Non-specific effects of the CINNAMATE-4-HYDROXYLASE inhibitor piperonylic acid.

Chemical inhibitors are often implemented for the functional characterization of genes to overcome the limitations associated with genetic approaches. Although it is well established that the specificity of the compound is key to success of a pharmacological approach, off-target effects are often overlooked or simply neglected in a complex biological setting. Here we illustrate the cause and implications of such secondary effects by focusing on piperonylic acid (PA), an inhibitor of CINNAMATE-4-HYDROXYLASE (C4H) that is frequently used to investigate the involvement of lignin during plant growth and development. When supplied to plants, we found that PA is recognized as a substrate by GRETCHEN HAGEN 3.6 (GH3.6), an amido synthetase involved in the formation of the indole-3-acetic acid (IAA) conjugate IAA-Asp. By competing for the same enzyme, PA interferes with IAA conjugation, resulting in an increase in IAA concentrations in the plant. In line with the broad substrate specificity of the GH3 family of enzymes, treatment with PA increased not only IAA levels but also those of other GH3-conjugated phytohormones, namely jasmonic acid and salicylic acid. Finally, we found that interference with the endogenous function of GH3s potentially contributes to phenotypes previously observed upon PA treatment. We conclude that deregulation of phytohormone homeostasis by surrogate occupation of the conjugation machinery in the plant is likely a general phenomenon when using chemical inhibitors. Our results hereby provide a novel and important basis for future reference in studies using chemical inhibitors.

Effect of ascorbate and hydrogen peroxide on hormone and metabolite levels during post-germination growth in wheat.

The modulation of hormone and metabolite levels by ascorbate (ASA) and hydrogen peroxide (H2 O2 ) was compared during post-germination growth in shoots of wheat. Treatment with ASA resulted in a greater reduction of growth than the addition of H2 O2 . ASA also had a larger effect on the redox state of the shoot tissues as shown by the higher ASA and glutathione (GSH) levels, lower glutathione disulfide (GSSG) content and GSSG/GSH ratio compared to the H2 O2 treatment. Apart from common responses (i.e., increase of cis-zeatin and its O-glucosides), the contents of several compounds related to cytokinin (CK) and abscisic acid (ABA) metabolism were greater after ASA application. These differences in the redox state and hormone metabolism following the two treatments may be responsible for their distinct influence on various metabolic pathways. Namely, the glycolysis and citrate cycle were inhibited by ASA and they were not affected by H2 O2 , while the amino acid metabolism was induced by ASA and repressed by H2 O2 based on the changes in the level of the related carbohydrates, organic and amino acids. The first two pathways produce reducing power, while the last one needs it; therefore ASA, as a reductant may suppress and induce them, respectively. H2 O2 as an oxidant had different effect, namely it did not alter glycolysis and citrate cycle, and inhibited the formation of amino acids.

Hormonomic Changes Driving the Negative Impact of Broomrape on Plant Host Interactions with Arbuscular Mycorrhizal Fungi.

Belowground interactions of plants with other organisms in the rhizosphere rely on extensive small-molecule communication. Chemical signals released from host plant roots ensure the development of beneficial arbuscular mycorrhizal (AM) fungi which in turn modulate host plant growth and stress tolerance. However, parasitic plants have adopted the capacity to sense the same signaling molecules and to trigger their own seed germination in the immediate vicinity of host roots. The contribution of AM fungi and parasitic plants to the regulation of phytohormone levels in host plant roots and root exudates remains largely obscure. Here, we studied the hormonome in the model system comprising tobacco as a host plant, Phelipanche spp. as a holoparasitic plant, and the AM fungus Rhizophagus irregularis. Co-cultivation of tobacco with broomrape and AM fungi alone or in combination led to characteristic changes in the levels of endogenous and exuded abscisic acid, indole-3-acetic acid, cytokinins, salicylic acid, and orobanchol-type strigolactones. The hormonal content in exudates of broomrape-infested mycorrhizal roots resembled that in exudates of infested non-mycorrhizal roots and differed from that observed in exudates of non-infested mycorrhizal roots. Moreover, we observed a significant reduction in AM colonization of infested tobacco plants, pointing to a dominant role of the holoparasite within the tripartite system.

