Adaptive significance of gall formation for a gall-inducing aphids on Japanese elm trees

Insect galls are abnormal plant tissues induced by external stimuli from parasitizing insects. It has been suggested that the stimuli include phytohormones such as auxin and cytokinins produced by the insects. In our study on the role of hormones in gall induction by the aphid Tetraneura nigriabdominalis, it was found that feedback regulation related to auxin and cytokinin activity is absent in gall tissues, even though the aphids contain higher concentrations of those phytohormones than do plant tissues. Moreover, jasmonic acid signaling appears to be compromised in gall tissue, and consequently, the production of volatile organic compounds, which are a typical defense response of host plants to herbivory, is diminished. These findings suggest that these traits of the gall tissue benefit aphids, because the gall tissue is highly sensitive to auxin and cytokinin, which induce and maintain it. The induced defenses against aphid feeding are also compromised. The abnormal responsiveness to phytohormones is regarded as a new type of extended phenotype of gall-inducing insects.     

Biosynthetic pathway of the phytohormone auxin in insects and screening of its inhibitors

Insect galls are abnormal plant tissues induced by galling insects. The galls are used for food and habitation, and the phytohormone auxin, produced by the insects, may be involved in their formation. We found that the silkworm, a non-galling insect, also produces an active form of auxin, indole-3-acetic acid (IAA), by de novo synthesis from tryptophan (Trp). A detailed metabolic analysis of IAA using IAA synthetic enzymes from silkworms indicated an IAA biosynthetic pathway composed of a three-step conversion: Trp → indole-3-acetaldoxime → indole-3-acetaldehyde (IAAld) → IAA, of which the first step is limiting IAA production. This pathway was shown to also operate in gall-inducing sawfly. Screening of a chemical library identified two compounds that showed strong inhibitory activities on the conversion step IAAld → IAA. The inhibitors can be efficiently used to demonstrate the importance of insect-synthesized auxin in gall formation in the future.     

Sexually dimorphic gall structures correspond to differential phytohormone contents in male and female wasp larvae

Sexually dimorphic galls are rare among gall‐inducing insects and the reason for their occurrence is unknown. The pteromalid wasp Trichilogaster acaciaelongifoliae, which induces galls on Acacia longifolia, is one such species. In the present study, the anatomical and physiological attributes of male and female galls of T. acaciaelongifoliae are examined and compared. Histological preparations are used to characterize anatomical differences between male and female gall chambers. Bioassays, high‐performance liquid chromatography‐mass spectrometry and an enzyme immunoassay are used to measure concentrations of auxin and cytokinin in normal buds, galled tissues, and larvae of both sexes. Female chambers are found to be 3.3‐fold larger, and are associated with 1.5‐fold more storage tissue and 3.5‐fold more vascular tissues than male chambers. Tissues from female chambers induce stronger cytokinin‐like bioactivity than tissues from male chambers. Female larvae have considerably higher concentrations of cytokinin free bases, ribosides, glucosides and monophosphates than male larvae; higher auxin‐like bioactivity than in normal or galled plant tissues; and almost twice the concentration of auxin than male larvae. Both male and female larvae contain much higher auxin concentrations than either galled or normal plant tissues. These findings suggest that differing levels of phytohormones are involved in the development of sexual dimorphism of gall structures in this species.     

Phytohormones in Japanese Mugwort Gall Induction by a Gall-Inducing Gall Midge

A variety of insect species induce galls on host plants. Liquid chromatographic/tandem mass spectrometric analyses showed that a gall midge (Rhopalomyia yomogicola) that induces galls on Artemisia princeps contained high levels of indole-3-acetic acid and cytokinins. The gall midge larvae also synthesized indole-3-acetic acid from tryptophan. Close observation of gall tissue sections indicated that the larval chamber was surrounded by layers of cells having secondary cell walls with extensive lignin deposition, except for the part of the gall that constituted the feeding nutritive tissue which was composed of small cells negatively stained for lignin. The differences between these two types of tissue were confirmed by an expression analysis of the genes involved in the synthesis of the secondary cell wall. Phytohormones may have functioned in maintaining the feeding part of the gall as fresh nutritive tissue. Together with the results in our previous study, those presented here suggest the importance of phytohormones in gall induction.     

