Recent Progress Regarding the Molecular Aspects of Insect Gall Formation.

Galls are characteristic plant structures formed by cell size enlargement and/or cell proliferation induced by parasitic or pathogenic organisms. Insects are a major inducer of galls, and insect galls can occur on plant leaves, stems, floral buds, flowers, fruits, or roots. Many of these exhibit unique shapes, providing shelter and nutrients to insects. To form unique gall structures, gall-inducing insects are believed to secrete certain effector molecules and hijack host developmental programs. However, the molecular mechanisms of insect gall induction and development remain largely unknown due to the difficulties associated with the study of non-model plants in the wild. Recent advances in next-generation sequencing have allowed us to determine the biological processes in non-model organisms, including gall-inducing insects and their host plants. In this review, we first summarize the adaptive significance of galls for insects and plants. Thereafter, we summarize recent progress regarding the molecular aspects of insect gall formation.

A new galling insect model enhances photosynthetic activity in an obligate holoparasitic plant.

Insect-induced galls are microhabitats distinct from the outer environment that support inhabitants by providing improved nutrients, defence against enemies, and other unique features. It is intriguing as to how insects reprogram and modify plant morphogenesis. Because most of the gall systems are formed on trees, it is difficult to maintain them in laboratories and to comprehend the mechanisms operative in them through experimental manipulations. Herein, we propose a new model insect, Smicronyx madaranus, for studying the mechanisms of gall formation. This weevil forms spherical galls on the shoots of Cuscuta campestris, an obligate parasitic plant. We established a stable system for breeding and maintaining this ecologically intriguing insect in the laboratory, and succeeded in detailed analyses of the gall-forming behaviour, gall formation process, and histochemical and physiological features. Parasitic C. campestris depends on host plants for its nutrients, and usually shows low chlorophyll content and photosynthetic activity. We demonstrate that S. madaranus-induced galls have significantly increased CO2 absorbance. Moreover, chloroplasts and starch accumulated in gall tissues at locations inhabited by the weevil larvae. These results suggest that the gall-inducing weevils enhance the photosynthetic activity in C. campestris, and modify the plant tissue to a nutrient-rich shelter for them.

Ecological rigidity and the hardness of selection in the wild.

Hutchinson's ecological theater and evolutionary play is a classical view of evolutionary ecology-ecology provides a template in which evolution occurs. An opposing view is that ecological and evolutionary changes are like two actors on a stage, intertwined by density and frequency dependence. These opposing views correspond to hard and soft selection, respectively. Although often presented as diametrically opposed, both types of selection can occur simultaneously, yet we largely lack knowledge of the relative importance of hard versus soft selection in the wild. I use a dataset of 3000 individual gall makers from 15 wild local populations over 5 years to investigate the hardness of selection. I show that enemy attack consistently favors some gall sizes over others (hard selection) but that these biases can be fine-tuned by density and frequency dependence (soft selection). As a result, selection is hard and soft in roughly equal measures, but the importance of each type varies as species interactions shift. I conclude that eco-evolutionary dynamics should occur when a mix of hard and soft selection acts on a population. This work contributes to the rapprochement of disparate views of evolutionary ecology-ecology is neither a rigid theater nor a flexible actor, but instead embodies components of both.

An insect nucleoside diphosphate kinase (NDK) functions as an effector protein in wheat - Hessian fly interactions.

Like pathogens, galling insects deliver effectors into plant tissues that induce gall formation. The gall midge Mayetiola destructor, also called Hessian fly, can convert a whole wheat seedling into a gall by inducing the formation of nutritive cells at the feeding site, inhibiting wheat growth, and reprogramming metabolic pathways of the attacked plants. Here we demonstrated the identification of a secreted Hessian fly protein, the nucleoside diphosphate kinase (NDK), in infested wheat plants through liquid chromatography-tandem mass spectrometry (LC-MS/MS) and western blots. In association with the NDK presence, enzymatic activity of NDK increased significantly in wheat tissues at the feeding site. In addition, there was a sudden increase in ATP abundance at the feeding site of infested susceptible wheat seedlings 24 h following Hessian fly larval infestation. Even though a direct link between cause and effect remains to be established, our data points to the direction that Hessian fly larvae injected NDK into wheat tissues as an effector protein, which plays a role in manipulating host plants and converting the plants into galls.