Light Quality and Intensity Modulate Cold Acclimation in Arabidopsis.

Plant survival in temperate zones requires efficient cold acclimation, which is strongly affected by light and temperature signal crosstalk, which converge in modulation of hormonal responses. Cold under low light conditions affected Arabidopsis responses predominantly in apices, possibly because energy supplies were too limited for requirements of these meristematic tissues, despite a relatively high steady-state quantum yield. Comparing cold responses at optimal light intensity and low light, we found activation of similar defence mechanisms-apart from CBF1-3 and CRF3-4 pathways, also transient stimulation of cytokinin type-A response regulators, accompanied by fast transient increase of trans-zeatin in roots. Upregulated expression of components of strigolactone (and karrikin) signalling pathway indicated involvement of these phytohormones in cold responses. Impaired response of phyA, phyB, cry1 and cry2 mutants reflected participation of these photoreceptors in acquiring freezing tolerance (especially cryptochrome CRY1 at optimal light intensity and phytochrome PHYA at low light). Efficient cold acclimation at optimal light was associated with upregulation of trans-zeatin in leaves and roots, while at low light, cytokinin (except cis-zeatin) content remained diminished. Cold stresses induced elevation of jasmonic acid and salicylic acid (in roots). Low light at optimal conditions resulted in strong suppression of cytokinins, jasmonic and salicylic acid.

Transcription of specific auxin efflux and influx carriers drives auxin homeostasis in tobacco cells.

Auxin concentration gradients are informative for the transduction of many developmental cues, triggering downstream gene expression and other responses. The generation of auxin gradients depends significantly on cell-to-cell auxin transport, which is supported by the activities of auxin efflux and influx carriers. However, at the level of individual plant cell, the co-ordination of auxin efflux and influx largely remains uncharacterized. We addressed this issue by analyzing the contribution of canonical PIN-FORMED (PIN) proteins to the carrier-mediated auxin efflux in Nicotiana tabacum L., cv. Bright Yellow (BY-2) tobacco cells. We show here that a majority of canonical NtPINs are transcribed in cultured cells and in planta. Cloning of NtPIN genes and their inducible overexpression in tobacco cells uncovered high auxin efflux activity of NtPIN11, accompanied by auxin starvation symptoms. Auxin transport parameters after NtPIN11 overexpression were further assessed using radiolabelled auxin accumulation and mathematical modelling. Unexpectedly, these experiments showed notable stimulation of auxin influx, which was accompanied by enhanced transcript levels of genes for a specific auxin influx carrier and by decreased transcript levels of other genes for auxin efflux carriers. A similar transcriptional response was observed upon removal of auxin from the culture medium, which resulted in decreased auxin efflux. Overall, our results revealed an auxin transport-based homeostatic mechanism for the maintenance of endogenous auxin levels. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at http://osf.io/ka97b/.

CgIPT1 is required for synthesis of cis-zeatin cytokinins and contributes to stress tolerance and virulence in Colletotrichum graminicola.

We have previously shown that the maize pathogen Colletotrichum graminicola is able to synthesise cytokinins (CKs). However, it remained unsettled whether fungal CK production is essential for virulence in this hemibiotrophic fungus. Here, we identified a candidate gene, CgIPT1, that is homologous to MOD5 of Saccharomyces cerevisiae and genes from other fungi and plants, which encode tRNA-isopentenyltransferases (IPTs). We show that the wild type strain mainly synthesises cis-zeatin-type (cisZ) CKs whereas ΔCgipt1 mutants are severely impeded to do so. The spectrum of CKs produced confirms bioinformatical analyses predicting that CgIpt1 is a tRNA-IPT. The virulence of the ΔCgipt1 mutants is moderately reduced. Furthermore, the mutants exhibit increased sensitivities to osmotic stress imposed by sugar alcohols and salts, as well as cell wall stress imposed by Congo red. Amendment of media with CKs did not reverse this phenotype suggesting that fungal-derived CKs do not explain the role of CgIpt1 in mediating abiotic stress tolerance. Moreover, the mutants still cause green islands on senescing maize leaves indicating that the cisZ-type CKs produced by the fungus do not cause this phenotype.

