Temperature and solar irradiation effects on secondary metabolism during ripening of field-grown everbearing strawberries.

The garden strawberry (Fragaria x ananassa Duch.) is cultivated and consumed worldwide because of the pleasant flavor and health-promoting phytochemicals of its false fruits. Monocrop cultivars produce fully ripe strawberries in about one month post-anthesis throughout the spring, while everbearing cultivars undergo additional strawberry production in autumn. In this work, we evaluated the impact of different season-dependent environmental conditions on the ripening program of an everbearing field-gown strawberry variety from autumn 2015 to spring 2016. We combined ad hoc sampling and environmental data collection with LC-MS-based untargeted metabolomics to dissect the effects of cumulative temperature and solar irradiation on fruit quality parameters and secondary metabolism during ripening. Different dynamics in specific sub-groups of metabolites were observed in strawberries experiencing distinct amounts of cumulative temperature and solar irradiation during spring and autumn. The integration of statistical analyses on collected data revealed that solar irradiation mainly affected fruit fresh weight and organic acid levels, whereas temperature had a more selective effect on the accumulation of specific flavonols, anthocyanins, and soluble sugar. These findings are of suitable interest to design further approaches for the study of the complex interactions among environmental conditions and ripening in strawberries grown in a real-world scenario.

RNA interference-based strategies to control Botrytis cinerea infection in cultivated strawberry.

KEY MESSAGE: Gene silencing of BcDCL genes improves gray mold disease control in the cultivated strawberry. Gene silencing technology offers new opportunities to develop new formulations or new pathogen-resistant plants for reducing impacts of agricultural systems. Recent studies offered the proof of concept that the symptoms of gray mold can be reduced by downregulating Dicer-like 1 (DCL1) and 2 (DCL2) genes of Botrytis cinerea. In this study, we demonstrate that both solutions based on dsRNA topical treatment and in planta expression targeting BcDCL1 and BcDCL2 genes can be used to control the strawberry gray mold, the most harmful disease for different fruit crops. 50, 70 and 100 ng µL-1 of naked BcDCL1/2 dsRNA, sprayed on plants of Fragaria x ananassa cultivar Romina in the greenhouse, displayed significant reduction of susceptibility, compared to the negative controls, but to a lesser extent than the chemical fungicide. Three independent lines of Romina cultivar were confirmed for their stable expression of the hairpin gene construct that targets the Bc-DCL1 and 2 sequences (hp-Bc-DCL1/2), and for the production of hp construct-derived siRNAs, by qRT-PCR and Northern blot analyses. In vitro and in vivo detached leaves, and fruits from the hp-Bc-DCL1/2 lines showed significantly enhanced tolerance to this fungal pathogen compared to the control. This decreased susceptibility was correlated to the reduced fungal biomass and the downregulation of the Bc-DCL1 and 2 genes in B. cinerea. These results confirm the potential of both RNAi-based products and plants for protecting the cultivated strawberry from B. cinerea infection, reducing the impact of chemical pesticides on the environment and the health of consumers.

Involvement of the tomato BBX16 and BBX17 microProteins in reproductive development.

BBXs are B-Box zinc finger proteins that can act as transcription factors and regulators of protein complexes. Several BBX proteins play important roles in plant development. Two Arabidopsis thaliana microProteins belonging to the BBX family, named miP1a and miP1b, homotypically interact with and modulate the activity of other BBX proteins, including CONSTANS, which transcriptionally activates the florigen, FLOWERING LOCUS T. Arabidopsis plants overexpressing miP1a and miP1b showed delayed flowering. In tomato, the closest homologs of miP1a and miP1b are the microProteins SlBBX16 and SlBBX17. This study was aimed at investigating whether the constitutive expression of SlBBX16/17 in Arabidopsis and tomato impacted reproductive development. The heterologous expression of the two tomato microProteins in Arabidopsis caused a delay in the flowering transition; however, the effect was weaker than that observed when the native miP1a/b were overexpressed. In tomato, overexpression of SlBBX17 prolonged the flowering period; this effect was accompanied by downregulation of the flowering inhibitors Self Pruning (SP) and SP5G. SlBBX16 and SlBBX17 can hetero-oligomerize with TCMP-2, a cystine-knot peptide involved in flowering pattern regulation and early fruit development in tomato. The increased expression of both microProteins also caused alterations in tomato fruit development: we observed in the case of SlBBX17 a decrease in the number and size of ripe fruits as compared to WT plants, while for SlBBX16, a delay in fruit production up to the breaker stage. These effects were associated with changes in the expression of GA-responsive genes.