Tetraneura ulmi (Hemiptera: Eriosomatinae) Induces Oxidative Stress and Alters Antioxidant Enzyme Activities in Elm Leaves.

Gall formation is induced by an insect, which changes normal plant development and results in the formation of a new organ, following distinct stages of metabolic and developmental alterations. Research on mechanisms of recognition and responses to biotic stress may help to understand the interactions between galling aphids and their host plants. In this study, Tetraneura ulmi L. (Hemiptera: Eriosomatinae) galls and Ulmus pumila L. (Rosales: Ulmaceae) leaves were used as a model. Concentrations of hydrogen peroxide (H2O2) and thiobarbituric acid reactive substances, electrolyte leakage, as well as the activity of ascorbate peroxidase, guaiacol peroxidase, and catalase (CAT) were determined in galls and two parts of galled leaves (with and without visible damage). Biochemical analyses were performed at three stages of gall development: initial, fully developed, and mature galls. A slight increment in H2O2 content with a strong enhancement of ascorbate peroxidase and CAT activities were observed in galls and galled leaves in the first stage. In subsequent stages of gall development, a progressing increase in H2O2 production and cell membrane damage was associated with declining antioxidant enzyme activities, especially in gall tissues. The stages of gall development are likely to be part of cell death triggered by aphid feeding. It seems that the gall is the result of a biochemical struggle between the host plant and the gall inducer.

Physiological Response of Pedunculate Oak Trees to Gall-Inducing Cynipidae.

Gall-inducing Cynipidae (Hymenoptera) manipulate the leaves of their host plants and induce local resistance, resulting in a diversity of physiological changes. In this study, three gall morphotypes caused by the asexual generation of Cynips quercusfolii L., Neuroterus numismalis (Fourc.) and Neuroterus quercusbaccarum L. (Hymenoptera: Cynipidae) on pedunculate oaks (Quercus robur L. (Fagales: Fagaceae)), were used as a model to examine physiological alterations in galls and foliar tissues, compared to non-galled tissues. Our goal was to investigate whether plant physiological response to insect feeding on the same host plant varies depending on gall-wasp species. In particular, the cytoplasmic membrane condition, hydrogen peroxide (H2O2) concentration and changes in antioxidative enzyme activities, including guaiacol peroxidase (GPX) and ascorbate peroxidase (APX) were examined in this study. All cynipid species increased H2O2 levels in the leaves with galls, while the level of H2O2 in galls depended on the species. The presence of galls of all species on oak leaves caused an increase of electrolyte leakage and lipid peroxidation level. A significant induction of GPX activity was observed in the leaves with galls of all species, indicating stress induction. Conversely, the decrease in APX activity in both leaves with galls and galled tissues exposed to feeding of all cynipid species.

Impact of the Asian Chestnut Gall Wasp, Dryocosmus kuriphilus (Hymenoptera, Cynipidae), on the Chestnut Component of Honey in the Southern Swiss Alps.

The Asian chestnut gall wasp (ACGW; Dryocosmus kuriphilus Yasumatsu, Hymenoptera, Cynipidae) is considered as one of the most dangerous pests of the genus Castanea. In southern Switzerland, repeated heavy ACGW attacks prevented chestnut trees from vegetating normally for years before the arrival and spread of the biological control agent Torymus sinensis (Kamijo, Hymenoptera, Torymidae). This resulted in a greatly reduced green biomass and flower production. In this paper, we analyze the impact of such an ecosystem alteration of the environment on the composition of produced honey. Six beekeepers were chosen from sites with different densities of chestnut trees, each of which providing series of honey samples from 2010 to 2016. We determined the chestnut component in the honeys via a combined chemical and sensory approach, and correlated the obtained results with the degree of yearly ACGW-induced crown damage and weather conditions during the period in question in the surrounding chestnut stands. The chestnut component in the analyzed honey sample series showed a strong correlation with the degree of ACGW-induced crown damage, whereas meteorological conditions of the corresponding year had a very marginal effect. Decreases in the chestnut component of the honey were statistically significant starting from a ACGW infestation level of 30%.