Distinct metabolism of N-glucosides of isopentenyladenine and trans-zeatin determines cytokinin metabolic spectrum in Arabidopsis.

The diversity of cytokinin (CK) metabolites suggests their interconversions are the predominant regulatory mechanism of CK action. Nevertheless, little is known about their directionality and kinetics in planta. CK metabolite levels were measured in 2-wk-old Arabidopsis thaliana plants at several time points up to 100 min following exogenous application of selected CKs. The data were then evaluated qualitatively and by mathematical modeling. Apart from elevated levels of trans-zeatin (tZ) metabolites upon application of N6 -(Δ2 -isopentenyl)adenine (iP), we observed no conversions between the individual CK-types - iP, tZ, dihydrozeatin (DHZ) and cis-zeatin (cZ). In particular, there was no sign of isomerization between tZ and cZ families. Also, no increase of DHZ-type CKs was observed after application of tZ, suggesting low baseline activity of zeatin reductase. Among N-glucosides, those of iP were not converted back to iP while tZ N-glucosides were cleaved to tZ bases, thus affecting the whole metabolic spectrum. We present the first large-scale study of short-term CK metabolism kinetics and show that tZ N7- and N9-glucosides are metabolized in vivo. We thus refute the generally accepted hypothesis that N-glucosylation irreversibly inactivates CKs. The subsequently constructed mathematical model provides estimates of the metabolic conversion rates.

A novel putative auxin carrier family regulates intracellular auxin homeostasis in plants.

The phytohormone auxin acts as a prominent signal, providing, by its local accumulation or depletion in selected cells, a spatial and temporal reference for changes in the developmental program. The distribution of auxin depends on both auxin metabolism (biosynthesis, conjugation and degradation) and cellular auxin transport. We identified in silico a novel putative auxin transport facilitator family, called PIN-LIKES (PILS). Here we illustrate that PILS proteins are required for auxin-dependent regulation of plant growth by determining the cellular sensitivity to auxin. PILS proteins regulate intracellular auxin accumulation at the endoplasmic reticulum and thus auxin availability for nuclear auxin signalling. PILS activity affects the level of endogenous auxin indole-3-acetic acid (IAA), presumably via intracellular accumulation and metabolism. Our findings reveal that the transport machinery to compartmentalize auxin within the cell is of an unexpected molecular complexity and demonstrate this compartmentalization to be functionally important for a number of developmental processes.

Hormonal Responses to Plasmodiophora brassicae Infection in Brassica napus Cultivars Differing in Their Pathogen Resistance.

Hormonal dynamics after Plasmodiophora brassicae infection were compared in two Brassica napus cultivars-more resistant SY Alister and more sensitive Hornet, in order to elucidate responses associated with efficient defense. Both cultivars responded to infection by the early transient elevation of active cytokinins (predominantly cis-zeatin) and auxin indole-3-acetic acid (IAA) in leaves and roots, which was longer in Hornet. Moderate IAA levels in Hornet roots coincided with a high expression of biosynthetic gene nitrilase NIT1 (contrary to TAA1, YUC8, YUC9). Alister had a higher basal level of salicylic acid (SA), and it stimulated its production (via the expression of isochorismate synthase (ICS1)) in roots earlier than Hornet. Gall formation stimulated cytokinin, auxin, and SA levels-with a maximum 22 days after inoculation (dai). SA marker gene PR1 expression was the most profound at the time point where gall formation began, in leaves, roots, and especially in galls. Jasmonic acid (JA) was higher in Hornet than in Alister during the whole experiment. To investigate SA and JA function, SA was applied before infection, and twice (before infection and 15 dai), and JA at 15 dai. Double SA application diminished gall formation in Alister, and JA promoted gall formation in both cultivars. Activation of SA/JA pathways reflects the main differences in clubroot resistance.

