Optimization of Anthocyanin Production in Tobacco Cells.

Plant cell cultures have emerged as a promising tool for producing active molecules due to their numerous advantages over traditional agricultural methods. Flavonols, and anthocyanin pigments in particular, together with other phenolic compounds such as chlorogenic acid, are known for their beneficial health properties, mainly due to their antioxidant, antimicrobial, and anti-inflammatory activities. The synthesis of these molecules is finely regulated in plant cells and controlled at the transcriptional level by specific MYB and bHLH transcription factors that coordinate the transcription of structural biosynthetic genes. The co-expression of peach PpMYB10.1 and PpbHLH3 in tobacco was used to develop tobacco cell lines showing high expression of both the peach transgenes and the native flavonol structural genes. These cell lines were further selected for fast growth. High production levels of chlorogenic acid, anthocyanins (mainly cyanidin 3-rutinoside), and other phenolics were also achieved in pre-industrial scale-up trials. A single-column-based purification protocol was developed to produce a lyophile called ANT-CA, which was stable over time, showed beneficial effects on cell viability, and had antioxidant, anti-inflammatory, antibacterial, and wound-healing activities. This lyophile could be a valuable ingredient for food or cosmetic applications.

Overexpression of Isoprene Synthase Affects ABA- and Drought-Related Gene Expression and Enhances Tolerance to Abiotic Stress.

Isoprene is the most abundant single biogenic volatile compound emitted by plants. Despite the relevance of this molecule to plant abiotic resistance and its impact on global atmospheric chemistry, little is known about the details of its mechanism of action. Here, we characterized through both physiological and molecular methods the mechanisms of action of isoprene using model transgenic arabidopsis lines overexpressing a monocot isoprene synthase gene. Our results demonstrated the effect that isoprene had on ABA signaling at different tissue-specific, spatial, and temporal scales. In particular, we found that isoprene enhanced stomatal sensitivity to ABA through upregulation of RD29B signaling gene. By contrast, isoprene decreased sensitivity to ABA in germinating seeds and roots, suggesting tissue-specific mechanisms of action. In leaves, isoprene caused the downregulation of COR15A and P5CS genes, suggesting that the enhanced tolerance to water-deprivation stress observed in isoprene-emitting plants may be mediated chiefly by an enhanced membrane integrity and tolerance to osmotic stress.

MYB107 and MYB9 Homologs Regulate Suberin Deposition in Angiosperms.

Suberin, a polymer composed of both aliphatic and aromatic domains, is deposited as a rough matrix upon plant surface damage and during normal growth in the root endodermis, bark, specialized organs (e.g., potato [Solanum tuberosum] tubers), and seed coats. To identify genes associated with the developmental control of suberin deposition, we investigated the chemical composition and transcriptomes of suberized tomato (Solanum lycopersicum) and russet apple (Malus x domestica) fruit surfaces. Consequently, a gene expression signature for suberin polymer assembly was revealed that is highly conserved in angiosperms. Seed permeability assays of knockout mutants corresponding to signature genes revealed regulatory proteins (i.e., AtMYB9 and AtMYB107) required for suberin assembly in the Arabidopsis thaliana seed coat. Seeds of myb107 and myb9 Arabidopsis mutants displayed a significant reduction in suberin monomers and altered levels of other seed coat-associated metabolites. They also exhibited increased permeability, and lower germination capacities under osmotic and salt stress. AtMYB9 and AtMYB107 appear to synchronize the transcriptional induction of aliphatic and aromatic monomer biosynthesis and transport and suberin polymerization in the seed outer integument layer. Collectively, our findings establish a regulatory system controlling developmentally deposited suberin, which likely differs from the one of stress-induced polymer assembly recognized to date.

Interference with ethylene perception at receptor level sheds light on auxin and transcriptional circuits associated with the climacteric ripening of apple fruit (Malus x domestica Borkh.).