Larvae and Nests of Aculeate Hymenoptera (Hymenoptera: Aculeata) Nesting in Reed Galls Induced by Lipara spp. (Diptera: Chloropidae) with a Review of Species Recorded. Part II.

The ability of aculeate Hymenoptera to utilize wetlands is poorly understood, and descriptions of their nests and developmental stages are largely absent. Here we present results based on our survey of hymenopterans using galls induced by Lipara spp. flies on common reed Phragmites australis in the years 2015-2016. We studied 20,704 galls, of which 9,446 were longitudinally cut and the brood from them reared in the laboratory, while the remaining 11,258 galls reared in rearing bags also in laboratory conditions. We recorded eight species that were previously not known to nest in reed galls: cuckoo wasps Chrysis rutilans and Trichrysis pumilionis, solitary wasps Stenodynerus chevrieranus and Stenodynerus clypeopictus, and bees Pseudoanthidium tenellum, Stelis punctulatissima, Hylaeus communis and Hylaeus confusus. Forty five species of Hymenoptera: Aculeata are known to be associated with reed galls, of which 36 make their nests there, and the other are six parasitoids of the family Chrysididae and three cuckoo bees of the genus Stelis. Of these species, Pemphredon fabricii and in southern Europe also Heriades rubicola are very common in reed galls, followed by Hylaeus pectoralis and two species of the genus Trypoxylon. We also found new host-parasite associations: Chrysis angustula in nests of Pemphredon fabricii, Chrysis rutilans in nests of Stenodynerus clypeopictus, Trichrysis pumilionis in nests of Trypoxylon deceptorium, and Stelis breviuscula in nests of Heriades rubicola. We provide new descriptions of the nests of seven species nesting in reed galls and morphology of mature larvae of eight species nesting in reed galls and two parasitoids and one nest cleptoparasite. The larvae are usually very similar to those of related species but possess characteristics that make them easy to distinguish from related species. Our results show that common reeds are not only expansive and harmful, but very important for many insect species associated with habitats dominated by this plant species.

Gall-Insect Community on Big Sagebrush Varies With Plant Size but not Plant Age.

There is astounding variation in the abundance and diversity of insect herbivores among plant individuals within plant species in natural systems. One of the most well studied hypotheses for this pattern, the plant architecture hypothesis, suggests that insect community patterns vary with plant structural complexity and plant traits associated with structure. An important limitation to our understanding of the plant architecture hypothesis has been that most studies on the topic confound plant size and plant age. This occurs because, for most plant species, larger individuals are older individuals. This is a limitation because it prevents us from knowing whether insect community patterns are more dependent on traits associated with plant size, like resource quantity or plant apparency, or traits associated with plant age, like ontogenetic changes in phytochemistry. To separate these effects, we characterized galling insect communities on sagebrush (Artemisia tridentata)-a shrub in which age and size are not tightly correlated. We identified gall insects and recorded morphological measurements from 60 plants that varied separately in size and age. We found that plant size explained significantly more variation in insect gall abundance and species richness than did plant age. These results suggest that processes supporting the plant architecture hypothesis in this system are driven primarily by plant size and not plant age per se. Resource qualities associated with host-plant ontogeny may be less important than resource quantity in the assembly of herbivorous insect communities.

An endoparasitoid avoids hyperparasitism by manipulating immobile host herbivore to modify host plant morphology.