Control of cytokinin and auxin homeostasis in cyanobacteria and algae.

BACKGROUND AND AIMS: The metabolism of cytokinins (CKs) and auxins in vascular plants is relatively well understood, but data concerning their metabolic pathways in non-vascular plants are still rather rare. With the aim of filling this gap, 20 representatives of taxonomically major lineages of cyanobacteria and algae from Cyanophyceae, Xanthophyceae, Eustigmatophyceae, Porphyridiophyceae, Chlorophyceae, Ulvophyceae, Trebouxiophyceae, Zygnematophyceae and Klebsormidiophyceae were analysed for endogenous profiles of CKs and auxins and some of them were used for studies of the metabolic fate of exogenously applied radiolabelled CK, [3H]trans-zeatin (transZ) and auxin ([3H]indole-3-acetic acid (IAA)), and the dynamics of endogenous CK and auxin pools during algal growth and cell division. METHODS: Quantification of phytohormone levels was performed by high-performance or ultrahigh-performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-MS/MS, UHPLC-MS/MS). The dynamics of exogenously applied [3H]transZ and [3H]IAA in cell cultures were monitored by HPLC with on-line radioactivity detection. KEY RESULTS: The comprehensive screen of selected cyanobacteria and algae for endogenous CKs revealed a predominance of bioactive and phosphate CK forms while O- and N-glucosides evidently did not contribute greatly to the total CK pool. The abundance of cis-zeatin-type CKs and occurrence of CK 2-methylthio derivatives pointed to the tRNA pathway as a substantial source of CKs. The importance of the tRNA biosynthetic pathway was proved by the detection of tRNA-bound CKs during the course of Scenedesmus obliquus growth. Among auxins, free IAA and its oxidation catabolite 2-oxindole-3-acetic acid represented the prevailing endogenous forms. After treatment with [3H]IAA, IAA-aspartate and indole-3-acetyl-1-glucosyl ester were detected as major auxin metabolites. Moreover, different dynamics of endogenous CKs and auxin profiles during S. obliquus culture clearly demonstrated diverse roles of both phytohormones in algal growth and cell division. CONCLUSIONS: Our data suggest the existence and functioning of a complex network of metabolic pathways and activity control of CKs and auxins in cyanobacteria and algae that apparently differ from those in vascular plants.

PIN6 auxin transporter at endoplasmic reticulum and plasma membrane mediates auxin homeostasis and organogenesis in Arabidopsis.

Plant development mediated by the phytohormone auxin depends on tightly controlled cellular auxin levels at its target tissue that are largely established by intercellular and intracellular auxin transport mediated by PIN auxin transporters. Among the eight members of the Arabidopsis PIN family, PIN6 is the least characterized candidate. In this study we generated functional, fluorescent protein-tagged PIN6 proteins and performed comprehensive analysis of their subcellular localization and also performed a detailed functional characterization of PIN6 and its developmental roles. The localization study of PIN6 revealed a dual localization at the plasma membrane (PM) and endoplasmic reticulum (ER). Transport and metabolic profiling assays in cultured cells and Arabidopsis strongly suggest that PIN6 mediates both auxin transport across the PM and intracellular auxin homeostasis, including the regulation of free auxin and auxin conjugates levels. As evidenced by the loss- and gain-of-function analysis, the complex function of PIN6 in auxin transport and homeostasis is required for auxin distribution during lateral and adventitious root organogenesis and for progression of these developmental processes. These results illustrate a unique position of PIN6 within the family of PIN auxin transporters and further add complexity to the developmentally crucial process of auxin transport.

