Benedetto Ruperti

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.

The endoplasmic reticulum localized PIN8 is a pollen-specific auxin carrier involved in intracellular auxin homeostasis

The plant hormone auxin is a mobile signal which affects nuclear transcription by regulating the stability of auxin/indole-3-acetic acid (IAA) repressor proteins. Auxin is transported polarly from cell to cell by auxin efflux proteins of the PIN family, but it is not as yet clear how auxin levels are regulated within cells and how access of auxin to the nucleus may be controlled. The Arabidopsis genome contains eight PINs, encoding proteins with a similar membrane topology. While five of the PINs are typically targeted polarly to the plasma membranes, the smallest members of the family, PIN5 and PIN8, seem to be located not at the plasma membrane but in endomembranes. Here we demonstrate by electron microscopy analysis that PIN8, which is specifically expressed in pollen, resides in the endoplasmic reticulum and that it remains internally localized during pollen tube growth. Transgenic Arabidopsis and tobacco plants were generated overexpressing or ectopically expressing functional PIN8, and its role in control of auxin homeostasis was studied. PIN8 ectopic expression resulted in strong auxin-related phenotypes. The severity of phenotypes depended on PIN8 protein levels, suggesting a rate-limiting activity for PIN8. The observed phenotypes correlated with elevated levels of free IAA and ester-conjugated IAA. Activation of the auxin-regulated synthetic DR5 promoter and of auxin response genes was strongly repressed in seedlings overexpressing PIN8 when exposed to 1-naphthalene acetic acid. Thus, our data show a functional role for endoplasmic reticulum-localized PIN8 and suggest a mechanism whereby PIN8 controls auxin thresholds and access of auxin to the nucleus, thereby regulating auxin-dependent transcriptional activity.

Peach fruit ripening and quality in relation to picking time, and hypoxic and high CO2 short-term postharvest treatments

Peach fruits (Prunus persica L. Batsch, cv Springcrest) were harvested at two ripening stages (flesh firmness of 60 N, first harvest, and 45 N, second harvest) and maintained at 20°C in air (control) or for 24 and 48 h in streams of ultra low (<1%) oxygen (ULO) or high (30%) CO2 concentration and then transferred to air for up to 8 days. The decline in flesh firmness was strongly reduced by ULO and CO2 treatments in fruits of both harvests, although the effect was stronger in fruits picked earlier in which ethylene biosynthesis remained at the basal level. In fruits of the second harvest, endo β-1,4-glucanase (EGase) activity was lower in ULO- and CO2-treated fruits than in control fruits at the end of the 24 h treatment and the following two days in air. Acetaldehyde (AA) gradually accumulated in control fruit and the highest concentrations were detected during late ripening. Both treatments induced a strong accumulation of AA but, with the exception of the 24 and 48 h CO2 treatments performed on fruits of the second harvest, a decrease in AA content was observed when the fruits were transferred to air. A slight increase in ethanol (EtOH) was found throughout the ripening process in control fruits; ULO and CO2 strongly stimulated EtOH production. When fruits were transferred to air, EtOH concentration declined rapidly. Alcohol dehydrogenase (ADH) activity significantly increased in control fruit only in the late stages of ripening. Greater ADH activity was found throughout the experimental period in fruits of the first harvest treated for 24 h in ULO and CO2, whereas, at day 8, control and treated fruits of the second harvest showed similar ADH activity values. Hypoxic and, to a lesser extent, CO2-enriched atmospheres stimulated Adh gene expression.

Genetic and environmental factors affecting allergen-related gene expression in apple fruit (Malus domestica L. Borkh)

Freshly consumed apples can cause allergic reactions because of the presence of four classes of allergens, namely, Mal d 1, Mal d 2, Mal d 3, and Mal d 4, and their cross-reactivity with sensitizing allergens of other species. Knowledge of environmental and endogenous factors affecting the allergenic potential of apples would provide important information to apple breeders, growers, and consumers for the selection of hypoallergenic genotypes, the adoption of agronomical practices decreasing the allergenic potential, and the consumption of fruits with reduced amount of allergens. In the present research, expression studies were performed by means of real-time PCR for all the known allergen-encoding genes in apple. Fruit samples were collected from 15 apple varieties and from fruits of three different trials, set up to assess the effect of shadowing, elevation, storage, and water stress on the expression of allergen genes. Principal components analysis (PCA) was performed for the classification of varieties according to gene expression values, pointing out that the cultivars Fuji and Brina were two good hypoallergenic candidates. Shadowing, elevation, and storage significantly affected the transcription of the allergen-encoding genes, whereas water stress slightly influenced the expression of only two genes, in spite of the dramatic effect on both fruit size and vegetative growth of the trees. In particular, shadowing may represent an important cultural practice aimed at reducing apple cortex allergenicity. Moreover, elevation and storage may be combined to reduce the allergenic potential of apple fruits. The possible implications of the results for breeders, growers, and consumers are discussed critically.

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.