Many parasitic organisms have an ability to manipulate their hosts to increase their own fitness. In parasitoids, behavioral changes of mobile hosts to avoid or protect against predation and hyperparasitism have been intensively studied, but host manipulation by parasitoids associated with endophytic or immobile hosts has seldom been investigated. We examined the interactions between a gall inducer Masakimyia pustulae (Diptera: Cecidomyiidae) and its parasitoids. This gall midge induces dimorphic leaf galls, thick and thin types, on Euonymus japonicus (Celastraceae). Platygaster sp. was the most common primary parasitoid of M. pustulae. In galls attacked by Platygaster sp., whole gall thickness as well as thicknesses of upper and lower gall wall was significantly larger than unparasitized galls, regardless of the gall types, in many localities. In addition, localities and tree individuals significantly affected the thickness of gall. Galls attacked by Platygaster sp. were seldom hyperparasitized in the two gall types. These results strongly suggest that Platygaster sp. manipulates the host plant's development to avoid hyperparasitism by thickening galls.

Phytohormone dynamics associated with gall insects, and their potential role in the evolution of the gall-inducing habit.

While plant galls can be induced by a variety of organisms, insects produce the most diverse and complex galls found in nature; yet, how these galls are formed is unknown. Phytohormones have long been hypothesized to play a key role in gall production, but their exact role, and how they influence galls, has been unclear. Research in the past decade has provided better insight into the role of plant hormones in gall growth and plant defenses. We review and synthesize recent literature on auxin, cytokinins, and abscisic, jasmonic, and salicylic acids to provide a broader understanding of how these phytohormones might effect gall production, help plants defend against galls, and/or allow insects to overcome host-plant defenses. After reviewing these topics, we consider the potential for phytohormones to have facilitated the evolution of insect galls. More specialized research is needed to provide a mechanistic understanding of how phytohormones operate in gall-insect-plant interactions, but current evidence strongly supports phytohormones as key factors determining the success and failure of insect galls.

Ectopic localization of auxin and cytokinin in tobacco seedlings by the plant-oncogenic AK-6b gene of Agrobacterium tumefaciens AKE10.

The oncogenic 6b gene of Agrobacterium tumefaciens induces a number of morphological and metabolic alterations in plants. Although molecular functions associated with the 6b genes have been proposed, including auxin transport, sugar transport, transcriptional regulation, and miRNA metabolism, so far an unequivocal conclusion has not been obtained. We investigated the association between auxin accumulation and tumor development of the tobacco seedlings expressing the AK-6b gene under the control of the dexamethasone-inducible promoter. Indole-3-acetic acid (IAA) localization was examined by immunochemical staining with monoclonal antibody against IAA and by histochemical analysis using the IAA-specific induced construct, DR5::GUS (ß-glucuronidase). Both procedures indicated that IAA preferentially accumulated in the tumorous protrusions as well as in newly developing vascular bundles in the tumors. Furthermore, true leaves also showed abaxial IAA localization, leading to altered leaves in which the adaxial and abaxial identities were no longer evident. Co-localization of cytokinin and auxin in the abaxial tumors was verified by immunochemical staining with an antibody against cytokinin. Treatment of AK-6b-seedlings with N-1-naphthylphthalamic acid, an inhibitor of polar auxin transport, promoted the morphological severity of phenotypes, whereas 1-naphthoxyacetic acid, a specific auxin influx carrier inhibitor, induced tumor regression on cotyledons and new tumorous proliferations on hypocotyls. Prominent accumulation of both auxin and cytokinin was observed in both regressed and newly developing tumors. We suggest from these results that modulation of auxin/cytokinin localization as a result of AK-6b gene expression is responsible for the tumorous proliferation.

Electrochemical and spectroscopic investigations of carboxylic acid ligand and its triorganotin complexes for their binding with ds.DNA: in vitro biological studies.

