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.

Fruit normally develops from the ovary after pollination and fertilization. However, the ovary can also generate seedless fruit without fertilization by parthenocarpy. Parthenocarpic fruit development has been obtained in tomato (Solanum lycopersicum) by genetic modification using auxin-synthesising gene(s) (DefH9-iaaM; DefH9-RI-iaaM) expressed specifically in the placenta and ovules. We have performed a cDNA Amplified Fragment Length Polymorphism (cDNA-AFLP) analysis on pre-anthesis tomato flower buds (0.5 cm long) collected from DefH9-iaaM and DefH9-RI-iaaM parthenocarpic and wild-type plants, with the aim to identify genes involved in very early phases of tomato fruit development. We detected 212 transcripts differentially expressed in auxin-ipersynthesising pre-anthesis flower buds, 65 of them (31%) have unknown function. Several differentially expressed genes show homology to genes involved in protein trafficking and protein degradation via proteasome. These processes are crucial for auxin cellular transport and signaling, respectively. The data presented might contribute to elucidate the molecular basis of the fruiting process and to develop new methods to confer parthenocarpy to species of agronomic interest. In a recently published work, we have demonstrated that one of the genes identified in this screening, corresponding to #109 cDNA clone, regulates auxin-dependent fruit initiation and its suppression causes parthenocarpic fruit development in tomato.

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.

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.

RNA interference (RNAi) is a post-translational regulatory mechanism that controls gene expression in plants. This process can be artificially induced by double-stranded RNA (dsRNA) molecules with sequence homology to target mRNAs. Exogenously applied dsRNA on leaves has been shown to silence virulence genes of fungi and viruses, conferring protection to plants. Coupling dsRNA to nanoparticles has been demonstrated to prolong the silencing effect. The ability of exogenous dsRNA to silence endogenous genes in plants is currently under debate, mainly due to the difficulty in delivering dsRNA into plant tissues and organs. Our study aims to develop a method based on the exogenous application of dsRNA on tomato flowers for silencing endogenous genes controlling ovary growth. Two methods of dsRNA delivery into tomato flower buds (i.e., pedicel soaking and injection) were compared to test their efficacy in silencing the tomato Aux/IAA9 (SlIAA9) gene, which encodes for a known repressor of ovary growth. We examined the silencing effect of dsRNA alone and coupled to layered double hydroxide (LDHs) nanoparticles. We found that injection into the pedicel led to the silencing of SlIAA9 and the efficacy of the method was confirmed by choosing a different ovary growth repressor gene (SlAGAMOUS-like 6; SlAGL6). The coupling of dsRNA to LDHs increased the silencing effect in the case of SlIAA9. Silencing of the two repressors caused an increase in ovary size only when flower buds were treated with dsRNA coupled to LDHs. RNA-Seq of small RNAs showed that induction of RNAi was caused by the processing of injected dsRNA. In this work, we demonstrate for the first time that exogenous dsRNA coupled to LDHs can induce post-transcriptional gene silencing in the young tomato ovary by injection into the flower pedicel. This method represents a silencing tool for the study of the molecular changes occurring during the early stages of ovary/fruit growth as a consequence of downregulation of target genes, without the need to produce transgenic plants stably expressing RNAi constructs.

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.