SUPERMAN genes: uncovering a new function in the development of complex inflorescences.

The Arabidopsis SUPERMAN (SUP) gene and its orthologs in eudicots are crucial in regulating the number of reproductive floral organs. In Medicago truncatula, in addition to this function, a novel role in controlling meristem activity during compound inflorescence development was assigned to the SUP-ortholog (MtSUP). These findings led us to investigate whether the role of SUP genes in inflorescence development was legume-specific or could be extended to other eudicots. To assess that, we used Solanum lycopersicum as a model system with a cymose complex inflorescence and Arabidopsis thaliana as the best-known example of simple inflorescence. We conducted a detailed comparative expression analysis of SlSUP and SUP from vegetative stages to flower transition. In addition, we performed an exhaustive phenotypic characterisation of two different slsup and sup mutants during the plant life cycle. Our findings reveal that SlSUP is required for precise regulation of the meristems that control shoot and inflorescence architecture in tomato. In contrast, in Arabidopsis, SUP performs no meristematic function, but we found a role of SUP in floral transition. Our findings suggest that the functional divergence of SUP-like genes contributed to the modification of inflorescence architecture during angiosperm evolution.

SUPERMAN strikes again in legumes.

The SUPERMAN (SUP) gene was described in Arabidopsis thaliana over 30 years ago. SUP was classified as a cadastral gene required to maintain the boundaries between reproductive organs, thus controlling stamen and carpel number in flowers. We summarize the information on the characterization of SUP orthologs in plant species other than Arabidopsis, focusing on the findings for the MtSUP, the ortholog in the legume Medicago truncatula. M. truncatula has been widely used as a model system to study the distinctive developmental traits of this family of plants, such as the existence of compound inflorescence and complex floral development. MtSUP participates in the complex genetic network controlling these developmental processes in legumes, sharing conserved functions with SUP. However, transcriptional divergence between SUP and MtSUP provided context-specific novel functions for a SUPERMAN ortholog in a legume species. MtSUP controls the number of flowers per inflorescence and the number of petals, stamens and carpels regulating the determinacy of ephemeral meristems that are unique in legumes. Results obtained in M. truncatula provided new insights to the knowledge of compound inflorescence and flower development in legumes. Since legumes are valuable crop species worldwide, with high nutritional value and important roles in sustainable agriculture and food security, new information on the genetic control of their compound inflorescence and floral development could be used for plant breeding.

The tapetal tissue is essential for the maintenance of redox homeostasis during microgametogenesis in tomato.

The tapetum is a specialized layer of cells within the anther, adjacent to the sporogenous tissue. During its short life, it provides nutrients, molecules and materials to the pollen mother cells and microsporocytes, being essential during callose degradation and pollen wall formation. The interaction between the tapetum and sporogenous cells in Solanum lycopersicum (tomato) plants, despite its importance for breeding purposes, is poorly understood. To investigate this process, gene editing was used to generate loss-of-function mutants that showed the complete and specific absence of tapetal cells. These plants were obtained targeting the previously uncharacterized Solyc03g097530 (SlTPD1) gene, essential for tapetum specification in tomato plants. In the absence of tapetum, sporogenous cells developed and callose deposition was observed. However, sporocytes failed to undergo the process of meiosis and finally degenerated, leading to male sterility. Transcriptomic analysis conducted in mutant anthers lacking tapetum revealed the downregulation of a set of genes related to redox homeostasis. Indeed, mutant anthers showed a reduction in the accumulation of reactive oxygen species (ROS) at early stages and altered activity of ROS-scavenging enzymes. The results obtained highlight the importance of the tapetal tissue in maintaining redox homeostasis during male gametogenesis in tomato plants.

PsEND1 Is a Key Player in Pea Pollen Development Through the Modulation of Redox Homeostasis.

Redox homeostasis has been linked to proper anther and pollen development. Accordingly, plant cells have developed several Reactive Oxygen Species (ROS)-scavenging mechanisms to maintain the redox balance. Hemopexins constitute one of these mechanisms preventing heme-associated oxidative stress in animals, fungi, and plants. Pisum sativum ENDOTHECIUM 1 (PsEND1) is a pea anther-specific gene that encodes a protein containing four hemopexin domains. We report the functional characterization of PsEND1 and the identification in its promoter region of cis-regulatory elements that are essential for the specific expression in anthers. PsEND1 promoter deletion analysis revealed that a putative CArG-like regulatory motif is necessary to confer promoter activity in developing anthers. Our data suggest that PsEND1 might be a hemopexin regulated by a MADS-box protein. PsEND1 gene silencing in pea, and its overexpression in heterologous systems, result in similar defects in the anthers consisting of precocious tapetum degradation and the impairment of pollen development. Such alterations were associated to the production of superoxide anion and altered activity of ROS-scavenging enzymes. Our findings demonstrate that PsEND1 is essential for pollen development by modulating ROS levels during the differentiation of the anther tissues surrounding the microsporocytes.