MicroRNAs associated with AGL6 and IAA9 function in tomato fruit set.

OBJECTIVE: Fruit set is triggered after ovule fertilization, as a consequence of the downregulation of ovary growth repressors, such as the tomato transcription factors Auxin/indole-3-acetic acid 9 (IAA9) and Agamous-like 6 (AGL6). In a recent work, we developed a method to silence IAA9 and AGL6 in tomato ovaries using exogenous dsRNAs. We also produced small RNA libraries from IAA9- and AGL6-silenced ovaries to confirm the presence of siRNAs, derived from exogenous dsRNA, targeting IAA9 and AGL6. The objective of this work is to exploit these sRNA libraries to identify miRNAs differentially expressed in IAA9- and AGL6-silenced ovaries as compared with unpollinated control ovaries. RESULTS: We identified by RNA sequencing 125 and 104 known and 509 and 516 novel miRNAs from reads mapped to mature or hairpin sequences, respectively. Of the known miRNAs, 7 and 45 were differentially expressed in IAA9- and AGL6-silenced ovaries compared to control ones, respectively. Six miRNAs were common to both datasets, suggesting their importance in the fruit set process. The expression pattern of two of these (miR393 and miR482e-5p) was verified by stem-loop qRT-PCR. The identified miRNAs represent a pool of regulatory sRNAs potentially involved in tomato fruit initiation.

Efficient protocol of de novo shoot organogenesis from somatic embryos for grapevine genetic transformation.

Plant genetic transformation is a powerful tool that can facilitate breeding programs for disease tolerance, abiotic stress, fruit production, and quality by preserving the characteristics of fruit tree elite genotypes. However, most grapevine cultivars worldwide are considered recalcitrant, and most available genetic transformation protocols involve regeneration by somatic embryogenesis, which often requires the continuous production of new embryogenic calli. Cotyledons and hypocotyls derived from flower-induced somatic embryos of the Vitis vinifera cultivars Ancellotta and Lambrusco Salamino, in comparison with the model cultivar Thompson Seedless, are here validated for the first time as starting explants for in vitro regeneration and transformation trials. Explants were cultured on two different MS-based culture media, one having a combination of 4.4 µM BAP and 0.49 µM IBA (M1), and the other only supplemented with 13.2 µM BAP (M2). The competence to regenerate adventitious shoots was higher in cotyledons than in hypocotyls on both M1 and M2. M2 medium increased significantly the average number of shoots only in Thompson Seedless somatic embryo-derived explants. This efficient regeneration strategy, that proposes a combination of somatic embryogenesis and organogenesis, has been successfully exploited in genetic engineering experiments. Ancellotta and Lambrusco Salamino cotyledons and hypocotyls produced the highest number of calli expressing eGFP when cultured on M2 medium, while for Thompson Seedless both media tested were highly efficient. The regeneration of independent transgenic lines of Thompson Seedless was observed from cotyledons cultured on both M1 and M2 with a transformation efficiency of 12 and 14%, respectively, and from hypocotyls on M1 and M2 with a transformation efficiency of 6 and 12%, respectively. A single eGFP fluorescent adventitious shoot derived from cotyledons cultured on M2 was obtained for Ancellotta, while Lambrusco Salamino showed no regeneration of transformed shoots. In a second set of experiments, using Thompson Seedless as the model cultivar, we observed that the highest number of transformed shoots was obtained from cotyledons explants, followed by hypocotyls and meristematic bulk slices, confirming the high regeneration/transformation competences of somatic embryo-derived cotyledons. The independent transformed shoots obtained from the cultivars Thompson Seedless and Ancellotta were successfully acclimatized in the greenhouse and showed a true-to-type phenotype. The novel in vitro regeneration and genetic transformation protocols optimized in this study will be useful for the application of new and emerging modern biotechnologies also to other recalcitrant grapevine genotypes.