Detection of cytokinins and auxin in plant tissues using histochemistry and immunocytochemistry

We report a new method for histochemical localization of cytokinins (CKs) in plant tissues based on bromophenol blue/silver nitrate staining. The method was validated by immunohistochemistry using anti-trans-zeatin riboside antibody. Indole-3-acetic acid (auxin, IAA) was localized by anti-IAA antibody in plant tissues as a proof for IAA histolocalization. We used root sections, because they are major sites of CKs synthesis, and insect galls of Piptadenia gonoacantha that accumulate IAA. Immunostaining confirmed the presence of zeatin and sites of accumulation of IAA indicated by histochemistry. The colors developed by histochemical reactions in free-hand sections of plant tissues were similar to those obtained by thin layer chromatography (TLC), which reinforced the reactive sites of zeatin. The histochemical method for detecting CKs is useful for galls and roots, whereas IAA detection is more efficient for gall tissues. Therefore, galls constitute a useful model for validating histochemical techniques due to their rapid cell cycles and relatively high accumulation of plant hormones.     

Feeding by a gall-inducing caterpillar species alters levels of indole-3-acetic and abscisic acid in Solidago altissima (Asteraceae) stems

Various plant antagonists appear to alter phytohormone levels for their own benefit. Among insects, gall-inducing species appear to influence phytohormones and it is widely believed that they alter levels of indole-3-acetic acid (IAA) to help produce their galls, but evidence exists for only a limited number of species. To further explore the role of phytohormones in gall formation, we measured levels of IAA and abscisic acid (ABA), a hormone involved in plant defenses and that can influence IAA, in tissues of control stems of Solidago altissima (Asteraceae) and those galled by Gnorimoschema gallaesolidaginis (Gelechiidae). This gall-inducing caterpillar species significantly altered the distribution of IAA in galls and the larvae themselves contained high concentrations of IAA. In contrast, the generalist caterpillar Heliothis virescens (Noctuidae) neither altered IAA nor accumulated significant concentrations of IAA, suggesting that G. gallaesolidaginis may have a distinctive influence over IAA. The gall-inducing caterpillars, particularly younger larvae, also contained high levels of ABA but did not increase levels of ABA, which is induced by herbivory of H. virescens. Because G. gallaesolidaginis also does not increase levels of other defense-related hormones, avoiding generalized plant defenses may facilitate gall induction and formation.     

Cytokinins and auxin communicate nitrogen availability as long-distance signal molecules in pineapple (Ananas comosus)

This work aimed at identifying a possible role of phytohormones in long-distance (root-shoot) signaling under nitrogen deficiency. Three-months old pineapple plants were transferred from Murashige and Skoog (MS) medium to nitrogen-free MS (-N). During the first 24h on -N, 20 plants were harvested every 4h. After 30 days in -N, the remaining plants were transferred back to regular MS (+N) and 20 plants harvested every 4h for the first 24h. Following the harvests, endogenous levels of nitrate (NO(3)(-)), indole-3-acetic acid (IAA), isopentenyladenine (iP), isopentenyladenine riboside (iPR), zeatin (Z) and zeatin riboside (ZR) were analyzed in roots and leaves. In N-starved plants, the NO(3)(-) level dropped by 20% in roots between the first (4h) and the second harvest (8h). In leaves a reduction of 20% was found 4h later. Accumulation of IAA peaked in leaves at 16h. In roots, the accumulation of IAA only started at 16h while the leaf content was already in decline, which suggests that the hormone might have traveled from the leaves to the roots, communicating N-shortage. The contents of the four cytokinins were generally low in both, shoot and roots, and remained almost unchanged during the 24h of analysis. After N re-supply, roots showed a NO(3)(-) peak at 8h whereas the foliar concentration increased 4h later. Hormone levels in roots climaxed at 8h, this coinciding with the highest NO(3)(-) concentration. In leaf tissue, a dramatic accumulation was only observed for Z and ZR, and the peak was seen 4h later than in roots, suggesting that Z-type cytokinins might have traveled from the roots to the leaves. These findings provide evidence that there is a signaling pathway for N availability in pineapple plants, communicated upwards through cytokinins (N-supplemented plants) and downwards through auxin (N-starved plants).     