Isolation and Functional Analysis of a PISTILLATA-like MADS-Box Gene from Argan Tree (Argania spinosa).

Argan trees (Argania spinosa) belong to a species native to southwestern Morocco, playing an important role in the environment and local economy. Argan oil extracted from kernels has a unique composition and properties. Argan trees were introduced in Tunisia, where hundreds of trees can be found nowadays. In this study, we examined reproductive development in Argan trees from four sites in Tunisia and carried out the functional characterization of a floral homeotic gene in this non-model species. Despite the importance of reproductive development, nothing is known about the genetic network controlling flower development in Argania spinosa. Results obtained in several plant species established that floral organ development is mostly controlled by MADS-box genes and, in particular, APETALA3 (AP3) and PISTILLATA (PI) homologs are required for proper petal and stamen identity. Here, we describe the isolation and functional characterization of a MADS-box gene from Argania spinosa. Phylogenetic analyses showed strong homology with PI-like proteins, and the expression of the gene was found to be restricted to the second and third whorls. Functional homology with Arabidopsis PI was demonstrated by the ability of AsPI to confer petal and stamen identity when overexpressed in a pi-1 mutant background. The identification and characterization of this gene support the strong conservation of PI homologs among distant angiosperm plants.

MtSUPERMAN plays a key role in compound inflorescence and flower development in Medicago truncatula.

Legumes have unique features, such as compound inflorescences and a complex floral ontogeny. Thus, the study of regulatory genes in these species during inflorescence and floral development is essential to understand their role in the evolutionary origin of developmental novelties. The SUPERMAN (SUP) gene encodes a C2H2 zinc-finger transcriptional repressor that regulates the floral organ number in the third and fourth floral whorls of Arabidopsis thaliana. In this work, we present the functional characterization of the Medicago truncatula SUPERMAN (MtSUP) gene based on gene expression analysis, complementation and overexpression assays, and reverse genetic approaches. Our findings provide evidence that MtSUP is the orthologous gene of SUP in M. truncatula. We have unveiled novel functions for a SUP-like gene in eudicots. MtSUP controls not only the number of floral organs in the inner two whorls, but also in the second whorl of the flower. Furthermore, MtSUP regulates the activity of the secondary inflorescence meristem, thus controlling the number of flowers produced. Our work provides insight into the regulatory network behind the compound inflorescence and flower development in this angiosperm family.

Engineered Male Sterility by Early Anther Ablation Using the Pea Anther-Specific Promoter PsEND1.

Genetic engineered male sterility has different applications, ranging from hybrid seed production to bioconfinement of transgenes in genetic modified crops. The impact of this technology is currently patent in a wide range of crops, including legumes, which has helped to deal with the challenges of global food security. Production of engineered male sterile plants by expression of a ribonuclease gene under the control of an anther- or pollen-specific promoter has proven to be an efficient way to generate pollen-free elite cultivars. In the last years, we have been studying the genetic control of flower development in legumes and several genes that are specifically expressed in a determinate floral organ were identified. Pisum sativum ENDOTHECIUM 1 (PsEND1) is a pea anther-specific gene displaying very early expression in the anther primordium cells. This expression pattern has been assessed in both model plants and crops (tomato, tobacco, oilseed rape, rice, wheat) using genetic constructs carrying the PsEND1 promoter fused to the uidA reporter gene. This promoter fused to the barnase gene produces full anther ablation at early developmental stages, preventing the production of mature pollen grains in all plant species tested. Additional effects produced by the early anther ablation in the PsEND1::barnase-barstar plants, with interesting biotechnological applications, have also been described, such as redirection of resources to increase vegetative growth, reduction of the need for deadheading to extend the flowering period, or elimination of pollen allergens in ornamental plants (Kalanchoe, Pelargonium). Moreover, early anther ablation in transgenic PsEND1::barnase-barstar tomato plants promotes the developing of the ovaries into parthenocarpic fruits due to the absence of signals generated during the fertilization process and can be considered an efficient tool to promote fruit set and to produce seedless fruits. In legumes, the production of new hybrid cultivars will contribute to enhance yield and productivity by exploiting the hybrid vigor generated. The PsEND1::barnase-barstar construct could be also useful to generate parental lines in hybrid breeding approaches to produce new cultivars in different legume species.