A carboxylic acid ligand, (Z)-4-(4-acetylphenylamino)-4-oxobut-2-enoic acid (APA-1), and its triphenyl-(APA-2) and tributyl-tin(IV) (APA-3) compounds have been synthesized and investigated for their binding with ds.DNA using UV-visible spectroscopy, fluorescence spectroscopy, cyclic voltammetry, and viscosity measurements under physiological conditions of pH and temperature. The experimental results from all techniques i.e. binding constant (Kb), binding site size (n) and free energy change (ΔG) were in good agreement and inferred spontaneous compound-DNA complexes formation via intercalation. Among all the compounds APA-3 showed comparatively greater binding at pH 4.7 as evident from its greater Kb values {APA-3: Kb: 5.63×10(4)M(-1) (UV); 7.94×10(4)M(-1) (fluorescence); 9.91×10(4)M(-1) (CV)}. Electrochemical processes of compounds before and after the addition of DNA were found diffusion controlled. Among all compounds, APA-3 exhibited best antitumor activity.

Richness and composition of gall-inducing arthropods at Coiba National Park, Panama

Interest in studying galls and their arthropods inducers has been growing rapidly in the last two decades. However, the Neotropical region is probably the least studied region for gall-inducing arthropods. A study of the richness and composition of gall-inducing arthropods was carried out at Coiba National Park in the Republic of Panama. Field data come from samples obtained between August 1997 and September 1999, with three (two-week long) more intensive samplings. Seventeen sites, representing the main land habitats of Coiba National Park were surveyed. 4942 galls of 50 insect and 9 mite species inducing galls on 50 vascular plants from 30 botanical families were colleted. 62.7% of the galls were induced by gall midges (Diptera, Cecidomyiidae), 15.3% by mites, Eriophyidae, 8.5% by Homoptera, Psyllidae, 6.8% by Coccidae and 5.1% by Phlaeothripidae (Tysanoptera). The host plant families with the most galls were Myrtaceae with seven, Bignoniaceae with five and Euphorbiaceae, Fabaceae and Melastomataceae with four. Leaf galls accounted for about 93% of collected galls. Most leaf galls were pit/blister galls followed by covering and pouch galls. Gall richness per collecting site was between 1 and 19 species. Coiba’s gall diversity is discussed in relation to data available from other tropical sites from continental Panama and the Neotropical region. Our results support the idea that it may be premature to conclude that species richness of gall inducers declines near the equator. Rev. Biol. Trop. 56 (3): 1269-1286. Epub 2008 September 30.

El interés por el estudio de las agallas y los artrópodos que las inducen ha crecido en todo el mundo en los últimos veinte años. Sin embargo, los artrópodos que inducen agallas en la región Neotropical son probablemente los menos estudiados. Un estudio de la riqueza y composición de artrópodos que inducen agallas fue desarrollado en el Parque Nacional Coiba en la Republica de Panamá. Los datos provienen de muestreos intensivos de dos semanas, efectuados entre agosto de 1997 y septiembre de 1999, en 17 sitios del área insular del Parque Nacional Coiba. Un total de 4942 agallas, que corresponden a 50 especies de insectos y nueve de ácaros, fueron colectadas en 50 especies de plantas vasculares de 30 familias. El 62.7% de las agallas correspondieron a especies de la familia Cecidomyiidae (Diptera), el 15.3% a ácaros de la familia Eriophyidae, el 8.5% a Psyllidae (Homoptera), un 6.8% a Coccidae y el 5.1% a Phlaeothripidae (Tysanoptera). Las familias de plantas con más especies de inductores de agallas fueron Myrtaceae con siete, Bignoniaceae con cinco y Euphorbiaceae, Fabaceae y Melastomataceae con cuatro. Las agallas formadas en hojas representaron el 93% del total. El número de inductores de agallas por sitio osciló entre uno y diecinueve. La riqueza de artrópodos inductores de agallas del Parque Nacional Coiba se discute con datos disponibles de la literatura para el área continental de Panamá y el Neotrópico. Nuestros resultados apoyan la idea de que es prematuro concluir que la riqueza de artrópodos que inducen agallas disminuye hacia el Ecuador.

Richness and composition of gall-inducing arthropods at Coiba National Park, Panama.