Exogenous phytohormones and the induction of plant galls by insects

The mechanism of gall induction by insects has remained elusive. Previous studies have met with limited success in attempting to induce galls by injection or application of chemical compounds. To determine whether an exogenous source of phytohormones plays a role in gall induction, we injected cytokinin (CK), auxin (IAA), gibberellic acid (GA), and abscisic acid (ABA) in various concentrations, ratios, and combinations into leaf petioles of Capsicum annuum L. cv. California Wonder (Solanaceae). We found that CK + IAA injections lead to gall-like growth in C. annuum. GA enhanced and ABA inhibited gall growth except in the presence of GA. Isopentenyl adenine (IP) was the most effective type of CK at inducing growth. Our work is consistent with the hypothesis that exogenous CK + IAA produced and supplied by insects leads to gall induction. We hypothesize that insects have obtained the capability to induce galls via acquisition of the biosynthetic pathways to produce IAA and trans-zeatin family CKs through microbial symbiosis or lateral gene transfer.     

Effect of ambiol and 2-chloroethylphosphonic acid on the content of phytohormones in potato leaves and tubers

The effects of the antioxidant Ambiol and 2-chlorethylphosphonic acid (2-CEPA) on individual concentrations and concentration ratios of phytohormones, photosynthesis and photophosphorylation rates, sucrose and starch content in tubers, and plant productivity were studied in potato (Solanum tuberosum L). Ambiol increased the ratio of indoleacetic acid (IAA) to abscisic acid (ABA), IAA/ABA, and that of zeatin (Z) and zeatin riboside (ZR) to ABA, (Z + ZR)/ABA. These effects were underlain by an increase in the content of auxins and cytokinins and a decrease in ABA. Unlike Ambiol, 2-CEPA increased the level of ABA, the effect being the most pronounced in the tubers. Ambiol increased the rates of photosynthesis and noncyclic photophosphorylation in chloroplasts isolated from potato leaves. The relation of this phenomenon to auxin and cytokinin accumulation, Ambiol- and 2-CEPA-induced changes in the hormonal balance of potato tubers, carbon metabolism, and plant productivity is discussed.     

Life history of <Emphasis Type="Italic">Stenopsylla nigricornis</Emphasis> (Hemiptera: Psylloidea: Triozidae) and phytohormones involved in its gall induction

Many phytophagous insects have an ability to manipulate plant tissue and induce galls, but the mechanism is not yet fully understood. Some insects have multivoltine life cycles, and each generation induces galls on different plant species or different organs in the same host. Such host-use patterns are interesting study subjects to clarify the gall-inducing mechanisms of insects. We focused on a multivoltine and gall-inducing psyllid Stenopsylla nigricornis Kuwayama (Hemiptera: Psylloidea: Triozidae), which is associated with Symplocos lucida Sieb. (Symplocaceae). Based on periodic field surveys in Kyushu, Japan, S. nigricornis is revealed to have a bivoltine life history. Then, we revealed that the spring generation induces galls on leaves, while the autumn generation does so on flower buds and overwintering leaf buds. We also analyzed phytohormones in normal plant tissue, S. nigricornis nymphs, and their galls. As a result, nymphs were discovered to contain much higher concentrations of isopentenyladenosine and its possible precursor, isopentenyladenosine riboside than plant tissues, strongly suggesting that the phytohormone is involved in gall induction by S. nigricornis. Because flower bud galls contained significantly lower concentrations of abscisic acid (ABA) than normal flower bud, the autumn generation nymphs are considered to regulate the ABA level and to promote the earlier opening of host flower buds.     

Leaf‐derived cecidomyiid galls are sinks in Machilus thunbergii (Lauraceae) leaves

Three relevant hypotheses – nutrition, environment and the enemies hypothesis – often invoked to explore source and sink relationships between galls and their host plants are still under dispute. In this research, chlorophyll fluorescence, gas exchange capacity, stomatal conductance, total carbon and nitrogen, total soluble sugars and starches, and scanning and transmission electron microscopy of two types of galls were used to investigate source–sink relationships. Compared with host leaves, these galls demonstrated slightly lower chlorophyll fluorescence; however, gas exchange capacity and stomatal conductance were not detected at all. Scanning electron micrographs demonstrated that the abaxial epidermis of host leaves contain normal amounts of stomata, whereas no stomata were observed on the exterior and interior surfaces of both types of galls. In addition, gall inner surfaces were covered with many kinds of fungal hyphae. Gall total carbon (C) and nitrogen (N) levels were lower but the C/N ratio was higher in galls than host leaves. Both types of galls accumulated higher total soluble sugars and starches than host leaves. Transmission electron micrographs also revealed that both types of galls contain plastoglobuli and giant starch granules during gall development. Results strongly indicate that leaf‐derived cecidomyiid galls are sinks in Machilus thunbergii leaves. However, it is perplexing how larvae cycle and balance CO₂ and O₂ in gall growth chambers without stomata.     

