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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., (© 2024 The Author(s). Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Rodas, Roque, Hamza, Gómez-Mena, Beltrán and Cañas.)

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., (© 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hamza, Roque, Gómez-Mena, Madueño, Beltrán and Cañas.)

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., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Key Message: The early flowering system HSP::AtFT allowed a fast evaluation of a gene containment system based on the construct PsEND1::barnase-barstar for poplar. Transgenic lines showed disturbed pollen development and sterility. Vertical gene transfer through pollen flow from transgenic or non-native plant species into their crossable natural relatives is a major concern. Gene containment approaches have been proposed to reduce or even avoid gene flow among tree species. However, evaluation of genetic containment strategies for trees is very difficult due to the long-generation times. Early flowering induction would allow faster evaluation of genetic containment in this case. Although no reliable methods were available for the induction of fertile flowers in poplar, recently, a new early flowering approach was developed. In this study, early flowering poplar lines containing the gene construct PsEND1::barnase-barstar were obtained. The PsEND1 promoter was chosen due to its early expression pattern, its versality and efficiency for generation of male-sterile plants fused to the barnase gene. RT-PCRs confirmed barnase gene activity in flowers, and pollen development was disturbed, leading to sterile flowers. The system developed in this study represents a valuable tool for gene containment studies in forest tree species.

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 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.

Enzymes play a key role in insect-plant relationships. For a better understanding of these interactions, we analyzed Tuta absoluta digestive enzymes. Here, we describe a detailed protocol for the detection of trypsin and papain-like enzymes in Tuta absoluta larvae by enzyme histochemistry. This assay uses frozen and unfixed samples to avoid the loss of enzymatic activity. We also describe a protocol for the quantification of trypsin and papain-like enzymes in the larvae of Tuta absoluta at different developmental instars., Competing Interests: Competing interestsThe authors declare that there are no conflicts of interest related with this work., (Copyright © 2018 The Authors; exclusive licensee Bio-protocol LLC.)

Background: Plants and insects have coexisted for million years and evolved a set of interactions which affect both organisms at different levels. Plants have developed various morphological and biochemical adaptations to cope with herbivores attacks. However, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) has become the major pest threatening tomato crops worldwide and without the appropriated management it can cause production losses between 80 to 100%., Results: The aim of this study was to investigate the in vivo effect of a serine proteinase inhibitor (BTI-CMe) and a cysteine proteinase inhibitor (Hv-CPI2) from barley on this insect and to examine the effect their expression has on tomato defensive responses. We found that larvae fed on tomato transgenic plants co-expressing both proteinase inhibitors showed a notable reduction in weight. Moreover, only 56% of these larvae reached the adult stage. The emerged adults showed wings deformities and reduced fertility. We also investigated the effect of proteinase inhibitors ingestion on the insect digestive enzymes. Our results showed a decrease in larval trypsin activity. Transgenes expression had no harmful effect on Nesidiocoris tenuis (Reuter) (Heteroptera: Miridae), a predator of Tuta absoluta, despite transgenic tomato plants attracted the mirid. We also found that barley cystatin expression promoted plant defense by inducing the expression of the tomato endogenous wound inducible Proteinase inhibitor 2 (Pin2) gene, increasing the production of glandular trichomes and altering the emission of volatile organic compounds., Conclusion: Our results demonstrate the usefulness of the co-expression of different proteinase inhibitors for the enhancement of plant resistance to Tuta absoluta.

Many researchers have sought along the last two decades a legume species that could serve as a model system for genetic studies to resolve specific developmental or metabolic processes that cannot be studied in other model plants. Nitrogen fixation, nodulation, compound leaf, inflorescence and plant architecture, floral development, pod formation, secondary metabolite biosynthesis, and other developmental and metabolic aspects are legume-specific or show important differences with those described in Arabidopsis thaliana, the most studied model plant. Mainly Medicago truncatula and Lotus japonicus were proposed in the 1990s as model systems due to their key attributes, diploid genome, autogamous nature, short generation times, small genome sizes, and both species can be readily transformed. After more than decade-long, the genome sequences of both species are essentially complete, and a series of functional genomics tools have been successfully developed and applied. Mutagens that cause insertions or deletions are being used in these model systems because these kinds of DNA rearrangements are expected to assist in the isolation of the corresponding genes by Target-Induced Local Lesions IN Genomes (TILLING) approaches. Different M. truncatula mutants have been obtained following γ-irradiation or fast neutron bombardment (FNB), ethyl-nitrosourea (ENU) or ethyl-methanesulfonate (EMS) treatments, T-DNA and activation tagging, use of the tobacco retrotransposon Tnt1 to produce insertional mutants, gene silencing by RNAi, and transient post-transcriptional gene silencing by virus-induced gene silencing (VIGS). Emerging technologies of targeted mutagenesis and gene editing, such as the CRISPR-Cas9 system, could open a new era in this field. Functional genomics tools and phenotypic analyses of several mutants generated in M. truncatula have been essential to better understand differential aspects of legumes development and metabolism.