Apple (Malus x domestica Borkh.) is a model species for studying the metabolic changes that occur at the onset of ripening in fruit crops, and the physiological mechanisms that are governed by the hormone ethylene. In this study, to dissect the climacteric interplay in apple, a multidisciplinary approach was employed. To this end, a comprehensive analysis of gene expression together with the investigation of several physiological entities (texture, volatilome and content of polyphenolic compounds) was performed throughout fruit development and ripening. The transcriptomic profiling was conducted with two microarray platforms: a dedicated custom array (iRIPE) and a whole genome array specifically enriched with ripening-related genes for apple (WGAA). The transcriptomic and phenotypic changes following the application of 1-methylcyclopropene (1-MCP), an ethylene inhibitor leading to important modifications in overall fruit physiology, were also highlighted. The integrative comparative network analysis showed both negative and positive correlations between ripening-related transcripts and the accumulation of specific metabolites or texture components. The ripening distortion caused by the inhibition of ethylene perception, in addition to affecting the ethylene pathway, stimulated the de-repression of auxin-related genes, transcription factors and photosynthetic genes. Overall, the comprehensive repertoire of results obtained here advances the elucidation of the multi-layered climacteric mechanism of fruit ripening, thus suggesting a possible transcriptional circuit governed by hormones and transcription factors.

On the role of ethylene, auxin and a GOLVEN-like peptide hormone in the regulation of peach ripening.

BACKGROUND: In melting flesh peaches, auxin is necessary for system-2 ethylene synthesis and a cross-talk between ethylene and auxin occurs during the ripening process. To elucidate this interaction at the transition from maturation to ripening and the accompanying switch from system-1 to system-2 ethylene biosynthesis, fruits of melting flesh and stony hard genotypes, the latter unable to produce system-2 ethylene because of insufficient amount of auxin at ripening, were treated with auxin, ethylene and with 1-methylcyclopropene (1-MCP), known to block ethylene receptors. The effects of the treatments on the different genotypes were monitored by hormone quantifications and transcription profiling. RESULTS: In melting flesh fruit, 1-MCP responses differed according to the ripening stage. Unexpectedly, 1-MCP induced genes also up-regulated by ripening, ethylene and auxin, as CTG134, similar to GOLVEN (GLV) peptides, and repressed genes also down-regulated by ripening, ethylene and auxin, as CTG85, a calcineurin B-like protein. The nature and transcriptional response of CTG134 led to discover a rise in free auxin in 1-MCP treated fruit. This increase was supported by the induced transcription of CTG475, an IAA-amino acid hydrolase. A melting flesh and a stony hard genotype, differing for their ability to synthetize auxin and ethylene amounts at ripening, were used to study the fine temporal regulation and auxin responsiveness of genes involved in the process. Transcriptional waves showed a tight interdependence between auxin and ethylene actions with the former possibly enhanced by the GLV CTG134. The expression of genes involved in the regulation of ripening, among which are several transcription factors, was similar in the two genotypes or could be rescued by auxin application in the stony hard. Only GLV CTG134 expression could not be rescued by exogenous auxin. CONCLUSIONS: 1-MCP treatment of peach fruit is ineffective in delaying ripening because it stimulates an increase in free auxin. As a consequence, a burst in ethylene production speeding up ripening occurs. Based on a network of gene transcriptional regulations, a model in which appropriate level of CTG134 peptide hormone might be necessary to allow the correct balance between auxin and ethylene for peach ripening to occur is proposed.

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

BACKGROUND: Postharvest 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. RESULTS: Approximately 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. CONCLUSION: Overall, 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.

Regulation of anthocyanin biosynthesis in peach fruits.