Interest in studying galls and their arthropods inducers has been growing rapidly in the last two decades. However, the Neotropical region is probably the least studied region for gall-inducing arthropods. A study of the richness and composition of gall-inducing arthropods was carried out at Coiba National Park in the Republic of Panama. Field data come from samples obtained between August 1997 and September 1999, with three (two-week long) more intensive samplings. Seventeen sites, representing the main land habitats of Coiba National Park were surveyed. 4942 galls of 50 insect and 9 mite species inducing galls on 50 vascular plants from 30 botanical families were colleted. 62.7% of the galls were induced by gall midges (Diptera, Cecidomyiidae), 15.3% by mites, Eriophyidae, 8.5% by Homoptera, Psyllidae, 6.8% by Coccidae and 5.1% by Phlaeothripidae (Tysanoptera). The host plant families with the most galls were Myrtaceae with seven, Bignoniaceae with five and Euphorbiaceae, Fabaceae and Melastomataceae with four. Leaf galls accounted for about 93% of collected galls. Most leaf galls were pit/blister galls followed by covering and pouch galls. Gall richness per collecting site was between 1 and 19 species. Coiba's gall diversity is discussed in relation to data available from other tropical sites from continental Panama and the Neotropical region. Our results support the idea that it may be premature to conclude that species richness of gall inducers declines near the equator.

Life history traits of Pseudasphondylia rokuharensis (Diptera: Cecidomyiidae) affecting emergence of adults and synchronization with host plant phenology.

To identify adaptive strategies of gall-inducing cecidomyiids, we studied the life history traits of Pseudasphondylia rokuharensis (Diptera: Cecidomyiidae), a univoltine species inducing fruit galls on a deciduous shrub, Viburnum dilatatum. Although univoltine gall midges that are associated with trees or shrubs are usually difficult to rear from immature stages under artificial conditions, we successfully reared P. rokuharensis from first instar to adult. Mature larvae entered diapause in autumn, and the diapause terminated after low temperature treatment (4 mo of incubation at 5 degrees C). The adults emerged within a shorter period of time than those without low temperature treatment. Thus, the diapause provides better synchronization of adult emergence in the spring. Because adults live for only a few days, this synchronization improves the chances of adults finding a mate. This is the first report on the mechanism of larval diapause termination for univoltine and tree- or shrub-associated gall midges. The numbers of days needed for adults to emerge under laboratory conditions after low temperature incubation were significantly different between two different localities. This result might suggest the possible existence of ecoclines in their life history parameters.

Willow regrowth after galling increases bud production through increased shoot survival.

Insect herbivory can negatively or positively affect plant performance. We examined how a stem gall midge Rabdophaga rigidae affects the survival, growth, and bud production of current year shoots of the willow Salix eriocarpa. In mid-May, the gall midge initiates stem galls on the apical regions of shoots. The following spring, galled shoots had thicker basal diameters and more lateral shoots than ungalled shoots. Although galled shoots were on average 1.6 times longer than ungalled shoots, there were no significant differences in shoot length or in the numbers of reproductive, vegetative, and dormant buds per shoot. However, the subsequent survival of galled shoots was significantly higher than that of ungalled shoots, probably because of the thicker basal diameter. This increased shoot survival resulted in approximately two times greater reproductive, vegetative, and dormant bud production on galled shoots compared with ungalled shoots in the following spring. These results suggest that the willow regrowth induced by galling can lead to an increase in bud production through increased shoot survival.

Hybrid necrosis: autoimmunity as a potential gene-flow barrier in plant species.

Ecological factors, hybrid sterility and differences in ploidy levels are well known for contributing to gene-flow barriers in plants. Another common postzygotic incompatibility, hybrid necrosis, has received comparatively little attention in the evolutionary genetics literature. Hybrid necrosis is associated with a suite of phenotypic characteristics that are similar to those elicited in response to various environmental stresses, including pathogen attack. The genetic architecture is generally simple, and complies with the Bateson-Dobzhansky-Muller model for hybrid incompatibility between species. We survey the extensive literature on this topic and present the hypothesis that hybrid necrosis can result from autoimmunity, perhaps as a pleiotropic effect of evolution of genes that are involved in pathogen response.