Humanity faces great challenges with respect to the use of energy, the production of food and feed, and the management of the Earth through sustainable practices. Agriculture can play a key role to give appropriate responses to these challenges. By the end of this century, human population will grow up to around 10,000 million people, meaning we must be able to produce food and feed for more than an additional number of 3300 million people. Legumes together with cereals have been combined to produce healthy food along the history of agriculture in all geographical areas of the planet. However, recently, the use of legumes, mainly in the developed countries, has been neglected therefore compromising human health and sustainable production of food and feed. Agronomy has always been driven by technology and innovation. The development of genomic tools in legume model systems such as Medicago truncatula will allow to make progress into the knowledge of critical processes of legumes biology such as nitrogen fixation, including the mechanisms controlling nodulation through soil nitrogen sensing, drought and flooding tolerances or the understanding of key factors governing the vegetative development of legumes, the control of inflorescences architecture or floral transition, and fruit set and seed development and composition. Traditional breeding combined with genome editing techniques will drive the production of grain and forage legume varieties for the future.

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.

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.

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., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Most predator-prey models extrapolate functional responses from small-scale experiments assuming spatially uniform within-plant predator-prey interactions. However, some predators focus their search in certain plant regions, and herbivores tend to select leaves to balance their nutrient uptake and exposure to plant defenses. Individual-based models that account for heterogeneous within-plant predator-prey interactions can be used to scale-up functional responses, but they would require the generation of explicit prey spatial distributions within-plant architecture models. The silverleaf whitefly, Bemisia tabaci biotype B (Gennadius) (Hemiptera: Aleyrodidae), is a significant pest of tomato crops worldwide that exhibits highly aggregated populations at several spatial scales, including within the plant. As part of an analytical framework to understand predator-silverleaf whitefly interactions, the objective of this research was to develop an algorithm to generate explicit spatial counts of silverleaf whitefly nymphs within tomato plants. The algorithm requires the plant size and the number of silverleaf whitefly individuals to distribute as inputs, and includes models that describe infestation probabilities per leaf nodal position and the aggregation pattern of the silverleaf whitefly within tomato plants and leaves. The output is a simulated number of silverleaf whitefly individuals for each leaf and leaflet on one or more plants. Parameter estimation was performed using nymph counts per leaflet censused from 30 artificially infested tomato plants. Validation revealed a substantial agreement between algorithm outputs and independent data that included the distribution of counts of both eggs and nymphs. This algorithm can be used in simulation models that explore the effect of local heterogeneity on whitefly-predator dynamics., (© The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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.

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., (© 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.)

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.

The soybean aphid, Aphis glycines Matsumura, is a pest of cultivated soybean, Glycine max (L.) Merr., in North America. Recent developments in host plant resistance studies have identified at least four soybean aphid resistance genes (Rag1-4) and two soybean aphid biotypes (biotype 1 and 2), defined by differential survivability on resistant soybean. Detached soybean leaves were tested as a more rapid and practical assay to assess host plant resistance and virulence. Two susceptible lines ('Wyandot' and 'Williams 82') and two resistant lines (PI 243540 and PI 567301B) were examined. Various life history characteristics were compared among aphids on whole plants and detached leaves. Results indicated that resistance to soybean aphid was lost using detached leaves of PI 567301B but retained with PI 243540. To test for aphid virulence, net fecundities were compared among biotype 1 and biotype 2 after rearing on detached leaves of the resistant 'Jackson' (to which biotype 2 is virulent). A significant difference was detected in net fecundities among biotypes on detached leaves of Jackson and used to predict growth rates and virulence from 30 field-collected individuals of unknown virulence. No field individuals matched biotype 2 predictions, but four individuals had higher net fecundities than biotype 2 predictions (13%) and could be considered moderately virulent. The results indicated that the retention of soybean aphid resistance in detached leaves is dependent on PI and resistant source, but if resistance is retained, detached leaves could be used to determine soybean aphid virulence.

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.

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.

