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.

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.

Arabidopsis calmodulin-like protein CML36 is a calcium (Ca2+) sensor that interacts with the plasma membrane Ca2+-ATPase isoform ACA8 and stimulates its activity.

Calmodulin-like (CML) proteins are major EF-hand-containing, calcium (Ca2+)-binding proteins with crucial roles in plant development and in coordinating plant stress tolerance. Given their abundance in plants, the properties of Ca2+ sensors and identification of novel target proteins of CMLs deserve special attention. To this end, we recombinantly produced and biochemically characterized CML36 from Arabidopsis thaliana We analyzed Ca2+ and Mg2+ binding to the individual EF-hands, observed metal-induced conformational changes, and identified a physiologically relevant target. CML36 possesses two high-affinity Ca2+/Mg2+ mixed binding sites and two low-affinity Ca2+-specific sites. Binding of Ca2+ induced an increase in the α-helical content and a conformational change that lead to the exposure of hydrophobic regions responsible for target protein recognition. Cation binding, either Ca2+ or Mg2+, stabilized the secondary and tertiary structures of CML36, guiding a large structural transition from a molten globule apo-state to a compact holoconformation. Importantly, through in vitro binding and activity assays, we showed that CML36 interacts directly with the regulative N terminus of the Arabidopsis plasma membrane Ca2+-ATPase isoform 8 (ACA8) and that this interaction stimulates ACA8 activity. Gene expression analysis revealed that CML36 and ACA8 are co-expressed mainly in inflorescences. Collectively, our results support a role for CML36 as a Ca2+ sensor that binds to and modulates ACA8, uncovering a possible involvement of the CML protein family in the modulation of plant-autoinhibited Ca2+ pumps.

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.

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.

The Arabidopsis nox mutant lacking carotene hydroxylase activity reveals a critical role for xanthophylls in photosystem I biogenesis.

Carotenes, and their oxygenated derivatives xanthophylls, are essential components of the photosynthetic apparatus. They contribute to the assembly of photosynthetic complexes and participate in light absorption and chloroplast photoprotection. Here, we studied the role of xanthophylls, as distinct from that of carotenes, by characterizing a no xanthophylls (nox) mutant of Arabidopsis thaliana, which was obtained by combining mutations targeting the four carotenoid hydroxylase genes. nox plants retained α- and ß-carotenes but were devoid in xanthophylls. The phenotype included depletion of light-harvesting complex (LHC) subunits and impairment of nonphotochemical quenching, two effects consistent with the location of xanthophylls in photosystem II antenna, but also a decreased efficiency of photosynthetic electron transfer, photosensitivity, and lethality in soil. Biochemical analysis revealed that the nox mutant was specifically depleted in photosystem I function due to a severe deficiency in PsaA/B subunits. While the stationary level of psaA/B transcripts showed no major differences between genotypes, the stability of newly synthesized PsaA/B proteins was decreased and translation of psaA/B mRNA was impaired in nox with respect to wild-type plants. We conclude that xanthophylls, besides their role in photoprotection and LHC assembly, are also needed for photosystem I core translation and stability, thus making these compounds indispensable for autotrophic growth.

Involvement of the Putative N-Acetylornithine Deacetylase from Arabidopsis thaliana in Flowering and Fruit Development.

In eukaryotic cells, the non-proteinogenic amino acid ornithine is the precursor of arginine and polyamines (PAs). The final step of ornithine biosynthesis occurs in plants via a cyclic pathway catalyzed by N(2)-acetylornithine:N-acetylglutamate acetyltransferase (NAOGAcT). An alternative route for ornithine formation, the linear pathway, has been reported for enteric bacteria and a few other organisms; the acetyl group of N(2)-acetylornithine is released as acetate by N(2)-acetylornithine deacetylase (NAOD). NAOD activity has never been demonstrated in plants, although many putative NAOD-like genes have been identified. In this investigation, we examined the effect of down-regulation of the putative Arabidopsis thaliana NAOD gene by using AtNAOD-silenced (sil#17) and T-DNA insertional mutant (atnaod) plants. The ornithine content was consistently reduced in sil#17 and atnaod plants compared with wild-type plants, suggesting that in addition to NAOGAcT action, AtNAOD contributes to the regulation of ornithine levels in plant cells. Ornithine depletion was associated with altered levels of putrescine and spermine. Reduced AtNAOD expression resulted in alterations at the reproductive level, causing early flowering and impaired fruit setting. In this regard, the highest level of AtNAOD expression was observed in unfertilized ovules. Our findings suggest that AtNAOD acts as a positive regulator of fruit setting and agree with those obtained in tomato auxin-synthesizing parthenocarpic plants, where induction of SlNAOD was associated with the onset of ovary growth. Thus, here we have uncovered the first hints of the functions of AtNAOD by connecting its role in flower and fruit development with the regulation of ornithine and PA levels.

Local and systemic proteomic changes in medicago truncatula at an early phase of Sinorhizobium meliloti infection.

A symbiotic association with N-fixing bacteria facilitates the growth of leguminous plants under nitrogen-limiting conditions. The establishment of the symbiosis requires signal exchange between the host and the bacterium, which leads to the formation of root nodules, inside which bacteria are hosted. The formation of nodules is controlled through local and systemic mechanisms, which involves root-shoot communication. Our study was aimed at investigating the proteomic changes occurring in shoots and concomitantly in roots of Medicago truncatula at an early stage of Sinorhizobium meliloti infection. The principal systemic effects consisted in alteration of chloroplast proteins, induction of proteins responsive to biotic stress, and changes in proteins involved in hormonal signaling and metabolism. The most relevant local effect was the induction of proteins involved in the utilization of photosynthates and C-consuming processes (such as sucrose synthase and fructose-bisphosphate aldolase). In addition, some redox enzymes such as peroxiredoxin and ascorbate peroxidase showed an altered abundance. The analysis of local and systemic proteome changes suggests the occurrence of a stress response in the shoots and the precocious alteration of energy metabolism in roots and shoots. Furthermore, our data indicate the possibility that ABA and ethylene participate in the communicative network between root and shoot in the control of rhizobial infection.

