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.

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.

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.

Biosafety considerations of RNAi-mediated virus resistance in fruit-tree cultivars and in rootstock.

A major application of RNA interference (RNAi) is envisaged for the production of virus-resistant transgenic plants. For fruit trees, this remains the most, if not the only, viable option for the control of plant viral disease outbreaks in cultivated orchards, due to the difficulties associated with the use of traditional and conventional disease-control measures. The use of RNAi might provide an additional benefit for woody crops if silenced rootstock can efficiently transmit the silencing signal to non-transformed scions, as has already been demonstrated in herbaceous plants. This would provide a great opportunity to produce non-transgenic fruit from transgenic rootstock. In this review, we scrutinise some of the concerns that might arise with the use of RNAi for engineering virus-resistant plants, and we speculate that this virus resistance has fewer biosafety concerns. This is mainly because RNAi-eliciting constructs only express small RNA molecules rather than proteins, and because this technology can be applied using plant rootstock that can confer virus resistance to the scion, leaving the scion untransformed. We discuss the main biosafety concerns related to the release of new types of virus-resistant plants and the risk assessment approaches in the application of existing regulatory systems (in particular, those of the European Union, the USA, and Canada) for the evaluation and approval of RNAi-mediated virus-resistant plants, either as transgenic varieties or as plant virus resistance induced by transgenic rootstock.

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.

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 Medicago truncatula N5 gene encoding a root-specific lipid transfer protein is required for the symbiotic interaction with Sinorhizobium meliloti.

The Medicago truncatula N5 gene is induced in roots after Sinorhizobium meliloti infection and it codes for a putative lipid transfer protein (LTP), a family of plant small proteins capable of binding and transferring lipids between membranes in vitro. Various biological roles for plant LTP in vivo have been proposed, including defense against pathogens and modulation of plant development. The aim of this study was to shed light on the role of MtN5 in the symbiotic interaction between M. truncatula and S. meliloti. MtN5 cDNA was cloned and the mature MtN5 protein expressed in Escherichia coli. The lipid binding capacity and antimicrobial activity of the recombinant MtN5 protein were tested in vitro. MtN5 showed the capacity to bind lysophospholipids and to inhibit M. truncatula pathogens and symbiont growth in vitro. Furthermore, MtN5 was upregulated in roots after infection with either the fungal pathogen Fusarium semitectum or the symbiont S. meliloti. Upon S. meliloti infection, MtN5 was induced starting from 1 day after inoculation (dpi). It reached the highest concentration at 3 dpi and it was localized in the mature nodules. MtN5-silenced roots were impaired in nodulation, showing a 50% of reduction in the number of nodules compared with control roots. On the other hand, transgenic roots overexpressing MtN5 developed threefold more nodules with respect to control roots. Here, we demonstrate that MtN5 possesses biochemical features typical of LTP and that it is required for the successful symbiotic association between M. truncatula and S. meliloti.

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.

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.

Auxin and nitric oxide control indeterminate nodule formation.

BACKGROUND: Rhizobia symbionts elicit root nodule formation in leguminous plants. Nodule development requires local accumulation of auxin. Both plants and rhizobia synthesise auxin. We have addressed the effects of bacterial auxin (IAA) on nodulation by using Sinorhizobium meliloti and Rhizobium leguminosarum bacteria genetically engineered for increased auxin synthesis. RESULTS: IAA-overproducing S. meliloti increased nodulation in Medicago species, whilst the increased auxin synthesis of R. leguminosarum had no effect on nodulation in Phaseolus vulgaris, a legume bearing determinate nodules. Indeterminate legumes (Medicago species) bearing IAA-overproducing nodules showed an enhanced lateral root development, a process known to be regulated by both IAA and nitric oxide (NO). Higher NO levels were detected in indeterminate nodules of Medicago plants formed by the IAA-overproducing rhizobia. The specific NO scavenger cPTIO markedly reduced nodulation induced by wild type and IAA-overproducing strains. CONCLUSION: The data hereby presented demonstrate that auxin synthesised by rhizobia and nitric oxide positively affect indeterminate nodule formation and, together with the observation of increased expression of an auxin efflux carrier in roots bearing nodules with higher IAA and NO content, support a model of nodule formation that involves auxin transport regulation and NO synthesis.