Maura Begheldo

Signaling Pathways Mediating the Induction of Apple Fruitlet Abscission

Apple (Malus × domestica) represents an interesting model tree crop for studying fruit abscission. The physiological fruitlet drop occurring in this species can be easily magnified by using thinning chemicals, such as benzyladenine (BA), to obtain fruits with improved quality and marketability. Despite the economic importance of this process, the molecular determinants of apple fruitlet abscission are still unknown. In this research, BA was used to obtain fruitlet populations with different abscission potentials to be analyzed by means of a newly released 30K oligonucleotide microarray. RNAs were extracted from cortex and seed of apple fruitlets sampled over a 4-d time course, during which BA triggers fruit drop, and used for microarray hybridization. Transcriptomic profiles of persisting and abscising fruitlets were tested for statistical association with abscission potential, allowing us to identify molecular signatures strictly related to fruit destiny. A hypothetical model for apple fruitlet abscission was obtained by putting together available transcriptomic and metabolomic data. According to this model, BA treatment would establish a nutritional stress within the tree that is primarily perceived by the fruitlet cortex whose growth is blocked by resembling the ovary growth inhibition found in other species. In weaker fruits, this stress is soon visible also at the seed level, likely transduced via reactive oxygen species/sugar and hormones signaling cross talk, and followed by a block of embryogenesis and the consequent activation of the abscission zone.

Transcriptome profiling of ripening nectarine (Prunus persica L. Batsch) fruit treated with 1-MCP

A large-scale transcriptome analysis has been conducted using μPEACH1.0 microarray on nectarine (Prunus persica L. Batsch) fruit treated with 1-methylcyclopropene (1-MCP). 1-MCP maintained flesh firmness but did not block ethylene biosynthesis. Compared with samples at harvest, only nine genes appeared to be differentially expressed when fruit were sampled immediately after treatment, while a total of 90 targets were up- or down-regulated in untreated fruit. The effect of 1-MCP was confirmed by a direct comparison of transcript profiles in treated and untreated fruit after 24 h of incubation with 106 targets differentially expressed. About 30% of these targets correspond to genes involved in primary metabolism and response processes related to ethylene, auxin, and other hormones. In treated fruit, altered transcript accumulation was detected for some genes with a role in ripening-related events such as softening, colour development, and sugar metabolism. A rapid decrease in flesh firmness and an increase in ethylene production were observed in treated fruit maintained for 48 h in air at 20 °C after the end of the incubation period. Microarray comparison of this sample with untreated fruit 24 h after harvest revealed that about 45% of the genes affected by 1-MCP at the end of the incubation period changed their expression during the following 48 h in air. Among these genes, an ethylene receptor (ETR2) and three ethylene-responsive factors (ERF) were present, together with other transcription factors and ethylene-dependent genes involved in quality parameter changes.

NO homeostasis is a key regulator of early nitrate perception and root elongation in maize

Nitrate reductase produces nitric oxide (NO) as an early response to nitrate, and the coordinated induction of ns-haemoglobins finely modulates NO level. The control of NO homeostasis regulates root elongation and represents a novel key component of nitrate signaling in maize

Transcriptome analysis reveals coordinated spatiotemporal regulation of hemoglobin and nitrate reductase in response to nitrate in maize roots

Given the importance of nitrogen for plant growth and the environmental costs of intense fertilization, an understanding of the molecular mechanisms underlying the root adaptation to nitrogen fluctuations is a primary goal for the development of biotechnological tools for sustainable agriculture. This research aimed to identify the molecular factors involved in the response of maize roots to nitrate. cDNA-amplified fragment length polymorphism was exploited for comprehensive transcript profiling of maize (Zea mays) seedling roots grown with varied nitrate availabilities; 336 primer combinations were tested and 661 differentially regulated transcripts were identified. The expression of selected genes was studied in depth through quantitative real-time polymerase chain reaction and in situ hybridization. Over 50% of the genes identified responded to prolonged nitrate starvation and a few were identified as putatively involved in the early nitrate signaling mechanisms. Real-time results and in situ localization analyses demonstrated co-regulated transcriptional patterns in root epidermal cells for genes putatively involved in nitric oxide synthesis/scavenging. Our findings, in addition to strengthening already known mechanisms, revealed the existence of a new complex signaling framework in which brassinosteroids (BRI1), the module MKK2-MAPK6 and the fine regulation of nitric oxide homeostasis via the co-expression of synthetic (nitrate reductase) and scavenging (hemoglobin) components may play key functions in maize responses to nitrate.

Differential expression of two lipid transfer protein genes in reproductive organs of peach (Prunus persica L. Batsch)

Abstract Two cDNA clones (named Pp-LTP1 and Pp-LTP2 ) corresponding to different lipid transfer protein (LTP) genes have been isolated from peach ( Prunus persica L. Batsch) epicarp and ovary, respectively. Sequence analysis revealed that the two fragments share 54% identity at nucleotide level and show common features of plant LTP genes, such as conserved cysteine residues and lipid-binding motifs. Phylogenetic analysis grouped Pp-LTP1 and Pp-LTP2 in two distinct clusters, the former with most of LTP genes sequenced in the Rosaceae family, the latter only with one almond LTP. Genomic Southern data indicated that a small LTP gene family is present in peach. Pp-LTP1 and Pp-LTP2 have been used as gene-specific probes to describe expression in flowers and fruits throughout development. In petals, sepals and stamen only Pp-LTP1 was expressed whereas transcripts of Pp-LTP2 strongly accumulated in non-pollinated and pollinated ovary with a decreasing trend in the period of four weeks after pollination. In fruits, a dramatic accumulation of Pp-LTP1 mRNA was detected in epicarp at all stages of fruit development and, with the exception of the early growth stage, no Pp-LTP1 transcripts have been detected in mesocarp. When Pp-LTP2 was used as a probe in the same fruit tissues, a faint hybridisation signal was observed only in epicarp of fruitlets collected at an early growth stage. Infection with Monilia induced only a slight increase of Pp-LTP1 transcript in epicarp of pre-climacteric and climacteric fruits. These results support the hypothesis of multiple roles played by LTPs and, considering that LTPs have been recognised as the major allergen of peach, indicate that Pp-LTP1 could be related to the allergenicity of peach.