The DOF Transcription Factor SlDOF10 Regulates Vascular Tissue Formation During Ovary Development in Tomato.

The formation of fruits is an important step in the life cycle of flowering plants. The process of fruit development is highly regulated and involves the interaction of a complex regulatory network of genes in both space and time. To identify regulatory genes involved in fruit initiation in tomato we analyzed the transcriptomic profile of ovaries from the parthenocarpic PsEND1:barnase transgenic line. This line was generated using the cytotoxic gene barnase targeted to the anthers with the PsEND1 anther-specific promoter from pea. Among the differentially expressed genes we identified SlDOF10, a gene coding a DNA-binding with one finger (DOF) transcription factor which is activated in unpollinated ovaries of the parthenocarpic plants. SlDOF10 is preferentially expressed in the vasculature of the cotyledons and young leaves and in the root tip. During floral development, expression is visible in the vascular tissue of the sepals, the flower pedicel and in the ovary connecting the placenta with the developing ovules. The induction of the gene was observed in response to exogenous gibberellins and auxins treatments. To evaluate the gene function during reproductive development, we have generated SlDOF10 overexpressing and silencing stable transgenic lines. In particular, down-regulation of SlDOF10 activity led to a decrease in the area occupied by individual vascular bundles in the flower pedicel. Associated with this phenotype we observed induction of parthenocarpic fruit set. In summary, expression and functional analyses revealed a role for SlDOF10 gene in the development of the vascular tissue specifically during reproductive development highlighting the importance of this tissue in the process of fruit set.

Non-isotopic RNA In Situ Hybridization for Functional Analyses in Medicago truncatula.

Different strategies have been developed and implemented during the last decades aiming to decipher the function of particular genes. Among the different techniques, in situ hybridization of mRNA remains an essential experiment to fully understand gene function. Here, we describe a protocol for the in situ localization of gene transcripts in plants. It is optimized for use of paraffin-embedded tissues and DIG-labeled probes and has successfully applied to floral bud tissues from Medicago truncatula. Using this protocol, we have analyzed the expression of MADS-box transcription factors where some of them have been preserved as duplicates in the genome. When duplicated genes are analyzed, the tissue and cellular location of the transcripts is the only technique that accounts for small variations in the pattern of gene expression that occurred after duplication and diversification. The use of a well-standardized in situ hybridization protocol is vital for the systematic analysis of the function of genes in Medicago truncatula.

Functional Genomics and Genetic Control of Flower and Fruit Development in Medicago truncatula: An Overview.

A-, B-, and C-class genes code for MADS-box transcription factors required for floral organ identity in angiosperms. Other members of the family are also crucial to ensure proper carpel and fruit development. Development of genetic and genomic tools for Medicago truncatula has allowed its use as model system to study the genetic control of flower and fruit development in legumes. M. truncatula contains a single A-class gene, four B-function genes, and three C-class genes in its genome. This has made possible to do extensive functional characterization of these MADS-box transcription factors using gene expression analyses, protein-protein interactions, and forward and reverse genetic approaches. We have demonstrated the functions of these MADS-box transcription factors and the respective contributions of paralogous gene pairs to M. truncatula floral development. We have also defined the evolutionary outcomes of each duplicated pairs thus testing theoretical framework of several models about the evolution by gene duplication. Moreover, we have also studied the function of MADS-box fruit genes and how they may have contributed to the diversification of pod morphology within the Medicago genus. Our findings not only have contributed to increase knowledge in the field of the genetic control of flower and fruit development but also have provided a more complete understanding of the complexity of evolution by gene duplication and protein sequence diversification.

Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp.

