Vera Quecini

The Rg1 allele as a valuable tool for genetic transformation of the tomato 'Micro-Tom' model system

BackgroundThe cultivar Micro-Tom (MT) is regarded as a model system for tomato genetics due to its short life cycle and miniature size. However, efforts to improve tomato genetic transformation have led to protocols dependent on the costly hormone zeatin, combined with an excessive number of steps.ResultsHere we report the development of a MT near-isogenic genotype harboring the allele Rg1 (MT-Rg1), which greatly improves tomato in vitro regeneration. Regeneration was further improved in MT by including a two-day incubation of cotyledonary explants onto medium containing 0.4 μM 1-naphthaleneacetic acid (NAA) before cytokinin treatment. Both strategies allowed the use of 5 μM 6-benzylaminopurine (BAP), a cytokinin 100 times less expensive than zeatin. The use of MT-Rg1 and NAA pre-incubation, followed by BAP regeneration, resulted in high transformation frequencies (near 40%), in a shorter protocol with fewer steps, spanning approximately 40 days from Agrobacterium infection to transgenic plant acclimatization.ConclusionsThe genetic resource and the protocol presented here represent invaluable tools for routine gene expression manipulation and high throughput functional genomics by insertional mutagenesis in tomato.

Identification of a novel α-L-arabinofuranosidase gene associated with mealiness in apple

In order to investigate the genetic bases of the physiological syndrome mealiness that causes abnormal fruit softening and juice loss in apples, an integrative approach was devised, consisting of sensory, instrumental, biochemical, genetic, and genomic methods. High levels of activity of α-L-arabinofuranosidase (α-AFase), a hydrolase acting on the pectic component of the cell walls, were found in individuals exhibiting the mealiness phenotype in a segregating population. The expression levels of the previously uncharacterized apple AF gene MdAF3 are higher in fruits from plants consistently showing mealiness symptons and high α-AFase activity. The transcription of MdAF3 is differentially regulated in distinct genomic contexts and appears to be independent of ethylene. Thus, it is likely to be controlled by endogenous developmental mechanisms associated with fruit ripening. The use of integrative approaches has allowed the identification of a novel contributor to the mealiness phenotype in apple and it has been possible to overcome the problems posed by the unavailability of near-isogenic lines to dissect the genetic bases of a complex physiological trait in woody perennial species.

A reverse genetics approach identifies novel mutants in light responses and anthocyanin metabolism in petunia

Flower color and plant architecture are important commercially valuable features for ornamental petunias (Petunia x hybrida Vilm.). Photoperception and light signaling are the major environmental factors controlling anthocyanin and chlorophyll biosynthesis and shade-avoidance responses in higher plants. The genetic regulators of these processes were investigated in petunia by in silico analyses and the sequence information was used to devise a reverse genetics approach to probe mutant populations. Petunia orthologs of photoreceptor, light-signaling components and anthocyanin metabolism genes were identified and investigated for functional conservation by phylogenetic and protein motif analyses. The expression profiles of photoreceptor gene families and of transcription factors regulating anthocyanin biosynthesis were obtained by bioinformatic tools. Two mutant populations, generated by an alkalyting agent and by gamma irradiation, were screened using a phenotype-independent, sequence-based method by high-throughput PCR-based assay. The strategy allowed the identification of novel mutant alleles for anthocyanin biosynthesis (CHALCONE SYNTHASE) and regulation (PH4), and for light signaling (CONSTANS) genes.

Mutagenesis in Petunia x hybrida Vilm. and isolation of a novel morphological mutant