MAIN CONCLUSION: MYB10.1 and MYB10.3, with bHLH3, are the likely regulators of anthocyanin biosynthesis in peach fruit. MYB10.1/2/3 forms a cluster on the same genomic fragment where the Anther color ( Ag ) trait is located. Anthocyanins are bioactive compounds responsible for the pigmentation of many plant parts such as leaves, flowers, fruits and roots, and have potential benefits to human health. In peach [Prunus persica (L.) Batsch], peel color is a key determinant for fruit quality and is regulated by flavonoids including anthocyanins. The R2R3 MYB transcription factors (TFs) control the expression of anthocyanin biosynthetic genes with the help of co-activators belonging to the basic-helix-loop-helix (bHLH) and WD40 repeat families. In the peach genome six MYB10-like and three bHLH-like TFs were identified as candidates to be the regulators of the anthocyanin accumulation, which, in yellow flesh fruits, is highest in the peel, abundant in the part of the mesocarp surrounding the stone and lowest in the mesocarp. The expression of MYB10.1 and MYB10.3 correlates with anthocyanin levels of different peach parts. They also have positive correlation with the expression of key structural genes of the anthocyanin pathway, such as CHS, F3H, and UFGT. Functions of peach MYB10s were tested in tobacco and shown to activate key genes in the anthocyanin pathway when bHLHs were co-expressed as partners. Overexpression of MYB10.1/bHLH3 and MYB10.3/bHLH3 activated anthocyanin production by up-regulating NtCHS, NtDFR and NtUFGT while other combinations were not, or much less, effective. As three MYB10 genes are localized in a genomic region where the Ag trait, responsible for anther pigmentation, is localized, it is proposed they are key determinant to introduce new peach cultivars with higher antioxidant level and pigmented fruit.

A candidate gene based approach validates Md-PG1 as the main responsible for a QTL impacting fruit texture in apple (Malus x domestica Borkh).

BACKGROUND: Apple is a widely cultivated fruit crop for its quality properties and extended storability. Among the several quality factors, texture is the most important and appreciated, and within the apple variety panorama the cortex texture shows a broad range of variability. Anatomically these variations depend on degradation events occurring in both fruit primary cell wall and middle lamella. This physiological process is regulated by an enzymatic network generally encoded by large gene families, among which polygalacturonase is devoted to the depolymerization of pectin. In apple, Md-PG1, a key gene belonging to the polygalacturonase gene family, was mapped on chromosome 10 and co-localized within the statistical interval of a major hot spot QTL associated to several fruit texture sub-phenotypes. RESULTS: In this work, a QTL corresponding to the position of Md-PG1 was validated and new functional alleles associated to the fruit texture properties in 77 apple cultivars were discovered. 38 SNPs genotyped by gene full length resequencing and 2 SSR markers ad hoc targeted in the gene metacontig were employed. Out of this SNP set, eleven were used to define three significant haplotypes statistically associated to several texture components. The impact of Md-PG1 in the fruit cell wall disassembly was further confirmed by the cortex structure electron microscope scanning in two apple varieties characterized by opposite texture performance, such as 'Golden Delicious' and 'Granny Smith'. CONCLUSIONS: The results here presented step forward into the genetic dissection of fruit texture in apple. This new set of haplotypes, and microsatellite alleles, can represent a valuable toolbox for a more efficient parental selection as well as the identification of new apple accessions distinguished by superior fruit quality features.

Efficacy of Trichoderma longibrachiatum Trichogin GA IV Peptaibol analogs against the Black Rot Pathogen Xanthomonas campestris pv. campestris and other Phytopathogenic Bacteria.