Effects of Ambiol and 2-Chlorethylphosphonic Acid on the Content of Phytohormones in Potato Leaves and Tubers

The effects of the antioxidant Ambiol and 2-chlorethylphosphonic acid (2-CEPA) on individual concentrations and concentration ratios of phytohormones, photosynthesis and photophosphorylation rates, sucrose and starch content in tubers, and plant productivity were studied in potato (Solanum tuberosum L). Ambiol increased the ratio of indoleacetic acid (IAA) to abscisic acid (ABA), IAA/ABA, and that of zeatin (Z) and zeatin riboside (ZR) to ABA, (Z + ZR)/ABA. These effects were underlain by an increase in the content of auxins and cytokinins and a decrease in ABA. Unlike Ambiol, 2-CEPA increased the level of ABA, the effect being the most pronounced in the tubers. Ambiol increased the rates of photosynthesis and noncyclic photophosphorylation in chloroplasts isolated from potato leaves. The relation of this phenomenon to auxin and cytokinin accumulation, as well as Ambiol- and 2-CEPA-induced changes in the hormonal balance of potato tubers, carbon metabolism, and plant productivity, is discussed.     

The effect of exogenous and endogenous phytohormones on the in vitro development of gametophyte and sporophyte in <Emphasis Type="Italic">Asplenium nidus</Emphasis> L.

The fern Asplenium nidus L. is in great demand as an ornamental plant. The aim of this work was to investigate the influence of phytohormones in promoting a gametophytic and sporophytic growth in homogenized sporophytes tissue. Exogenous application of 0.5 and 5 μM N ⁶-benzyladenine, 0.05 and 0.5 μM indole-3-acetic acid (IAA), and 0.3 and 3 μM gibberellic acid (GA₃) favoured sporophyte regeneration, whereas gametophyte regeneration took place when plant material was cultured in a hormone-free liquid MS medium. The endogenous contents of the auxin IAA, the cytokinins trans-zeatin, trans-zeatin riboside, dihydrozeatin, dihydrozeatin riboside, isopentenyladenine and isopentenyladenosine, and the gibberellins GA₁, GA₃, GA₄, GA₇, GA₉ and GA₂₀ in growing gametophytes and sporophytes were evaluated. Similar levels of the auxin and cytokinins and qualitative differences in the gibberellins were found between both generations.     

Gall-inducing Pachypsylla celtidis (Psyllidae) infiltrate hackberry trees with high concentrations of phytohormones

Abstract

In order to identify phytohormones involved in the initiation and maintenance of galls on the hackberry tree, Celtis occidentalis (Ulmaceae), in the presence of the insect Pachypsylla celtidis (Psyllidae); endogenous levels of cytokinins (CKs) and abscisic acid (ABA) were measured in the tissues of leaves, galls, and larval insects using liquid-chromatography-tandem-mass-spectrometry. The CKs isopentenyl adenine, isopentenyl adenosine, trans-zeatin and cis-zeatin, were extremely concentrated in insect larvae compared to surrounding tissue of leaves and galls. ABA concentrations in the insects were also relatively high at about 3268 pmol?g-1 fresh mass – approximately 25 times higher than in leaves, and 17 times higher than in galls. This represents a novel case of high concentrations of ABA being found in a larval insect. These findings indicate that manipulation of CK and ABA are involved in the initiation and maintenance of hackberry galls in the presence of larval P. celtidis.     