Traditionally, mutagenesis has been used to introduce novel genetic variability in ornamental crops. More recently, it has become a powerful tool in gene discovery and functional analyses in reverse genetics approaches. The present work aimed to compare the efficiency of physical and chemical agents in generating mutant populations of petunia. We have indirectly evaluated the genomic damage by analyzing developmental characteristics of the plantlets derived from treated seeds employing gamma radiation at 0, 20, 40, 60, 80 and 100 Gy and the alkylating agent ethyl-methanesulfonate (EMS) at 0, 0.05, 0.1, 0.15, 0.2 and 0.25% (v/v). Gamma rays and EMS caused developmental defects and decreased seedling viability in plants obtained from the mutagenized seeds. High mutagen doses reduced in approximately 44% the number of plants with primary leaves at 15 days after sowing (DAS) and decreased seedling survival rates to 55% (gamma) and 28% (EMS), in comparison to untreated controls. Seedling height decrease was proportional to increasing EMS dosage, whereas 40 and 60 Gy of gamma irradiation caused the most significant reduction in height. Moderate DNA damage allowing a high saturation of mutant alleles in the genome and the generation of viable plants for reverse genetics studies was correlated to the biological parameter LD50, the dose required to kill half of the tested population. It corresponded to 100 Gy for gamma radiation and 0.1% for EMS treatment. The optimized mutagen treatments were used to develop petunia mutant populations (M1 and M2) and novel morphological mutants were identified.

Identifying water stress-response mechanisms in citrus by in silico transcriptome analysis

Water deficit is one of the most critical environmental stresses to which plants are submitted during their life cycle. The evolutionary and economic performance of the plant is affected directly by reducing its survival in the natural environment and its productivity in agriculture. Plants respond to water stress with biochemical and physiological modifications that may be involved in tolerance or adaptation mechanisms. A great number of genes have been identified as transcriptionally regulated for water deficit. EST sequencing projects provide a significant contribution to the discovery of expressed genes. The identification and determination of gene expression patterns is important not only to understand the molecular bases of plant responses but also to improve water stress tolerance. In our citrus transcriptome survey we have attempted to identify homologs to genes known to be induced and regulated under water stress conditions. We have identified 89 transcripts whose deduced amino acid sequences share similarities with proteins involved in uptake and transport of water and ion, 34 similar to components of the osmolyte metabolism, 67 involved in processes of membranes and proteins protection and 115 homologs of reactive oxygen species scavenger. Many drought-inducible genes identified are known to be regulated by development, salt, osmotic and low temperature. Their possible roles in specific or general mechanisms of water stress citrus responses are discussed.

Identification of a novel reference gene for apple transcriptional profiling under postharvest conditions.

Reverse Transcription quantitative PCR (RT-qPCR) is one of the most important techniques for gene expression profiling due to its high sensibility and reproducibility. However, the reliability of the results is highly dependent on data normalization, performed by comparisons between the expression profiles of the genes of interest against those of constitutively expressed, reference genes. Although the technique is widely used in fruit postharvest experiments, the transcription stability of reference genes has not been thoroughly investigated under these experimental conditions. Thus, we have determined the transcriptional profile, under these conditions, of three genes commonly used as reference—ACTIN (MdACT), PROTEIN DISULPHIDE ISOMERASE (MdPDI) and UBIQUITIN-CONJUGATING ENZYME E2 (MdUBC)—along with two novel candidates—HISTONE 1 (MdH1) and NUCLEOSSOME ASSEMBLY 1 PROTEIN (MdNAP1). The expression profile of the genes was investigated throughout five experiments, with three of them encompassing the postharvest period and the other two, consisting of developmental and spatial phases. The transcriptional stability was comparatively investigated using four distinct software packages: BestKeeper, NormFinder, geNorm and DataAssist. Gene ranking results for transcriptional stability were similar for the investigated software packages, with the exception of BestKeeper. The classic reference gene MdUBC ranked among the most stably transcribed in all investigated experimental conditions. Transcript accumulation profiles for the novel reference candidate gene MdH1 were stable throughout the tested conditions, especially in experiments encompassing the postharvest period. Thus, our results present a novel reference gene for postharvest experiments in apple and reinforce the importance of checking the transcription profile of reference genes under the experimental conditions of interest.