Black rot caused by the Gram-negative bacterial pathogen Xanthomonas campestris pv. campestris (Xcc) is considered one of the most destructive diseases affecting crucifers. Xcc is a seedborne pathogen able to infect the host at any growth stage. The management of the pathogen mainly relies on the use of copper-based products with possible negative effects on human health and the environment. Searching for protection alternatives is crucial for achieving a sustainable management of Xcc. Trichoderma spp. has been largely used as a biocontrol agent against several phytopathogens. Among Trichoderma species, Trichoderma longibrachiatum produces the peptaibol trichogin GA IV, a secondary metabolite with antimicrobial activity against Gram-positive bacteria, as well as filamentous and yeast-like fungi. In this work, we tested, at micromolar concentrations, 25 synthetic analogs of the peptaibol trichogin GA IV for their bacteriostatic and bactericidal activity toward the bacterium Xcc. One of the most effective peptides (4r) was also tested against the Gram-negative bacteria Xanthomonas arboricola, Pseudomonas corrugata, Pseudomonas savastanoi pv. savastanoi, Agrobacterium tumefaciens, Ralstonia solanacearum, and Erwinia carotovora subsp. carotovora, as well as the Gram-positive bacterium Bacillus subtilis. The peptide 4r reduced black rot symptoms on cauliflower plants when administered both before and 24 h after inoculation with Xcc. The cytotoxic activity of the peptide 4r was also evaluated towards suspensions of tobacco cells by Evans Blue assay.

Transcriptomic and photosynthetic analyses of Synechocystis sp. PCC6803 and Chlorogloeopsis fritschii sp. PCC6912 exposed to an M-dwarf spectrum under an anoxic atmosphere.

Introduction: Cyanobacteria appeared in the anoxic Archean Earth, evolving for the first time oxygenic photosynthesis and deeply changing the atmosphere by introducing oxygen. Starting possibly from UV-protected environments, characterized by low visible and far-red enriched light spectra, cyanobacteria spread everywhere on Earth thanks to their adaptation capabilities in light harvesting. In the last decade, few cyanobacteria species which can acclimate to far-red light through Far-Red Light Photoacclimation (FaRLiP) have been isolated. FaRLiP cyanobacteria were thus proposed as model organisms to study the origin of oxygenic photosynthesis as well as its possible functionality around stars with high far-red emission, the M-dwarfs. These stars are astrobiological targets, as their longevity could sustain life evolution and they demonstrated to host rocky terrestrial-like exoplanets within their Habitable Zone. Methods: We studied the acclimation responses of the FaRLiP strain Chlorogloeopsis fritschii sp. PCC6912 and the non-FaRLiP strain Synechocystis sp. PCC6803 to the combination of three simulated light spectra (M-dwarf, solar and far-red) and two atmospheric compositions (oxic, anoxic). We first checked their growth, O2 production and pigment composition, then we studied their transcriptional responses by RNA sequencing under each combination of light spectrum and atmosphere conditions. Results and discussion: PCC6803 did not show relevant differences in gene expression when comparing the responses to M-dwarf and solar-simulated lights, while far-red caused a variation in the transcriptional level of many genes. PCC6912 showed, on the contrary, different transcriptional responses to each light condition and activated the FaRLiP response under the M-dwarf simulated light. Surprisingly, the anoxic atmosphere did not impact the transcriptional profile of the 2 strains significantly. Results show that both cyanobacteria seem inherently prepared for anoxia and to harvest the photons emitted by a simulated M-dwarf star, whether they are only visible (PCC6803) or also far-red photons (PCC6912). They also show that visible photons in the simulated M-dwarf are sufficient to keep a similar metabolism with respect to solar-simulated light. Conclusion: Results prove the adaptability of the cyanobacterial metabolism and enhance the plausibility of finding oxygenic biospheres on exoplanets orbiting M-dwarf stars.

Spermidine application to young developing peach fruits leads to a slowing down of ripening by impairing ripening-related ethylene and auxin metabolism and signaling.