Proanthocyanidins (PAs), or condensed tannins, are powerful antioxidants that remove harmful free oxygen radicals from cells. To engineer the anthocyanin and proanthocyanidin biosynthetic pathways to de novo produce PAs in two Nicotiana species, we incorporated four transgenes to the plant chassis. We opted to perform a simultaneous transformation of the genes linked in a multigenic construct rather than classical breeding or retransformation approaches. We generated a GoldenBraid 2.0 multigenic construct containing two Antirrhinum majus transcription factors (AmRosea1 and AmDelila) to upregulate the anthocyanin pathway in combination with two Medicago truncatula genes (MtLAR and MtANR) to produce the enzymes that will derivate the biosynthetic pathway to PAs production. Transient and stable transformation of Nicotiana benthamiana and Nicotiana tabacum with the multigenic construct were respectively performed. Transient expression experiments in N. benthamiana showed the activation of the anthocyanin pathway producing a purple color in the agroinfiltrated leaves and also the effective production of 208.5 nmol (-) catechin/g FW and 228.5 nmol (-) epicatechin/g FW measured by the p-dimethylaminocinnamaldehyde (DMACA) method. The integration capacity of the four transgenes, their respective expression levels and their heritability in the second generation were analyzed in stably transformed N. tabacum plants. DMACA and phoroglucinolysis/HPLC-MS analyses corroborated the activation of both pathways and the effective production of PAs in T0 and T1 transgenic tobacco plants up to a maximum of 3.48 mg/g DW. The possible biotechnological applications of the GB2.0 multigenic approach in forage legumes to produce "bloat-safe" plants and to improve the efficiency of conversion of plant protein into animal protein (ruminal protein bypass) are discussed.

The parthenocarpic hydra mutant reveals a new function for a SPOROCYTELESS-like gene in the control of fruit set in tomato.

Fruit set is an essential process to ensure successful sexual plant reproduction. The development of the flower into a fruit is actively repressed in the absence of pollination. However, some cultivars from a few species are able to develop seedless fruits overcoming the standard restriction of unpollinated ovaries to growth. We report here the identification of the tomato hydra mutant that produces seedless (parthenocarpic) fruits. Seedless fruit production in hydra plants is linked to the absence of both male and female sporocyte development. The HYDRA gene is therefore essential for the initiation of sporogenesis in tomato. Using positional cloning, virus-induced gene silencing and expression analysis experiments, we identified the HYDRA gene and demonstrated that it encodes the tomato orthologue of SPOROCYTELESS/NOZZLE (SPL/NZZ) of Arabidopsis. We found that the precocious growth of the ovary is associated with changes in the expression of genes involved in gibberellin (GA) metabolism. Our results support the conservation of the function of SPL-like genes in the control of sporogenesis in plants. Moreover, this study uncovers a new function for the tomato SlSPL/HYDRA gene in the control of fruit initiation.

Evolution by gene duplication of Medicago truncatula PISTILLATA-like transcription factors.

PISTILLATA (PI) is a member of the B-function MADS-box gene family, which controls the identity of both petals and stamens in Arabidopsis thaliana. In Medicago truncatula (Mt), there are two PI-like paralogs, known as MtPI and MtNGL9. These genes differ in their expression patterns, but it is not known whether their functions have also diverged. Describing the evolution of certain duplicated genes, such as transcription factors, remains a challenge owing to the complex expression patterns and functional divergence between the gene copies. Here, we report a number of functional studies, including analyses of gene expression, protein-protein interactions, and reverse genetic approaches designed to demonstrate the respective contributions of each M. truncatula PI-like paralog to the B-function in this species. Also, we have integrated molecular evolution approaches to determine the mode of evolution of Mt PI-like genes after duplication. Our results demonstrate that MtPI functions as a master regulator of B-function in M. truncatula, maintaining the overall ancestral function, while MtNGL9 does not seem to have a role in this regard, suggesting that the pseudogenization could be the functional evolutionary fate for this gene. However, we provide evidence that purifying selection is the primary evolutionary force acting on this paralog, pinpointing the conservation of its biochemical function and, alternatively, the acquisition of a new role for this gene.

Two euAGAMOUS genes control C-function in Medicago truncatula.