Chemical Characterization of a Collagen-Derived Protein Hydrolysate and Biostimulant Activity Assessment of Its Peptidic Components.

Protein hydrolysates (PHs) are plant biostimulants consisting of oligopeptides and free amino acids exploited in agriculture to increase crop productivity. This work aimed to fractionate a commercial collagen-derived protein hydrolysate (CDPH) according to the molecular mass of the peptides and evaluate the bioactivity of different components. First, the CDPH was dialyzed and/or filtrated and analyzed on maize, showing that smaller compounds were particularly active in stimulating lateral root growth. The CDPH was then fractionated through fast protein liquid chromatography and tested on in vitro grown tomatoes proving that all the fractions were bioactive. Furthermore, these fractions were characterized by liquid chromatography-electrospray ionization-tandem mass spectrometry revealing a consensus sequence shared among the identified peptides. Based on this sequence, a synthetic peptide was produced. We assessed its structural similarity with the CDPH, the collagen, and polyproline type II helix by comparing the respective circular dichroism spectra and for the first time, we proved that a signature peptide was as bioactive as the whole CDPH.

Evaluation of the Potential Use of a Collagen-Based Protein Hydrolysate as a Plant Multi-Stress Protectant.

Protein hydrolysates (PHs) are a class of plant biostimulants used in the agricultural practice to improve crop performance. In this study, we have assessed the capacity of a commercial PH derived from bovine collagen to mitigate drought, hypoxic, and Fe deficiency stress in Zea mays. As for the drought and hypoxic stresses, hydroponically grown plants treated with the PH exhibited an increased growth and absorption area of the roots compared with those treated with inorganic nitrogen. In the case of Fe deficiency, plants supplied with the PH mixed with FeCl3 showed a faster recovery from deficiency compared to plants supplied with FeCl3 alone or with FeEDTA, resulting in higher SPAD values, a greater concentration of Fe in the leaves and modulation in the expression of genes related to Fe. Moreover, through the analysis of circular dichroism spectra, we assessed that the PH interacts with Fe in a dose-dependent manner. Various hypothesis about the mechanisms of action of the collagen-based PH as stress protectant particularly in Fe-deficiency, are discussed.

How Hormones and MADS-Box Transcription Factors Are Involved in Controlling Fruit Set and Parthenocarpy in Tomato.

Fruit set is the earliest phase of fruit growth and represents the onset of ovary growth after successful fertilization. In parthenocarpy, fruit formation is less affected by environmental factors because it occurs in the absence of pollination and fertilization, making parthenocarpy a highly desired agronomic trait. Elucidating the genetic program controlling parthenocarpy, and more generally fruit set, may have important implications in agriculture, considering the need for crops to be adaptable to climate changes. Several phytohormones play an important role in the transition from flower to fruit. Further complexity emerges from functional analysis of floral homeotic genes. Some homeotic MADS-box genes are implicated in fruit growth and development, displaying an expression pattern commonly observed for ovary growth repressors. Here, we provide an overview of recent discoveries on the molecular regulatory gene network underlying fruit set in tomato, the model organism for fleshy fruit development due to the many genetic and genomic resources available. We describe how the genetic modification of components of this network can cause parthenocarpy, discussing the contribution of hormonal signals and MADS-box transcription factors.

TCMP-2 affects tomato flowering and interacts with BBX16, a homolog of the arabidopsis B-box MiP1b.