Peach (Prunus persica var. laevis Gray) was chosen to unravel the molecular basis underlying the ability of spermidine (Sd) to influence fruit development and ripening. Field applications of 1 mM Sd on peach fruit at an early developmental stage, 41 days after full bloom (dAFB), i.e. at late stage S1, led to a slowing down of fruit ripening. At commercial harvest (125 dAFB, S4II) Sd-treated fruits showed a reduced ethylene production and flesh softening. The endogenous concentration of free and insoluble conjugated polyamines (PAs) increased (0.3-2.6-fold) 1 day after treatment (short-term response) butsoon it declined to control levels; starting from S3/S4, when soluble conjugated forms increased (up to five-fold relative to controls at ripening), PA levels became more abundant in treated fruits, (long-term response). Real-time reverse transcription-polymerase chain reaction analyses revealed that peaks in transcript levels of fruit developmental marker genes were shifted ahead in accord with a developmental slowing down. At ripening (S4I-S4II) the upregulation of the ethylene biosynthetic genes ACO1 and ACS1 was dramatically counteracted by Sd and this led to a strong downregulation of genes responsible for fruit softening, such as PG and PMEI. Auxin-related gene expression was also altered both in the short term (TRPB) and in the long term (GH3, TIR1 and PIN1), indicating that auxin plays different roles during development and ripening processes. Messenger RNA amounts of other hormone-related ripening-regulated genes, such as NCED and GA2-OX, were strongly downregulated at maturity. Results suggest that Sd interferes with fruit development/ripening by interacting with multiple hormonal pathways.

A microarray approach to identify genes involved in seed-pericarp cross-talk and development in peach.

BACKGROUND: Field observations and a few physiological studies have demonstrated that peach embryogenesis and fruit development are tightly coupled. In fact, attempts to stimulate parthenocarpic fruit development by means of external tools have failed. Moreover, physiological disturbances during early embryo development lead to seed abortion and fruitlet abscission. Later in embryo development, the interactions between seed and fruit development become less strict. As there is limited genetic and molecular information about seed-pericarp cross-talk and development in peach, a massive gene approach based on the use of the µPEACH 1.0 array platform and quantitative real time RT-PCR (qRT-PCR) was used to study this process. RESULTS: A comparative analysis of the transcription profiles conducted in seed and mesocarp (cv Fantasia) throughout different developmental stages (S1, S2, S3 and S4) evidenced that 455 genes are differentially expressed in seed and fruit. Among differentially expressed genes some were validated as markers in two subsequent years and in three different genotypes. Seed markers were a LTP1 (lipid transfer protein), a PR (pathogenesis-related) protein, a prunin and LEA (Late Embryogenesis Abundant) protein, for S1, S2, S3 and S4, respectively. Mesocarp markers were a RD22-like protein, a serin-carboxypeptidase, a senescence related protein and an Aux/IAA, for S1, S2, S3 and S4, respectively.The microarray data, analyzed by using the HORMONOMETER platform, allowed the identification of hormone-responsive genes, some of them putatively involved in seed-pericarp crosstalk. Results indicated that auxin, cytokinins, and gibberellins are good candidates, acting either directly (auxin) or indirectly as signals during early development, when the cross-talk is more active and vital for fruit set, whereas abscisic acid and ethylene may be involved later on. CONCLUSIONS: In this research, genes were identified marking different phases of seed and mesocarp development. The selected genes behaved as good seed markers, while for mesocarp their reliability appeared to be dependent upon developmental and ripening traits. Regarding the cross-talk between seed and pericarp, possible candidate signals were identified among hormones.Further investigations relying upon the availability of whole genome platforms will allow the enrichment of a marker genes repertoire and the elucidation of players other than hormones that are involved in seed-pericarp cross-talk (i.e. hormone peptides and microRNAs).

Cell-specific expression of tryptophan decarboxylase and 10-hydroxygeraniol oxidoreductase, key genes involved in camptothecin biosynthesis in Camptotheca acuminata Decne (Nyssaceae).