Both hypersensitive and non-hypersensitive responses are associated with resistance in Salix viminalis against the gall midge Dasineura marginemtorquens.

Hypersensitivity responses (HR) play a major role in plant resistance to pathogens. It is often claimed that HR is also important in plant resistance to insects, although there is little unambiguous documentation. Large genotypic variation in resistance against the gall midge Dasineura marginemtorquens is found in Salix viminalis. Variation in larval performance and induced responses within a full-sib S. viminalis family is reported here; 36 sibling plants were completely resistant (larvae died within 48 h after egg hatch, no gall induction), 11 plants were totally susceptible, 25 plants were variable (living and dead larvae present on the same plant). Resistance was associated with HR, but to different degrees; 21 totally resistant genotypes showed typical HR symptoms (many distinct necrotic spots) whereas the remaining 15 genotypes showed no, or very few, such symptoms. Hydrogen peroxide, used as a marker for HR, was induced in genotypes expressing HR symptoms but not in resistant genotypes without symptoms, or in susceptible genotypes. These data suggest that production of hydrogen peroxide, and accompanying cell death, cannot explain larval mortality in the symptomless reaction. Another, as yet unknown, mechanism of resistance may be present. If so, then it is possible that this unknown mechanism also contributes to resistance in plants displaying HR. The apparent complexity observed in this interaction, with both visible and invisible plant responses associated with resistance against an adapted insect species, may have implications for the study of resistance factors in other plant-insect interactions.

Flavonoid-related regulation of auxin accumulation in Agrobacterium tumefaciens-induced plant tumors.

Agrobacterium tumefaciens-induced plant tumors accumulate considerable concentrations of free auxin. To determine possible mechanisms by which high auxin concentrations are maintained, we examined the pattern of auxin and flavonoid distribution in plant tumors. Tumors were induced in transformants of Trifolium repens (L.), containing the beta-glucuronidase ( GUS)-fused auxin-responsive promoter ( GH3) or chalcone synthase ( CHS2) genes, and in transformants of Arabidopsis thaliana (L.) Heynh., containing the GUS-fused synthetic auxin response element DR5. Expression of GH3::GUS and DR5::GUS was strong in proliferating metabolically active tumors, thus suggesting high free-auxin concentrations. Immunolocalization of total auxin with indole-3-acetic acid antibodies was consistent with GH3::GUS expression indicating the highest auxin concentration in the tumor periphery. By in situ staining with diphenylboric acid 2-aminoethyl ester, by thin-layer chromatography, reverse-phase high-performance liquid chromatography, and two-photon laser-scanning microscopy spectrometry, tumor-specific flavones, isoflavones and pterocarpans were detected, namely 7,4'-dihydroxyflavone (DHF), formononetin, and medicarpin. DHF was the dominant flavone in high free-auxin-accumulating stipules of Arabidopsis leaf primordia. Flavonoids were localized at the sites of strongest auxin-inducible CHS2::GUS expression in the tumor that was differentially modulated by auxin in the vascular tissue. CHS mRNA expression changes corresponded to the previously analyzed auxin concentration profile in tumors and roots of tumorized Ricinus plants. Application of DHF to stems, apically pretreated with alpha-naphthaleneacetic acid, inhibited GH3::GUS expression in a fashion similar to 1-N-naphthyl-phthalamic acid. Tumor, root and shoot growth was poor in inoculated tt4(85) flavonoid-deficient CHS mutants of Arabidopsis. It is concluded that CHS-dependent flavonoid aglycones are possibly endogenous regulators of the basipetal auxin flux, thereby leading to free-auxin accumulation in A. tumefaciens-induced tumors. This, in turn, triggers vigorous proliferation and vascularization of the tumor tissues and suppresses their further differentiation.