C-function MADS-box transcription factors belong to the AGAMOUS (AG) lineage and specify both stamen and carpel identity and floral meristem determinacy. In core eudicots, the AG lineage is further divided into two branches, the euAG and PLE lineages. Functional analyses across flowering plants strongly support the idea that duplicated AG lineage genes have different degrees of subfunctionalization of the C-function. The legume Medicago truncatula contains three C-lineage genes in its genome: two euAG genes (MtAGa and MtAGb) and one PLENA-like gene (MtSHP). This species is therefore a good experimental system to study the effects of gene duplication within the AG subfamily. We have studied the respective functions of each euAG genes in M. truncatula employing expression analyses and reverse genetic approaches. Our results show that the M. truncatula euAG- and PLENA-like genes are an example of subfunctionalization as a result of a change in expression pattern. MtAGa and MtAGb are the only genes showing a full C-function activity, concomitant with their ancestral expression profile, early in the floral meristem, and in the third and fourth floral whorls during floral development. In contrast, MtSHP expression appears late during floral development suggesting it does not contribute significantly to the C-function. Furthermore, the redundant MtAGa and MtAGb paralogs have been retained which provides the overall dosage required to specify the C-function in M. truncatula.

PsPMEP, a pollen-specific pectin methylesterase of pea (Pisum sativum L.).

Pectin methylesterases (PMEs) are a family of enzymes involved in plant reproductive processes such as pollen development and pollen tube growth. We have isolated and characterized PsPMEP, a pea (Pisum sativum L.) pollen-specific gene that encodes a protein with homology to PMEs. Sequence analysis showed that PsPMEP belongs to group 2 PMEs, which are characterized by the presence of a processable amino-terminal PME inhibitor domain followed by the catalytic PME domain. Moreover, PsPMEP contains several motifs highly conserved among PMEs with the essential amino acid residues involved in enzyme substrate binding and catalysis. Northern blot and in situ hybridization analyses showed that PsPMEP is expressed in pollen grains from 4 days before anthesis till anther dehiscence and in pollinated carpels. In the PsPMEP promoter region, we have identified several conserved cis-regulatory elements that have been associated with gene pollen-specific expression. Expression analysis of PsPMEP promoter fused to the uidA reporter gene in Arabidopsis thaliana plants showed a similar expression pattern when compared with pea, indicating that this promoter is also functional in a non-leguminous plant. GUS expression was detected in mature pollen grains, during pollen germination, during pollen tube elongation along the transmitting tract, and when the pollen tube reaches the embryo sac in the ovule.

Early anther ablation triggers parthenocarpic fruit development in tomato.

Fruit set and fruit development in tomato is largely affected by changes in environmental conditions, therefore autonomous fruit set independent of fertilization is a highly desirable trait in tomato. Here, we report the production and characterization of male-sterile transgenic plants that produce parthenocarpic fruits in two tomato cultivars (Micro-Tom and Moneymaker). We generated male-sterility using the cytotoxic gene barnase targeted to the anthers with the PsEND1 anther-specific promoter. The ovaries of these plants grew in the absence of fertilization producing seedless, parthenocarpic fruits. Early anther ablation is essential to trigger the developing of the transgenic ovaries into fruits, in the absence of the signals usually generated during pollination and fertilization. Ovaries are fully functional and can be manually pollinated to obtain seeds. The transgenic plants obtained in the commercial cultivar Moneymaker show that the parthenocarpic development of the fruit does not have negative consequences in fruit quality. Throughout metabolomic analyses of the tomato fruits, we have identified two elite lines which showed increased levels of several health promoting metabolites and volatile compounds. Thus, early anther ablation can be considered a useful tool to promote fruit set and to obtain seedless and good quality fruits in tomato plants. These plants are also useful parental lines to be used in hybrid breeding approaches.

Functional specialization of duplicated AP3-like genes in Medicago truncatula.

The B-class of MADS box genes has been studied in a wide range of plant species, but has remained largely uncharacterized in legumes. Here we investigate the evolutionary fate of the duplicated AP3-like genes of a legume species. To obtain insight into the extent to which B-class MADS box gene functions are conserved or have diversified in legumes, we isolated and characterized the two members of the AP3 lineage in Medicago truncatula: MtNMH7 and MtTM6 (euAP3 and paleoAP3 genes, respectively). A non-overlapping and complementary expression pattern of both genes was observed in petals and stamens. MtTM6 was expressed predominantly in the outer cell layers of both floral organs, and MtNMH7 in the inner cell layers of petals and stamens. Functional analyses by reverse genetics approaches (RNAi and Tnt1 mutagenesis) showed that the contribution of MtNMH7 to petal identity is more important than that of MtTM6, whereas MtTM6 plays a more important role in stamen identity than its paralog MtNMH7. Our results suggest that the M. truncatula AP3-like genes have undergone a functional specialization process associated with complete partitioning of gene expression patterns of the ancestral gene lineage. We provide information regarding the similarities and differences in petal and stamen development among core eudicots.