Transcriptional regulatory networks controlling woolliness in peach in response to preharvest gibberellin application and cold storage

BackgroundPostharvest fruit conservation relies on low temperatures and manipulations of hormone metabolism to maintain sensory properties. Peaches are susceptible to chilling injuries, such as ‘woolliness’ that is caused by juice loss leading to a ‘wooly’ fruit texture. Application of gibberellic acid at the initial stages of pit hardening impairs woolliness incidence, however the mechanisms controlling the response remain unknown. We have employed genome wide transcriptional profiling to investigate the effects of gibberellic acid application and cold storage on harvested peaches.ResultsApproximately half of the investigated genes exhibited significant differential expression in response to the treatments. Cellular and developmental process gene ontologies were overrepresented among the differentially regulated genes, whereas sequences in cell death and immune response categories were underrepresented. Gene set enrichment demonstrated a predominant role of cold storage in repressing the transcription of genes associated to cell wall metabolism. In contrast, genes involved in hormone responses exhibited a more complex transcriptional response, indicating an extensive network of crosstalk between hormone signaling and low temperatures. Time course transcriptional analyses demonstrate the large contribution of gene expression regulation on the biochemical changes leading to woolliness in peach.ConclusionOverall, our results provide insights on the mechanisms controlling the complex phenotypes associated to postharvest textural changes in peach and suggest that hormone mediated reprogramming previous to pit hardening affects the onset of chilling injuries.

Ethylene-dependent regulation of an α-L-arabinofuranosidase is associated to firmness loss in 'Gala' apples under long term cold storage.

Fruit texture changes impair the quality of apples submitted to long term storage, especially under cold. The changes are due to cell wall modifications during ripening and senescence and are associated to ethylene. We have investigated the activity of α-l-arabinofuranosidase, a glycosyl hydrolase acting on the side chains of pectin in the cell wall and middle lamella. The transcription of arabinofuranosidase coding sequences 1 and 3 was investigated in plant organs and in response to ethylene, employing hormone application and 1-methylcyclopropene. The transcription of arabinofuranosidase genes is not restricted to fruits, although upregulated by ripening and ethylene. Transcripts of the genes were detected under cold storage up to 180 days. Similarly, arabinofuranosidase activity increased with rising levels of ethylene and under cold storage. Levels of arabinofuranosidase3 transcripts were higher than those of arabinofuranosidase1, suggesting that the first is an important contributor to enzyme activity and texture changes during cold storage.

Identification of a Simple Sequence Repeat molecular-marker set for large-scale analyses of pear germplasm

Simple Sequence Repeats (SSR) are molecular markers suitable to assess the genetic variation of germplasm resources; however, large-scale SSR use requires protocol optimization. The present work aimed to identify SSR markers, developed for pear and other fruit species that are effective in characterizing pear germplasm collections and in demonstrating their use in providing support for genetic breeding programs. From a total of 62 SSR markers investigated, 23 yielding reproducible and polymorphic patterns were used to genotype a sample of 42 pear accessions of the Brazilian Pear Germplasm Bank (PGB). When compared to these 23 SSR markers, a subset of eleven markers, selected based on He, PIC and PId, was used to distinguish individual accessions and perform cluster analysis with similar efficacy. Genetic diversity analysis clustered the European, Japanese and Chinese accessions in distinct groups. This markers subset constitutes a valuable tool for several applications related to pear genetic resources management and breeding.

In silico analysis of phytohormone metabolism and communication pathways in citrus transcriptome

Plant hormones play a crucial role in integrating endogenous and exogenous signals and in determining developmental responses to form the plant body throughout its life cycle. In citrus species, several economically important processes are controlled by phytohormones, including seed germination, secondary growth, fruit abscission and ripening. Integrative genomics is a powerful tool for linking newly researched organisms, such as tropical woody species, to functional studies already carried out on established model organisms. Based on gene orthology analyses and expression patterns, we searched the Citrus Genome Sequencing Consortium (CitEST) database for Expressed Sequence Tags (EST) consensus sequences sharing similarity to known components of hormone metabolism and signaling pathways in model species. More than 600 homologs of functionally characterized hormone metabolism and signal transduction members from model species were identified in citrus, allowing us to propose a framework for phytohormone signaling mechanisms in citrus. A number of components from hormone-related metabolic pathways were absent in citrus, suggesting the presence of distinct metabolic pathways. Our results demonstrated the power of comparative genomics between model systems and economically important crop species to elucidate several aspects of plant physiology and metabolism.