The mechanism of gall induction makes galls red

We propose that the commonly observed red coloration of insect-induced plant galls is due to the production of exogenous cytokinins by gall-inducing insects. A growing body of evidence indicates that gall-inducing insects, bacteria, and fungi produce cytokinins. We hypothesize that gall induction generally requires an exogenous source of cytokinin and auxin. Plant galls are mobilizing sinks induced by cytokinin and reinforced by transport and accumulation of sugar. Exogenous cytokinins lead to a cascade of effects including the up-regulation of anthocyanin synthesis, the source of red coloration. Experiments demonstrate that exogenous cytokinins and sugars up-regulate the phenylpropanoid and flavonoid pathways, leading to localized anthocyanin accumulation. We suggest that red coloration in plant galls is merely a consequence of the mechanism of gall induction, and therefore an example of fabricational noise rather than aposematic coloration. Only color manipulation experiments can determine whether gall color is also secondarily aposematic.     

Mixed significance of plant vigor: two species of galling Pontania in a hybridizing willow complex

Distributions of leaf galls and offspring performance of two Pontania sawfly species were explored in individual willows of the subarctic Salix caprea – starkeana hybrid complex. The more common sawfly, an undescribed species near the dolichura group (P1), had the highest gall numbers in trees with long shoots both in S. caprea and hybrids. While numbers were high on vigorously growing hybrids, offspring of P1 were aborted significantly more often on hybrids than on pure hosts. Further, non-aborted galls were smaller on hybrids. Fast shoot growth may be important for P1 sawflies, because females oviposit early in summer and larvae develop rapidly compared with the other species, Pontania pedunculi (P2). Distributions of P2 galls were related to tree height and not to shoot length in both parental and hybrid groups of willows. Like P1, also P2 offspring were frequently aborted on hybrids, but not significantly more often than on pure hosts, and P2 galls were equally large among the host groups. Survival of both species was related to abortion rates, while larvae were parasitized equally in all host groups. This study demonstrates that the significance of plant vigor may vary even for closely related galling sawflies exploiting the same hosts, or for the same species on different host plants. Vigorous growth may mislead gallers to oviposit on suboptimal plants.     

Development of Agrobacterium tumefaciens C58-induced plant tumors and impact on host shoots are controlled by a cascade of jasmonic acid,auxin, cytokinin,ethylene and abscisic acid

The development of Agrobacterium tumefaciens-induced plant tumors primarily depends on the excessive production of auxin and cytokinin by enzymes encoded on T-DNA genes integrated into the plant genome. The aim of the present study was to investigate the involvement of additional phytohormone signals in the vascularization required for rapid tumor proliferation. In stem tumors of Ricinus communis L., free auxin and zeatin riboside concentrations increased within 2 weeks to 15-fold the concentrations in control stem tissue. Auxin and cytokinin immunolocalization revealed the highest concentrations within and around tumor vascular bundles with concentration gradients. The time-course of changes in free auxin concentration in roots was inversely correlated with that in the tumors. The high ethylene emission induced by increased auxin- and cytokinin correlated with a 36-fold accumulation of abscisic acid in tumors. Ethylene emitted from tumors and exogenously applied ethylene caused an increase in abscisic acid concentrations also in the host leaves, with a diminution in leaf water vapor conductance. Jasmonic acid concentration reached a maximum already within the first week of bacterial infection. A wound effect could be excluded. The results demonstrate the concerted interaction of a cascade of transiently induced, non-T-DNA-encoded phytohormones jasmonic acid, ethylene and abscisic acid with T-DNA-encoded auxin and zeatin riboside plus trans-zeatin, all of which are required for successful plant tumor vascularization and development together with inhibition of host plant growth.     

Comprehensive transcriptome analysis of phytohormone biosynthesis and signaling genes in microspore/pollen and tapetum of rice