Production of engineered long-life and male sterile Pelargonium plants.

BACKGROUND: Pelargonium is one of the most popular garden plants in the world. Moreover, it has a considerable economic importance in the ornamental plant market. Conventional cross-breeding strategies have generated a range of cultivars with excellent traits. However, gene transfer via Agrobacterium tumefaciens could be a helpful tool to further improve Pelargonium by enabling the introduction of new genes/traits. We report a simple and reliable protocol for the genetic transformation of Pelargonium spp. and the production of engineered long-life and male sterile Pelargonium zonale plants, using the pSAG12::ipt and PsEND1::barnase chimaeric genes respectively. RESULTS: The pSAG12::ipt transgenic plants showed delayed leaf senescence, increased branching and reduced internodal length, as compared to control plants. Leaves and flowers of the pSAG12::ipt plants were reduced in size and displayed a more intense coloration. In the transgenic lines carrying the PsEND1::barnase construct no pollen grains were observed in the modified anther structures, which developed instead of normal anthers. The locules of sterile anthers collapsed 3-4 days prior to floral anthesis and, in most cases, the undeveloped anther tissues underwent necrosis. CONCLUSION: The chimaeric construct pSAG12::ipt can be useful in Pelargonium spp. to delay the senescence process and to modify plant architecture. In addition, the use of engineered male sterile plants would be especially useful to produce environmentally friendly transgenic plants carrying new traits by preventing gene flow between the genetically modified ornamentals and related plant species. These characteristics could be of interest, from a commercial point of view, both for pelargonium producers and consumers.

Efficient transformation of Kalanchoe blossfeldiana and production of male-sterile plants by engineered anther ablation.

Engineered male sterility in ornamental plants has many applications such as facilitate hybrid seed production, eliminate pollen allergens, reduce the need for deadheading to extend the flowering period, redirect resources from seeds to vegetative growth, increase flower longevity and prevent gene flow between genetically modified and related native plants. We have developed a reliable and efficient Agrobacterium-mediated protocol for the genetic transformation of different Kalanchoe blossfeldiana commercial cultivars. Transformation efficiency for cv. 'Hillary' was 55.3% whereas that of cv. 'Tenorio' reached 75.8%. Selection was carried out with the nptII gene and increasing the kanamycin concentration from 25 to 100 mg l(-1) allowed to reduced escapes from 50 to 60% to virtually 0%. This method was used to produce male-sterile plants through engineered anther ablation. In our approach, we tested a male sterility chimaeric gene construct (PsEND1::barnase) to evaluate its effectiveness and effect on phenotype. No significant differences were found in the growth patterns between the transgenic lines and the wild-type plants. No viable pollen grains were observed in the ablated anthers of any of the lines carrying the PsEND1::barnase construct, indicating that the male sterility was complete. In addition, seed set was completely abolished in all the transgenic plants obtained. Our engineered male-sterile approach could be used, alone or in combination with a female-sterility system, to reduce the invasive potential of new ornamentals, which has become an important environmental problem in many countries.

Analysis of B function in legumes: PISTILLATA proteins do not require the PI motif for floral organ development in Medicago truncatula.

The B-class gene PISTILLATA (PI) codes for a MADS-box transcription factor required for floral organ identity in angiosperms. Unlike Arabidopsis, it has been suggested that legume PI genes contribute to a variety of processes, such as the development of floral organs, floral common petal-stamen primordia, complex leaves and N-fixing root nodules. Another interesting feature of legume PI homologues is that some of them lack the highly conserved C-terminal PI motif suggested to be crucial for function. Therefore, legume PI genes are useful for addressing controversial questions on the evolution of B-class gene function, including how they may have diverged in both function and structure to affect different developmental processes. However, functional analysis of legume PI genes has been hampered because no mutation in any B-class gene has been identified in legumes. Here we fill this gap by studying the PI function in the model legume species Medicago truncatula using mutant and RNAi approaches. Like other legume species, M. truncatula has two PI homologues. The expression of the two genes, MtPI and MtNGL9, has strongly diverged, suggesting differences in function. Our analyses show that these genes are required for petal and stamen identity, where MtPI appears to play a predominant role. However, they appear not to be required for development of the nodule, the common primordia or the complex leaf. Moreover, both M. truncatula PI homologues lack the PI motif, which indicates that the C-terminal motif is not essential for PI activity.