PsEND1 is a pea anther-specific gene that displays very early expression in the anther primordium cells. Later on, PsEND1 expression becomes restricted to the epidermis, connective, endothecium and middle layer, but it is never observed in tapetal cells or microsporocytes. We fused the PsEND1 promoter region to the cytotoxic barnase gene to induce specific ablation of the cell layers where the PsEND1 is expressed and consequently to produce male-sterile plants. Expression of the chimaeric PsEND1::barnase gene in two Solanaceae (Nicotiana tabacum and Solanum lycopersicon) and two Brassicaceae (Arabidopsis thaliana and Brassica napus) species, impairs anther development from very early stages and produces complete male-sterile plants. The PsEND1::barnase gene is quite different to other chimaeric genes previously used in similar approaches to obtain male-sterile plants. The novelty resides in the use of the PsEND1 promoter, instead of a tapetum-specific promoter, to produce the ablation of specific cell lines during the first steps of the anther development. This chimaeric construct arrests the microsporogenesis before differentiation of the microspore mother cells and no viable pollen grains are produced. This strategy represents an excellent alternative to generate genetically engineered male-sterile plants, which have proved useful in breeding programmes for the production of hybrid seeds. The PsEND1 promoter also has high potential to prevent undesirable horizontal gene flow in many plant species.

Comparative studies help shed light on how the huge diversity in plant forms found in nature has been produced. We use legume species to study developmental differences in inflorescence architecture and flower ontogeny with classical models such as Arabidopsis thaliana or Antirrhinum majus. Whereas genetic control of these processes has been analyzed mostly in pea (Pisum sativum), Medicago truncatula is emerging as a promising alternative system for these studies due to the availability of a range of genetic tools. To assess the use of the retrotransposon Tnt1 for reverse genetics in M. truncatula, we screened a small Tnt1-mutagenized population using degenerate primers for MADS-box genes, known controllers of plant development. We describe here the characterization of mtpim, a new mutant caused by the insertion of Tnt1 in a homolog to the PROLIFERATING INFLORESCENCE MERISTEM (PIM)/APETALA1 (AP1)/SQUAMOSA genes. mtpim shows flower-to-inflorescence conversion and altered flowers with sepals transformed into leaves, indicating that MtPIM controls floral meristem identity and flower development. Although more extreme, this phenotype resembles the pea pim mutants, supporting the idea that M. truncatula could be used to complement analysis of reproductive development already initiated in pea. In fact, our study reveals aspects not shown by analysis of pea mutants: that the mutation in the AP1 homolog interferes with the specification of floral organs from common primordia and causes conversion of sepals into leaves, in addition to true conversion of flowers into inflorescences. The isolation of mtpim represents a proof of concept demonstrating that Tnt1 populations can be efficiently used in reverse genetics screenings in M. truncatula.

Current understanding of floral development is mainly based on what we know from Arabidopsis (Arabidopsis thaliana) and Antirrhinum majus. However, we can learn more by comparing developmental mechanisms that may explain morphological differences between species. A good example comes from the analysis of genes controlling flower development in pea (Pisum sativum), a plant with more complex leaves and inflorescences than Arabidopsis and Antirrhinum, and a different floral ontogeny. The analysis of UNIFOLIATA (UNI) and STAMINA PISTILLOIDA (STP), the pea orthologs of LEAFY and UNUSUAL FLORAL ORGANS, has revealed a common link in the regulation of flower and leaf development not apparent in Arabidopsis. While the Arabidopsis genes mainly behave as key regulators of flower development, where they control the expression of B-function genes, UNI and STP also contribute to the development of the pea compound leaf. Here, we describe the characterization of P. sativum PISTILLATA (PsPI), a pea MADS-box gene homologous to B-function genes like PI and GLOBOSA (GLO), from Arabidopsis and Antirrhinum, respectively. PsPI encodes for an atypical PI-type polypeptide that lacks the highly conserved C-terminal PI motif. Nevertheless, constitutive expression of PsPI in tobacco (Nicotiana tabacum) and Arabidopsis shows that it can specifically replace the function of PI, being able to complement the strong pi-1 mutant. Accordingly, PsPI expression in pea flowers, which is dependent on STP, is identical to PI and GLO. Interestingly, PsPI is also transiently expressed in young leaves, suggesting a role of PsPI in pea leaf development, a possibility that fits with the established role of UNI and STP in the control of this process.