Protocol: an improved and universal procedure for whole-mount immunolocalization in plants

Rapid advances in microscopy have boosted research on cell biology. However sample preparation enabling excellent reproducible tissue preservation and cell labeling for in depth microscopic analysis of inner cell layers, tissues and organs still represents a major challenge for immunolocalization studies. Here we describe a protocol for whole-mount immunolocalization of proteins which is applicable to a wide range of plant species. The protocol is improved and robust for optimal sample fixation, tissue clearing and multi-protein staining procedures and can be used in combination with simultaneous detection of specific sequences of nucleic acids. In addition, cell wall and nucleus labelling can be implemented in the protocol, thereby allowing a detailed analysis of morphology and gene expression patterns with single-cell resolution. Besides enabling accurate, high resolution and reproducible protein detection in expression and localization studies, the procedure takes a single working day to complete without the need for robotic equipment.

A Grapevine TTG2-Like WRKY Transcription Factor Is Involved in Regulating Vacuolar Transport and Flavonoid Biosynthesis

A small set of TTG2-like homolog proteins from different species belonging to the WRKY family of transcription factors were shown to share a similar mechanism of action and to control partially conserved biochemical/developmental processes in their native species. In particular, by activating P-ATPases residing on the tonoplast, PH3 from Petunia hybrida promotes vacuolar acidification in petal epidermal cells whereas TTG2 from Arabidopsis thaliana enables the accumulation of proanthocyanidins in the seed coat. In this work we functionally characterized VvWRKY26 identified as the closest grapevine homolog of PhPH3 and AtTTG2. When constitutively expressed in petunia ph3 mutant, VvWRKY26 can fulfill the PH3 function in the regulation of vacuolar pH and restores the wild type pigmentation phenotype. By a global correlation analysis of gene expression and by transient over-expression in Vitis vinifera, we showed transcriptomic relationships of VvWRKY26 with many genes related to vacuolar acidification and transport in grapevine. Moreover, our results indicate an involvement in flavonoid pathway possibly restricted to the control of proanthocyanidin biosynthesis that is consistent with its expression pattern in grape berry tissues. Overall, the results show that, in addition to regulative mechanisms and biological roles shared with TTG2-like orthologs, VvWRKY26 can play roles in fleshy fruit development that have not been previously reported in studies from dry fruit species. This study paves the way toward the comprehension of the regulatory network controlling vacuolar acidification and flavonoid accumulation mechanisms that contribute to the final berry quality traits in grapevine.

Ethylene negatively regulates transcript abundance of ROP-GAP rheostat-encoding genes and affects apoplastic reactive oxygen species homeostasis in epicarps of cold stored apple fruits

Highlight In cold-stressed apple skins, the ethylene-dependent negative regulation of ROP-GAP rheostat transcripts and apoplastic H2O2 homeostasis suggests an unprecedented putative crosstalk between ROS and ethylene in abiotic stress.

Whole-mount in situ detection of microRNAs on Arabidopsis tissues using Zip Nucleic Acid probes

MicroRNAs (miRNAs) affect fundamental processes of development. In plants miRNAs regulate organ development, transition to flowering, and responses to abiotic/biotic stresses. To understand the biological role of miRNAs, in addition to identifying their targeted transcripts, it is necessary to characterize the spatiotemporal regulation of their expression. Many methods have been used to define the set of organ-specific miRNAs by tissue dissection and miRNA profiling but none of them can describe their tissue and cellular distribution at the high resolution provided by in situ hybridization (ISH). This article describes the setup and optimization of a whole-mount ISH protocol to target endogenous miRNAs on intact Arabidopsis seedlings using DIG-labeled Zip Nucleic Acid (ZNA) oligonucleotide probes. Automation of the main steps of the procedure by robotized liquid handling has also been implemented in the protocol for best reproducibility of results, enabling running of ISH experiments at high throughput.

The miRNA-mediated post-transcriptional regulation of maize response to nitrate

Stress responses depend on the correct regulation of gene expression. The discovery that abiotic as well as biotic stresses can regulate miRNA levels, coupled with the identification and functional analyses of stress-associated genes as miRNA targets, provided clues about the vital role that several miRNAs may play in modulating plant resistance to stresses. Nitrogen availability seriously affects crops productivity and environment and the understanding of the miRNA-guided stress regulatory networks should provide new tools for the genetic improvement of nitrogen use efficiency of crops. A recent study revealed the potential role of a number of nitrate-responsive miRNAs in the maize adaptation to nitrate fluctuations. In particular, results obtained suggested that a nitrate depletion might regulate the expression of genes involved in the starvation adaptive response, by affecting the spatio-temporal expression patterns of specific miRNAs.