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.

The non-specific lipid transfer protein N5 of Medicago truncatula is implicated in epidermal stages of rhizobium-host interaction.

BACKGROUND: The symbiotic interaction between leguminous plants and rhizobia involves two processes: bacterial infection, resulting in the penetration of bacteria in epidermal and cortical cells, and root nodule organogenesis. Root nodule symbiosis is activated by rhizobial signalling molecules, called Nodulation factors (NFs). NF perception induces the expression of several genes called early nodulins. The early nodulin N5 of Medicago truncatula is a lipid transfer protein that has been shown to positively regulate nodulation although it displays in vitro inhibitory activity against Sinorhizobium meliloti. The purpose of this work was to investigate the role of MtN5 by studying its spatial and temporal pattern of expression during the symbiotic interaction, also in relation to known components of the symbiotic signalling pathway, and by analysing the phenotypic alterations displayed by rhizobia-inoculated MtN5-silenced roots. RESULTS: We show here that MtN5 is a NF-responsive gene expressed at a very early phase of symbiosis in epidermal cells and root hairs. MtN5 expression is induced in vitro by rhizobial effector molecules and by auxin and cytokinin, phytohormones involved in nodule organogenesis. Furthermore, lipid signaling is implicated in the response of MtN5 to rhizobia, since the activity of phospholipase D is required for MtN5 induction in S. meliloti-inoculated roots. MtN5-silenced roots inoculated with rhizobia display an increased root hair curling and a reduced number of invaded primordia compared to that in wild type roots, but with no impairment to nodule primordia formation. This phenotype is associated with the stimulation of ENOD11 expression, an early marker of infection, and with the down-regulation of Flotillin 4 (FLOT4), a protein involved in rhizobial entry. CONCLUSIONS: These data indicate that MtN5 acts downstream of NF perception and upstream of FLOT4 in regulating pre-infection events. The positive effect of MtN5 on nodule primordia invasion is linked to the restriction of bacterial spread at the epidermal level. Furthermore, MtN5 seems to be dispensable for nodule primordia formation. These findings provide new information about the complex mechanism that controls the competence of root epidermal cells for rhizobial invasion.

Double-Stranded RNA Targeting Dicer-Like Genes Compromises the Pathogenicity of Plasmopara viticola on Grapevine.

Downy mildew caused by Plasmopara viticola is one of the most devastating diseases of grapevine, attacking all green parts of the plant. The damage is severe when the infection at flowering stage is left uncontrolled. P. viticola management consumes a significant amount of classical pesticides applied in vineyards, requiring efficient and environmentally safe disease management options. Spray-induced gene silencing (SIGS), through the application of exogenous double-stranded RNA (dsRNA), has shown promising results for the management of diseases in crops. Here, we developed and tested the potential of dsRNA targeting P. viticola Dicer-like (DCL) genes for SIGS-based crop protection strategy. The exogenous application of PvDCL1/2 dsRNA, a chimera of PvDCL1 and PvDCL2, highly affected the virulence of P. viticola. The reduced expression level of PvDCL1 and PvDCL2 transcripts in infected leaves, treated with PvDCL1/2 dsRNA, was an indication of an active RNA interference mechanism inside the pathogen to compromise its virulence. Besides the protective property, the PvDCL1/2 dsRNA also exhibited a curative role by reducing the disease progress rate of already established infection. Our data provide a promising future for PvDCL1/2 dsRNA as a new generation of RNA-based resistant plants or RNA-based agrochemical for the management of downy mildew disease in grapevine.

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.

Molecular dissection of the role of auxin in fruit initiation.

Fruit set and growth usually requires fertilization. Fruit set and development without fertilization is called parthenocarpy. Feeding auxin to virgin flowers induces fruit development without fertilization. Recent studies by Hua Wang et al. and Marc Goetz et al. have identified molecular events leading to fruit initiation in the absence of fertilization, showing that parthenocarpy can be achieved by altering different steps of the auxin signaling pathway. Thus, independent evidence indicates that auxin plays a key role in fruit initiation.

Collective radioresistance of T47D breast carcinoma cells is mediated by a Syncytin-1 homologous protein.

It is generally accepted that radiotherapy must target clonogenic cells, i.e., those cells in a tumour that have self-renewing potential. Focussing on isolated clonogenic cells, however, may lead to an underestimate or even to an outright neglect of the importance of biological mechanisms that regulate tumour cell sensitivity to radiation. We develop a new statistical and experimental approach to quantify the effects of radiation on cell populations as a whole. In our experiments, we change the proximity relationships of the cells by culturing them in wells with different shapes, and we find that the radiosensitivity of T47D human breast carcinoma cells in tight clusters is different from that of isolated cells. Molecular analyses show that T47D cells express a Syncytin-1 homologous protein (SyHP). We observe that SyHP translocates to the external surface of the plasma membrane of cells killed by radiation treatment. The data support the fundamental role of SyHP in the formation of intercellular cytoplasmic bridges and in the enhanced radioresistance of surviving cells. We conclude that complex and unexpected biological mechanisms of tumour radioresistance take place at the cell population level. These mechanisms may significantly bias our estimates of the radiosensitivity of breast carcinomas in vivo and thereby affect treatment plans, and they call for further investigations.

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.

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.

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.