Flowering and fruiting are processes subject to complex control by environmental and endogenous signals. Endogenous signals comprise, besides classical phytohormones, also signaling peptides and miniproteins. Tomato cystine-knot miniproteins (TCMPs), which belong to a Solanaceous-specific group of Cys-rich protein family, have been recently involved in fruit development. TCMP-1 and TCMP-2 display a highly modulated expression pattern during flower and fruit development. A previous study reported that a change in the ratio of the two TCMPs affects the timing of fruit production. In this work, to investigate TCMP-2 mode of action, we searched for its interacting partners. One of the interactors identified by a yeast two hybrid screen, was the B-box domain-containing protein 16 (SlBBX16), whose closest homolog is the Arabidopsis microProtein 1b implicated in flowering time control. We demonstrated the possibility for the two proteins to interact in vivo in tobacco epidermal cells. Arabidopsis plants ectopically overexpressing the TCMP-2 exhibited an increased level of FLOWERING LOCUS T (FT) mRNA and anticipated flowering. Similarly, in previously generated transgenic tomato plants with increased TCMP-2 expression in flower buds, we observed an augmented expression of SINGLE-FLOWER TRUSS gene, the tomato ortholog of FT, whereas the expression of the antiflorigen SELF-PRUNING was unchanged. Consistently, these transgenic plants showed alterations in the flowering pattern, with an accelerated termination of the sympodial units. Overall, our study reveals a novel function for TCMP-2 as regulatory factor that might integrate, thanks to its capacity to interact with SlBBX16, into the signaling pathways that control flowering, and converge toward florigen regulation.

The Tomato Metallocarboxypeptidase Inhibitor I, which Interacts with a Heavy Metal-Associated Isoprenylated Protein, Is Implicated in Plant Response to Cadmium.

Metallocarboxypeptidases are metal-dependent enzymes, whose biological activity is regulated by inhibitors directed on the metal-containing active site. Some metallocarboxypeptidase inhibitors are induced under stress conditions and have a role in defense against pests. This paper is aimed at investigating the response of the tomato metallocarboxypeptidase inhibitor (TCMP)-1 to Cd and other abiotic stresses. To this aim, the tomato TCMP-1 was ectopically expressed in the model species Arabidopsis thaliana, and a yeast two-hybrid analysis was performed to identify interacting proteins. We demonstrate that TCMP-1 is responsive to Cd, NaCl, and abscisic acid (ABA) and interacts with the tomato heavy metal-associated isoprenylated plant protein (HIPP)26. A. thaliana plants overexpressing TCMP-1 accumulate lower amount of Cd in shoots, display an increased expression of AtHIPP26 in comparison with wild-type plants, and are characterized by a modulation in the expression of antioxidant enzymes. Overall, these results suggest a possible role for the TCMP-1/HIPP26 complex in Cd response and compartmentalization.

The different tolerance to magnesium deficiency of two grapevine rootstocks relies on the ability to cope with oxidative stress.

BACKGROUND: Magnesium (Mg) deficiency causes physiological and molecular responses, already dissected in several plant species. The study of these responses among genotypes showing a different tolerance to the Mg shortage can allow identifying the mechanisms underlying the resistance to this nutritional disorder. To this aim, we compared the physiological and molecular responses (e.g. changes in root metabolome and transcriptome) of two grapevine rootstocks exhibiting, in field, different behaviors with respect to Mg shortage (1103P, tolerant and SO4 susceptible). RESULTS: The two grapevine rootstocks confirmed, in a controlled growing system, their behavior in relation to the tolerance to Mg deficiency. Differences in metabolite and transcriptional profiles between the roots of the two genotypes were mainly linked to antioxidative compounds and the cell wall constituents. In addition, differences in secondary metabolism, in term of both metabolites (e.g. alkaloids, terpenoids and phenylpropanoids) and transcripts, assessed between 1103P and SO4 suggest a different behavior in relation to stress responses particularly at early stages of Mg deficiency. CONCLUSIONS: Our results suggested that the higher ability of 1103P to tolerate Mg shortage is mainly linked to its capability of coping, faster and more efficiently, with the oxidative stress condition caused by the nutritional disorder.

Overexpression of the Anthocyanidin Synthase Gene in Strawberry Enhances Antioxidant Capacity and Cytotoxic Effects on Human Hepatic Cancer Cells.