END1 was isolated by an immunosubtractive approach intended to identify specific proteins present in the different pea (Pisum sativum L.) floral organs and the genes encoding them. Following this strategy we obtained a monoclonal antibody (mAbA1) that specifically recognized a 26-kDa protein (END1) only detected in anther tissues. Northern blot assays showed that END1 is expressed specifically in the anther. In situ hybridization and immunolocalization assays corroborated the specific expression of END1 in the epidermis, connective, endothecium and middle layer cells during the different stages of anther development. END1 is the first anther-specific gene isolated from pea. The absence of a practicable pea transformation method together with the fact that no END1 homologue gene exists in Arabidopsis prevented us from carrying out END1 functional studies. However, we designed functional studies with the END1 promoter in different dicot species, as the specific spatial and temporal expression pattern of END1 suggested, among other things, the possibility of using its promoter region for biotechnological applications. Using different constructs to drive the uidA (beta-glucuronidase) gene controlled by the 2.7-kb isolated promoter sequence we have proven that the END1 promoter is fully functional in the anthers of transgenic Arabidopsis thaliana (L.) Heynh., Nicotiana tabacum L. (tobacco) and Lycopersicon esculentum Mill. (tomato) plants. The presence in the -330-bp region of the promoter sequence of three putative CArG boxes also suggests that END1 could be a target gene of MADS-box proteins and that, subsequently, it would be activated by genes controlling floral organ identity.

Plants are commonly attacked by more than one species of herbivore, potentially causing the induction of multiple, and possibly competing, plant defense systems. In the present paper, we determined the interaction between feeding by the phloem feeder silverleaf whitefly (SWF), Bemisia tabaci Gennadius (B-biotype = B. argentifolii Bellows and Perring), and the leaf-chewing beet armyworm (BAW), Spodoptera exigua Hübner, with regard to the induction of volatile compounds from cotton plants. Compared to undamaged control plants, infestation with SWF did not induce volatile emissions or affect the number and density of pigment glands that store volatile and nonvolatile terpenoid compounds, whereas infestation by BAW strongly induced plant volatile emission. When challenged by the two insect herbivores simultaneously, volatile emission was significantly less than for plants infested with only BAW. Our results suggest that tritrophic level interactions between cotton, BAW, and natural enemies of BAW, that are known to be mediated by plant volatile emissions, may be perturbed by simultaneous infestation by SWF. Possible mechanisms by which the presence of whiteflies may attenuate volatile emissions from caterpillar-damaged cotton plants are discussed.

APETALA1 (AP1) and its homologue SQUAMOSA (SQUA) are key regulatory genes specifying floral meristem identity in the model plants Arabidopsis and Antirrhinum. Despite many similarities in their sequence, expression and functions, only AP1 appears to have the additional role of specifying sepal and petal identity. No true AP1/SQUA-functional homologues from any other plant species have been functionally studied in detail, therefore the question of how the different functions of AP1-like genes are conserved between species has not been addressed. We have isolated and characterized PEAM4, the AP1/SQUA-functional homologue from pea, a plant with a different floral morphology and inflorescence architecture to that of Arabidopsis or Antirrhinum. PEAM4 encodes for a polypeptide 76% identical to AP1, but lacks the C-terminal prenylation motif, common to AP1 and SQUA, that has been suggested to control the activity of AP1. Nevertheless, constitutive expression of PEAM4 caused early flowering in tobacco and Arabidopsis. In Arabidopsis, PEAM4 also caused inflorescence-to-flower transformations similar to constitutive AP1 expression, and was able to rescue the floral organ defects of the strong ap1-1 mutant. Our results suggest that the control of both floral meristem and floral organ identity by AP1 is not restricted to Arabidopsis, but is extended to species with diverse floral morphologies, such as pea.

Protoplasts from Olea europaea L. have been compared in terms of their yield, viability, cell division and callus differentiation. Viable protoplasts were isolated from in vitro cultured leaves and cotyledons by an overnight incubation in an enzyme solution containing 1-1.5% driselase and 0.5M sucrose. This method allowed high yield of purified protoplasts, which floated and formed a dark green band at the meniscus, after centrifugation. Purified protoplasts were diluted to 3×10(4) protoplasts·ml(-1) in culture medium. After cell wall regeneration, protoplasts gradually increased their volumes under appropriate conditions. The first divisions occurred during the second week in culture. Division efficiency ranged from 5.2 to 9.8% after 20 days in culture. Two weeks later visible microcolonies developed only from cotyledon protoplasts. After 6 weeks in culture, the microcalli were transferred to a solidified culture medium with 0.6% agarose, which induced active callus growth.