Neutral invertases in grapevine and comparative analysis with Arabidopsis, poplar and rice

Neutral invertases (NIs, EC 3.2.1.26) cleave sucrose to glucose and fructose. They are encoded by a small gene family of 9 members in the Arabidopsis genome, 8 in rice, 16 in poplar and 9 in Vitis vinifera (L.). The grapevine NIs were identified in the 8.4X genome assembly of the quasi-homozygous line PN40024. In addition, alleles of three NIs were sequenced in the heterozygous cultivar ‘Cabernet Sauvignon’. Analyses of sequence variation between alleles, homoeologous and paralogous copies in grapevine and their orthologues in Arabidopsis, poplar and rice are provided. In grapevine, NIs were classified into four α NIs and five β NIs and subsequently grouped into hierarchical clades using a combination of evidence including amino acid identity, exon/intron structure, rate of synonymous substitutions (Ks) and chromosomal distribution. Estimation of Ks proved the ancient origin of all NIs and the lack of expansion by gene duplication past the event of polyploidisation. We then focused on transcription analysis of five NIs for which evidence of expression was available from expressed sequence tag databases. Among these, four NIs consisted of pairs of homoeologous copies, each pair lying on a pair of chromosomes duplicated by polyploidy. Unequal expression of homoeologous genes was observed by quantitative RT-PCR in leaf, flower, seed and root tissues. Since NIs might play significant roles in fruit and wine quality, NIs expression was monitored in flesh and skin of ‘Merlot’ berries and shown in parallel with the suite of changes that accompany fruit ripening, including glucose and fructose accumulation.

Characterization of a major latex protein (MLP) gene down-regulated by ethylene during peach fruitlet abscission☆

We report the isolation of a new peach gene, Pp-MLP1 , that shows significant similarity to a family of fruit- and flower-specific genes, designated as major latex protein (MLP) homologues. Transcript of Pp-MLP1 highly accumulated in cells of fruit pedicel, similar to lacticifers, adjacent to the abscission zone (non-abscission zone) and, to a lesser extent, in epicotyls, stems and roots, while no accumulation was detected in leaves. In contrast to the MLP homologues isolated so far, the Pp-MLP1 transcript was detected during fruit cells expansion, though its expression appeared unrelated to fruit ripening. Propylene treatment caused a decrease in mRNA accumulation of Pp-MLP1 in all tested tissues. The function of Pp-MLP1, as with all previously described MLP homologues, is unknown. MLPs are associated with fruit and flower development in addition to plant pathogenesis responses. Expression in tissues associated with abscission would be consistent with a role in implementing this aspect of floral development or possibly protective responses to plant pathogens which may infect post-abscission wounds. In addition, the high similarity between proteins encoding by Pp-MLP1 and Csf2 , an MLP gene associated with the early development of cucumber fruit, could suggest an alternativ ed evelopmental role such as cell and tissue expansion. # 2002 Elsevier Science Ireland Ltd. All rights reserved.

Identification and differential expression dynamics of peach small GTPases encoding genes during fruit development and ripening

The function of monomeric GTPases of the RAS superfamily in fruit development and ripening has been partially characterized. Here the identification of peach (Prunus persica) small GTPases of the RAS superfamily expressed in fruit and the characterization of their expression profiles during fruit development are described. Extensive searches on expressed sequence tag (EST) databases led to the selection of a total of 24 genes from peach encoding proteins with significant similarity to Arabidopsis small GTPases. Sequence similarity analyses and identification of conserved motifs, diagnostic of specific RAS families and subfamilies, enabled bona fide assignment of fourteen PpRAB, seven PpARF/ARL/SAR, two PpROP and one PpRAN GTPases. Transcriptional expression profiles of peach monomeric GTPases, analysed by real-time quantitative reverse transcription-PCR, were obtained for mesocarp samples, collected in two consecutive years. Reproducible patterns of expression could be identified for five peach RAB-encoding genes (PpRABA1-1, PpRABA2, PpRABD2-1, PpRABD2-2, and PpRABC2), two ARFs (PpARFA1-1 and PpARLB1), and two ROPs (PpROP3 and PpROP4). Interestingly, the transient transcriptional up-regulation of PpARF genes and of PpRAB genes of the A and D clades, putatively controlling the exocytic delivery of cell wall components and modifying enzymes, appeared to coincide with peaks of growth speed and sugar accumulation and with the final phases of ripening. To our knowledge, this is the first description of the co-ordinated differential expression of a set of genes encoding small GTPases of the ARF and RAB families which takes place during key moments of fruit development and maturation.

Modelling polar auxin transport in developmental patterning.

Auxin interacts with its own polar transport to influence cell polarity and tissue patterning. Research over the past decade has started to deliver new insights into the molecular mechanisms that drive and regulate polar auxin transport. The most prominent auxin efflux protein, PIN1, has subsequently become a crucial component of auxin transport models because it is now known to direct auxin flow and maintain local auxin gradients. Recent molecular and genetic experiments have allowed the formulation of conceptual models that are able to interpret the role of (i) auxin, (ii) its transport, and (iii) the dynamics of PIN1 in generating temporal and spatial patterns. Here we review the current mathematical models of patterning in two specific developmental contexts: lateral shoot and vein formation, focusing on how these models can help to untangle the details of auxin transport-mediated patterning.

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.