Food fortification through the increase and/or modulation of bioactive compounds has become a major goal for preventing several diseases, including cancer. Here, strawberry lines of cv. Calypso transformed with a construct containing an anthocyanidin synthase (ANS) gene were produced to study the effects on anthocyanin biosynthesis, metabolism, and transcriptome. Three strawberry ANS transgenic lines (ANS L5, ANS L15, and ANS L18) were analyzed for phytochemical composition and total antioxidant capacity (TAC), and their fruit extracts were assessed for cytotoxic effects on hepatocellular carcinoma. ANS L18 fruits had the highest levels of total phenolics and flavonoids, while those of ANS L15 had the highest anthocyanin concentration; TAC positively correlated with total polyphenol content. Fruit transcriptome was also specifically affected in the polyphenol biosynthesis and in other related metabolic pathways. Fruit extracts of all lines exerted cytotoxic effects in a dose/time-dependent manner, increasing cellular apoptosis and free radical levels and impairing mitochondrial functionality.

Genome-Wide Transcriptional Changes and Lipid Profile Modifications Induced by Medicago truncatula N5 Overexpression at an Early Stage of the Symbiotic Interaction with Sinorhizobium meliloti.

Plant lipid-transfer proteins (LTPs) are small basic secreted proteins, which are characterized by lipid-binding capacity and are putatively involved in lipid trafficking. LTPs play a role in several biological processes, including the root nodule symbiosis. In this regard, the Medicago truncatula nodulin 5 (MtN5) LTP has been proved to positively regulate the nodulation capacity, controlling rhizobial infection and nodule primordia invasion. To better define the lipid transfer protein MtN5 function during the symbiosis, we produced MtN5-downregulated and -overexpressing plants, and we analysed the transcriptomic changes occurring in the roots at an early stage of Sinorhizobium meliloti infection. We also carried out the lipid profile analysis of wild type (WT) and MtN5-overexpressing roots after rhizobia infection. The downregulation of MtN5 increased the root hair curling, an early event of rhizobia infection, and concomitantly induced changes in the expression of defence-related genes. On the other hand, MtN5 overexpression favoured the invasion of the nodules by rhizobia and determined in the roots the modulation of genes that are involved in lipid transport and metabolism as well as an increased content of lipids, especially galactolipids that characterize the symbiosome membranes. Our findings suggest the potential participation of LTPs in the synthesis and rearrangement of membranes occurring during the formation of the infection threads and the symbiosome membrane.

Prospect on Ionomic Signatures for the Classification of Grapevine Berries According to Their Geographical Origin.

The determination of food geographical origin has been an important subject of study over the past decade, with an increasing number of analytical techniques being developed to determine the provenance of agricultural products. Agricultural soils can differ for the composition and the relative quantities of mineral nutrients and trace elements whose bioavailability depends on soil properties. Therefore, the ionome of fruits, vegetables and derived products can reflect the mineral composition of the growth substrate. Multi-elemental analysis has been successfully applied to trace the provenance of wines from different countries or different wine-producing regions. However, winemaking process and environmental and cultural conditions may affect a geographical fingerprint. In this article, we discuss the possibility of applying ionomics in wines classification on a local scale and also by exploiting grape berry analyses. In this regard, we present the ionomic profile of grapevine berries grown within an area of approximately 300 km2 and the subsequent application of chemometric methods for the assignment of their geographical origin. The best discrimination was obtained by using a dataset composed only of rare earth elements. Considering the experiences reported in the literature and our results, we concluded that sample representativeness and the application of a preliminary Principal Component Analysis, as pattern recognition techniques, might represent two necessary starting points for the geographical determination of the geographical origin of grape berries; therefore, on the basis of these observations we also include some recommendations to be considered for future application of these techniques for grape and wines classification.

Growth Stimulatory Effects and Genome-Wide Transcriptional Changes Produced by Protein Hydrolysates in Maize Seedlings.

Protein hydrolysates are an emerging class of crop management products utilized for improving nutrient assimilation and mitigating crop stress. They generally consist of a mixture of peptides and free amino acids derived from the hydrolysis of plant or animal sources. The present work was aimed at studying the effects and the action mechanisms of a protein hydrolysate derived from animal residues on maize root growth and physiology in comparison with the effects induced by either free amino acids or inorganic N supply. The application of the protein hydrolysate caused a remarkable enhancement of root growth. In particular, in the protein hydrolysate-treated plants the length and surface area of lateral roots were about 7 and 1.5 times higher than in plants treated with inorganic N or free amino acids, respectively. The root growth promoting effect of the protein hydrolysate was associated with an increased root accumulation of K, Zn, Cu, and Mn when compared with inorganic N and amino acids treatments. A microarray analysis allowed to dissect the transcriptional changes induced by the different treatments demonstrating treatment-specific effects principally on cell wall organization, transport processes, stress responses and hormone metabolism.