BACKGROUND: Camptotheca acuminata is a major natural source of the terpenoid indole alkaloid camptothecin (CPT). At present, little is known about the cellular distribution of the biosynthesis of CPT, which would be useful knowledge for developing new strategies and technologies for improving alkaloid production. RESULTS: The pattern of CPT accumulation was compared with the expression pattern of some genes involved in CPT biosynthesis in C. acuminata [i.e., Ca-TDC1 and Ca-TDC2 (encoding for tryptophan decarboxylase) and Ca-HGO (encoding for 10-hydroxygeraniol oxidoreductase)]. Both CPT accumulation and gene expression were investigated in plants at different degrees of development and in plantlets subjected to drought-stress. In all organs, CPT accumulation was detected in epidermal idioblasts, in some glandular trichomes, and in groups of idioblast cells localized in parenchyma tissues. Drought-stress caused an increase in CPT accumulation and in the number of glandular trichomes containing CPT, whereas no increase in epidermal or parenchymatous idioblasts was observed. In the leaf, Ca-TDC1 expression was detected in some epidermal cells and in groups of mesophyll cells but not in glandular trichomes; in the stem, it was observed in parenchyma cells of the vascular tissue; in the root, no expression was detected. Ca-TDC2 expression was observed exclusively in leaves of plantlets subjected to drought-stress, in the same sites described for Ca-TDC1. In the leaf, Ca-HGO was detected in all chlorenchyma cells; in the stem, it was observed in the same sites described for Ca-TDC1; in the root, no expression was detected. CONCLUSIONS: The finding that the sites of CPT accumulation are not consistently the same as those in which the studied genes are expressed demonstrates an organ-to-organ and cell-to-cell translocation of CPT or its precursors.

Genetic Variation in Damaged Populations of Pistacia atlantica Desf.

The Atlas Pistachio tree, Pistacia atlantica Desf., has great importance in the ecological landscape of North Africa, due to its adaptive plasticity, as well as its use as a rootstock in the cultivation of the economically important species, Pistacia vera L. The conservation and valuation of this species require sampling and an assessment of its genetic variability. For the first time in North Africa, the inter-simple sequence repeats (ISSR) molecular marker has been used in genetic-diversity assessment and in the population relationships of P. atlantica subsp. atlantica. The ISSR markers tested showed 74.1% polymorphism, while molecular variance (AMOVA) analysis revealed a high percentage of the total genetic diversity of 55.7% among the four populations studied. Cluster analysis with neighbor-joining (NJ) and principal coordinate analysis (PCO) divided the study sites into four distinct groups according to their geographical locations (Tiaret, Batna, Djelfa, and Bechar). Isolation by distance or Mantel test gave a positive correlation of r = 0.86 between geographical and genetic distances. The results in this study indicate an absence of gene flow, implying that conservation efforts should be taken separately for each population.

A PLENA-like gene of peach is involved in carpel formation and subsequent transformation into a fleshy fruit.

MADS-box genes have been shown to play a role in the formation of fruits, both in Arabidopsis and in tomato. In peach, two C-class MADS-box genes have been isolated. Both of them are expressed during flower and mesocarp development. Here a detailed analysis of a gene that belongs to the PLENA subfamily of MADS-box genes is shown. The expression of this PLENA-like gene (PpPLENA) increases during fruit ripening, and its ectopic expression in tomato plants causes the transformation of sepals into carpel-like structures that become fleshy and ripen like real fruits. Interestingly, the transgenic berries constitutively expressing the PpPLENA gene show an accelerated ripening, as judged by the expression of genes that are important for tomato fruit ripening. It is suggested that PpPLENA might interfere with the endogenous activity of TAGL1, thereby activating the fruit ripening pathway earlier compared with wild-type tomato plants.

Effects on Plant Growth and Reproduction of a Peach R2R3-MYB Transcription Factor Overexpressed in Tobacco.

