A/genética , Frutas/genética , Frutas/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Ácidos Indolacéticos/farmacologia , Solanum lycopersicum/genética , Solanum lycopersicum/crescimento & desenvolvimento , Mutação , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética
10.
Nitric Oxide ; 76: 164-173, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28963074

RESUMO

High salinity greatly impacts agriculture, particularly in tomato (Solanum lycopersicum), a crop that is a model to study this abiotic stress. This work investigated whether hydrogen sulfide (H2S) acts upstream or downstream of nitric oxide (NO) in the signaling cascade during tomato response to salt stress. An NO-donor incremented H2S levels by 12-18.9% while an H2S-donor yielded 10% more NO in roots. The NO accumulated in roots one-hour after NaCl treatment while H2S accumulation started two-hour later. The NO stimulated H2S accumulation in roots/leaves, but not the opposite (i.e H2S was unable to stimulate NO accumulation) two-hour post NaCl treatment. Also, NO accumulation was accompanied by an increment of transcript levels of genes that encode for H2S-synthesizing enzymes. Our results indicate that H2S acts downstream of NO in the mitigation of oxidative stress, which helps tomato plants to tolerate high salinity.


Assuntos
Sulfeto de Hidrogênio/metabolismo , Óxido Nítrico/metabolismo , Salinidade , Estresse Salino/fisiologia , Solanum lycopersicum/metabolismo , Solanum lycopersicum/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Folhas de Planta/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacos
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