To investigate the involvement of phytohormones during rice microspore/pollen (MS/POL) development, endogenous levels of IAA, gibberellins (GAs), cytokinins (CKs) and abscisic acid (ABA) in the mature anther were analyzed. We also analyzed the global expression profiles of genes related to seven phytohormones, namely auxin, GAs, CKs, brassinosteroids, ethylene, ABA and jasmonic acids, in MS/POL and tapetum (TAP) using a 44K microarray combined with a laser microdissection technique (LM-array analysis). IAA and GA(4) accumulated in a much higher amount in the mature anther compared with the other tissues, while CKs and ABA did not. LM-array analysis revealed that sets of genes required for IAA and GA synthesis were coordinately expressed during the later stages of MS/POL development, suggesting that these genes are responsible for the massive accumulation of IAA and GA(4) in the mature anther. In contrast, genes for GA signaling were preferentially expressed during the early developmental stages of MS/POL and throughout TAP development, while their expression was down-regulated at the later stages of MS/POL development. In the case of auxin signaling genes, such mirror-imaged expression observed in GA synthesis and signaling genes was not observed. IAA receptor genes were mostly expressed during the late stages of MS/POL development, and various sets of AUX/IAA and ARF genes were expressed during the different stages of MS/POL or TAP development. Such cell type-specific expression profiles of phytohormone biosynthesis and signaling genes demonstrate the validity and importance of analyzing the expression of phytohormone-related genes in individual cell types independently of other cells/tissues.     

Transgenic tobacco plants co-expressing Agrobacterium iaa and ipt genes have wild-type hormone levels but display both auxin- and cytokinin-overproducing phenotypes

Transgenic tobacco lines simultaneously expressing the Agrobacterium iaaM, iaaH and ipt genes, obtained by crossing lines expressing ipt with lines expressing iaaM and iaaH, were used to study in planta interactions between auxin and cytokinins. All phenotypic traits of the respective parental lines characteristic of cytokinin and auxin overproduction were present in the cross. Indole-3-acetic acid (IAA) and combined zeatin riboside (ZR) and zeatin riboside-5'-monophosphate (ZRMP) contents were analysed by mass spectrometry in young, developing leaves from the cross, the parental lines and the wild type. Unexpectedly, hormone levels in the cross were very similar to wild-type levels. Thus IAA levels in the cross were much lower throughout vegetative development than in the parental IAA overproducing line, although expression of the bacterial IAA biosynthesis genes was not reduced. The results suggest that effects on apical dominance, adventitious root formation, leaf morphology and other traits commonly +/- associated with IAA and cytokinin overproduction, and observed in the iaa E ipt cross, cannot be explained solely by analysis of auxin and cytokinin contents in individual organs. As traits associated with both hormones are expressed in close spatial and temporal proximity, it is likely that cellular resolution of hormone contents is essential to explain physiological responses to auxins and cytokinins.     

Levels and immunolocalization of endogenous cytokinins in thidiazuron-induced shoot organogenesis in carnation

We evaluated the capacity of the plant growth regulator thidiazuron (TDZ), a substituted phenylurea with high cytokinin-like activity, to promote organogenesis in petals and leaves of several carnation cultivars (Dianthus spp.), combined with 1-naphthaleneacetic acid (NAA). The involvement of the endogenous auxin indole-3-acetic acid (IAA) and purine-type cytokinins was also studied. Shoot differentiation was found to depend on the explant, cultivar and balance of growth regulators. TDZ alone (0.5 and 5.0 micromol/L) as well as synergistically with NAA (0.5 and 5.0 micromol/L) promoted shoot organogenesis in petals, and was more active than N6-benzyladenine. In petals of the White Sim cultivar, TDZ induced cell proliferation in a concentration-dependent manner and, on day 7 of culture, the proportion of meristematic regions in those petals allowed the prediction of shoot regeneration capacity after 30 days of culture. Immunolocalization of CK ribosides, N6-(delta2-isopentenyl)adenosine, zeatin riboside (ZR) and dihydrozeatin riboside (DHZR), in organogenic petals showed them to be highly concentrated in the tips of bud primordia and in the regions with proliferation capacity. All of them may play a role in cell proliferation, and possibly in differentiation, during the organogenic process. After seven days of culture of White Sim petals, NAA may account for the changes found in the levels of IAA and DHZR, whereas TDZ may be responsible for the remarkable increases in N6-(delta2-isopentenyl)adenine (iP) and ZR. ZR is induced by low TDZ concentrations (0.0-0.005 micromol/L), whereas iP, that correlates with massive cell proliferation and the onset of shoot differentiation, is associated with high TDZ levels (0.5 micromol/L). In addition to the changes observed in quantification and in situ localization of endogenous phytohormones during TDZ-induced shoot organogenesis, we propose that TDZ also promotes growth directly, through its own biological activity. To our knowledge, this study is the first to evaluate the effect of TDZ on endogenous phytohormones in an organogenic process.