In plants, anthocyanin production is controlled by MYB and bHLH transcription factors. In peach, among the members of these families, MYB10.1 and bHLH3 have been shown to be the most important genes for production of these pigments during fruit ripening. Anthocyanins are valuable molecules, and the overexpression of regulatory genes in annual fast-growing plants has been explored for their biotechnological production. The overexpression of peach MYB10.1 in tobacco plants induced anthocyanin pigmentation, which was particularly strong in the reproductive parts. Pigment production was the result of an up-regulation of the expression level of key genes of the flavonoid biosynthetic pathway, such as NtCHS, NtCHI, NtF3H, NtDFR, NtANS, and NtUFGT, as well as of the proanthocyanidin biosynthetic pathway such as NtLAR. Nevertheless, phenotypic alterations in transgenic tobacco lines were not only limited to anthocyanin production. Lines showing a strong phenotype (type I) exhibited irregular leaf shape and size and reduced plant height. Moreover, flowers had reduced length of anther's filament, nondehiscent anthers, reduced pistil length, aborted nectary glands, and impaired capsule development, but the reproductive parts including androecium, gynoecium, and petals were more pigmented that in wild type. Surprisingly, overexpression of peach MYB10.1 led to suppression of NtMYB305, which is required for floral development and, of one of its target genes, NECTARIN1 (NtNCE1), involved in the nectary gland formation. MYB10.1 overexpression up-regulated JA biosynthetic (NtAOS) and signaling (NtJAZd) genes, as well as 1-aminocyclopropane-1-carboxylate oxidase (NtACO) in flowers. The alteration of these hormonal pathways might be among the causes of the observed floral abnormalities with defects in both male and female gametophyte development. In particular, approximately only 30% of pollen grains of type I lines were viable, while during megaspore formation, there was a block during FG1 (St3-II). This block seemed to be associated to an excessive accumulation of callose. It can be concluded that the overexpression of peach MYB10.1 in tobacco not only regulates flavonoid biosynthesis (anthocyanin and proanthocyanidin) in the reproductive parts but also plays a role in other processes such as vegetative and reproductive development.

Jasmonate-induced transcriptional changes suggest a negative interference with the ripening syndrome in peach fruit.

Peach (Prunus persica L. Batsch) was chosen as a model to shed light on the physiological role of jasmonates (JAs) during fruit ripening. To this aim, the effects of methyl jasmonate (MJ, 0.40 mM) and propyl dihydrojasmonate (PDJ, 0.22 mM), applied in planta at different fruit developmental stages, on the time-course of ethylene production and fruit quality traits were evaluated. MJ-induced changes in fruit transcriptome at harvest and the expression profiling of relevant JA-responsive genes were analysed in control and JA-treated fruit. Exogenously applied JAs affected the onset of ripening depending upon the fruit developmental stage, with PDJ being more active than MJ. Both compounds enhanced the transcription of allene oxide synthase (PpAOS1), the first specific enzyme in the biosynthesis of jasmonic acid, and altered the pattern of jasmonic acid accumulation. Microarray transcriptome profiling showed that MJ down-regulated some ripening-related genes, such as 1-aminocyclopropane-1-carboxylic acid oxidase (PpACO1) and polygalacturonase (PG), and the transcriptional modulator IAA7. MJ also altered the expression of cell wall-related genes, namely pectate lyase (PL) and expansins (EXPs), and up-regulated several stress-related genes, including some of those involved in JA biosynthesis. Time-course expression profiles of PpACO1, PL, PG, PpExp1, and the transcription factor LIM confirmed the array results. Thus, in peach fruit, exogenous JAs led to a ripening delay due to an interference with ripening- and stress/defence-related genes, as reflected in the transcriptome of treated fruit at harvest.

Climacteric ripening of apple fruit is regulated by transcriptional circuits stimulated by cross-talks between ethylene and auxin.