The Arabidopsis N-Acetylornithine Deacetylase Controls Ornithine Biosynthesis via a Linear Pathway with Downstream Effects on Polyamine Levels.

Arabidopsis thaliana At4g17830 codes for a protein showing sequence similarity with the Escherichia coli N-acetylornithine deacetylase (EcArgE), an enzyme implicated in the linear ornithine (Orn) biosynthetic pathway. In plants, N-acetylornithine deacetylase (NAOD) activity has yet to be demonstrated; however, At4g17830-silenced and mutant (atnaod) plants display an impaired reproductive phenotype and altered foliar levels of Orn and polyamines (PAs). Here, we showed the direct connection between At4g17830 function and Orn biosynthesis, demonstrating biochemically that At4g17830 codes for a NAOD. These results are the first experimental proof that Orn can be produced in Arabidopsis via a linear pathway. In this study, to identify the role of AtNAOD in reproductive organs, we carried out a transcriptomic analysis on atnaod mutant and wild-type flowers. In the atnaod mutant, the most relevant effects were the reduced expression of cysteine-rich peptide-coding genes, known to regulate male-female cross-talk during reproduction, and variation in the expression of genes involved in nitrogen:carbon (N:C) status. The atnaod mutant also exhibited increased levels of sucrose and altered sensitivity to glucose. We hypothesize that AtNAOD participates in Orn and PA homeostasis, contributing to maintain an optimal N:C balance during reproductive development.

Tomato cystine-knot miniproteins possessing anti-angiogenic activity exhibit in vitro gastrointestinal stability, intestinal absorption and resistance to food industrial processing.

The cystine-knot miniproteins present in tomato fruit (TCMPs) have been shown to exert anti-angiogenic effects by inhibiting endothelial cell migration and to display resistance to gastrointestinal proteolytic attack. To better define the pharmacological potential of TCMPs, their oral bioavailability and their resistance to industrial processing must be assessed. To explore the intestinal transport of TCMPs we used the differentiated Caco-2 cells model. After 24h incubation, 37.73±9.34% of TCMPs crossed the epithelium, without altering the integrity of the cell layer. To assess the effects of the industrial processing on the biochemical features and the biological activity of TCMPs, we developed a method for purifying the proteins from tomato paste. The tomato-paste purified TCMPs retained the resistance to gastrointestinal digestion and the inhibitory activity towards endothelial cell migration. Our previous and present results collectively demonstrate that TCMPs possess interesting features for drug development.

Plant cystine-knot peptides: pharmacological perspectives.

Cystine-knot miniproteins are a class of 30-50 amino acid long peptides widespread in eukaryotic organisms. Due to their very peculiar three-dimensional structure, they exhibit high resistance to heat and peptidase attack. The cystine-knot peptides are well represented in several plant species including medicinal herbs and crops. The pharmacological interest in plant cystine-knot peptides derives from their broad biological activities, mainly cytotoxic, antimicrobial and peptidase inhibitory and in the possibility to engineer them to incorporate pharmacophoric information for oral delivery or disease biomonitoring. The mechanisms of action of plant cystine-knot peptides are still largely unknown, although the capacity to interfere with plasma membranes seems a feature common to several cystine-knot peptides. In some cases, such as potato carboxypetidase inhibitor (PCI) and tomato cystine-knot miniproteins (TCMPs), the cystine-knot peptides target human growth factor receptors either by acting as growth factor antagonist or by altering their signal transduction pathway. The possibility to identify specific molecular targets of plant cystine-knot peptides in human cells opens novel possibilities for the pharmacological use of these peptides besides their use as scaffold to develop stable disease molecular markers and therapeutic agents.