Apple is a fleshy fruit distinguished by a climacteric type of ripening, since most of the relevant physiological changes are triggered and governed by the action of ethylene. After its production, this hormone is perceived by a series of receptors to regulate, through a signaling cascade, downstream ethylene related genes. The possibility to control the effect of ethylene opened new horizons to the improvement of the postharvest fruit quality. To this end, 1-methylcyclopropene (1-MCP), an ethylene antagonist, is routinely used to modulate the ripening progression increasing storage life. In a recent work published in The Plant Journal, the whole transcriptome variation throughout fruit development and ripening, with the adjunct comparison between normal and impaired postharvest ripening, has been illustrated. In particular, besides the expected downregulation of ethylene-regulated genes, we shed light on a regulatory circuit leading to de-repressing the expression of a specific set of genes following 1-MCP treatment, such as AUX/IAA, NAC and MADS. These findings suggested the existence of a possible ethylene/auxin cross-talk in apple, regulated by a transcriptional circuit stimulated by the interference at the ethylene receptor level.

The Peach RGF/GLV Signaling Peptide pCTG134 Is Involved in a Regulatory Circuit That Sustains Auxin and Ethylene Actions.

In vascular plants the cell-to-cell interactions coordinating morphogenetic and physiological processes are mediated, among others, by the action of hormones, among which also short mobile peptides were recognized to have roles as signals. Such peptide hormones (PHs) are involved in defense responses, shoot and root growth, meristem homeostasis, organ abscission, nutrient signaling, hormone crosstalk and other developmental processes and act as both short and long distant ligands. In this work, the function of CTG134, a peach gene encoding a ROOT GROWTH FACTOR/GOLVEN-like PH expressed in mesocarp at the onset of ripening, was investigated for its role in mediating an auxin-ethylene crosstalk. In peach fruit, where an auxin-ethylene crosstalk mechanism is necessary to support climacteric ethylene synthesis, CTG134 expression peaked before that of ACS1 and was induced by auxin and 1-methylcyclopropene (1-MCP) treatments, whereas it was minimally affected by ethylene. In addition, the promoter of CTG134 fused with the GUS reporter highlighted activity in plant parts in which the auxin-ethylene interplay is known to occur. Arabidopsis and tobacco plants overexpressing CTG134 showed abnormal root hair growth, similar to wild-type plants treated with a synthetic form of the sulfated peptide. Moreover, in tobacco, lateral root emergence and capsule size were also affected. In Arabidopsis overexpressing lines, molecular surveys demonstrated an impaired hormonal crosstalk, resulting in a re-modulated expression of a set of genes involved in both ethylene and auxin synthesis, transport and perception. These data support the role of pCTG134 as a mediator in an auxin-ethylene regulatory circuit and open the possibility to exploit this class of ligands for the rational design of new and environmental friendly agrochemicals able to cope with a rapidly changing environment.

Prunus transcription factors: breeding perspectives.

Many plant processes depend on differential gene expression, which is generally controlled by complex proteins called transcription factors (TFs). In peach, 1533 TFs have been identified, accounting for about 5.5% of the 27,852 protein-coding genes. These TFs are the reference for the rest of the Prunus species. TF studies in Prunus have been performed on the gene expression analysis of different agronomic traits, including control of the flowering process, fruit quality, and biotic and abiotic stress resistance. These studies, using quantitative RT-PCR, have mainly been performed in peach, and to a lesser extent in other species, including almond, apricot, black cherry, Fuji cherry, Japanese apricot, plum, and sour and sweet cherry. Other tools have also been used in TF studies, including cDNA-AFLP, LC-ESI-MS, RNA, and DNA blotting or mapping. More recently, new tools assayed include microarray and high-throughput DNA sequencing (DNA-Seq) and RNA sequencing (RNA-Seq). New functional genomics opportunities include genome resequencing and the well-known synteny among Prunus genomes and transcriptomes. These new functional studies should be applied in breeding programs in the development of molecular markers. With the genome sequences available, some strategies that have been used in model systems (such as SNP genotyping assays and genotyping-by-sequencing) may be applicable in the functional analysis of Prunus TFs as well. In addition, the knowledge of the gene functions and position in the peach reference genome of the TFs represents an additional advantage. These facts could greatly facilitate the isolation of genes via QTL (quantitative trait loci) map-based cloning in the different Prunus species, following the association of these TFs with the identified QTLs using the peach reference genome.