SlDREB2, a tomato dehydration-responsive element-binding 2 transcription factor, mediates salt stress tolerance in tomato and Arabidopsis.

To counter environmental cues, cultivated tomato (Solanum lycopersicum L.) has evolved adaptive mechanisms requiring regulation of downstream genes. The dehydration-responsive element-binding protein 2 (DREB2) transcription factors regulate abiotic stresses responses in plants. Herein, we isolated a novel DREB2-type regulator involved in salinity response, named SlDREB2. Spatio-temporal expression profile together with investigation of its promoter activity indicated that SlDREB2 is expressed during early stages of seedling establishment and in various vegetative and reproductive organs of adult plants. SlDREB2 is up-regulated in roots and young leaves following exposure to NaCl, but is also induced by KCl and drought. Its overexpression in WT Arabidopsis and atdreb2a mutants improved seed germination and plant growth in presence of different osmotica. In tomato, SlDREB2 affected vegetative and reproductive organs development and the intronic sequence present in the 5' UTR drives its expression. Physiological, biochemical and transcriptomic analyses showed that SlDREB2 enhanced plant tolerance to salinity by improvement of K(+) /Na(+) ratio, and proline and polyamines biosynthesis. Exogenous hormonal treatments (abscisic acid, auxin and cytokinins) and analysis of WT and 35S::SlDREB2 tomatoes hormonal contents highlighted SlDREB2 involvement in abscisic acid biosynthesis/signalling. Altogether, our results provide an overview of SlDREB2 mode of action during early salt stress response.

Stress-induced memory alters growth of clonal offspring of white clover (Trifolium repens).

PREMISE OF THE STUDY: The phenotype of an individual can be modified by the environment experienced by its predecessors, a phenomenon called transgenerational or maternal effects. These effects are studied mostly across sexual generations and are thought to be mediated also by epigenetic variation. However, we do not know how important transgenerational effects are across asexual generations of clonal plants. METHODS: We investigated the role of different drought intensities and durations experienced by parental plants of Trifolium repens on the growth of offspring ramets after transplantation of clonal cuttings to control conditions. We also treated half of the plants with 5-azacytidine, which is a demethylating agent, to test the potential role of DNA methylation on transgenerational effects. KEY RESULTS: Transgenerational effects were manifested as increased biomass of offspring ramets if parental plants experienced medium drought applied for a short period and decreased biomass of offspring ramets if parental plants experienced intense drought for a short period. These transgenerational effects were not observed for offspring of parents from the same treatments if these were treated with 5-azacytidine, whose application significantly decreased the amount of 5-methyl-2'-deoxycytidine in plants. CONCLUSIONS: Transgenerational effects might play an important role in the clonal plant Trifolium repens and are probably mediated by epigenetic variation. The growth and behavior of clonal plants might be affected not only by the ambient environment but also by environments that are no longer present at the time of clonal reproduction. This phenomenon can have yet unacknowledged ecological and evolutionary implications for clonal plants.

Tomato (Solanum lycopersicum L.) SlIPT3 and SlIPT4 isopentenyltransferases mediate salt stress response in tomato.

BACKGROUND: Cytokinins (CKs) are involved in response to various environmental cues, including salinity. It has been previously reported that enhancing CK contents improved salt stress tolerance in tomato. However, the underlying mechanisms of CK metabolism and signaling under salt stress conditions remain to be deciphered. RESULTS: Two tomato isopentenyltransferases, SlIPT3 and SlIPT4, were characterized in tomato and Arabidopsis. Both proteins displayed isopentenyltransferase (IPT) activity in vitro, while their encoding genes exhibited different spatio-temporal expression patterns during tomato plant development. SlIPT3 and SlIPT4 were affected by the endogenous CK status, tightly connected with CKs feedback regulation, as revealed by hormonal treatements. In response to salt stress, SlIPT3 and SlIPT4 were strongly repressed in tomato roots, and differently affected in young and old leaves. SlIPT3 overexpression in tomato resulted in high accumulation of different CK metabolites, following modifications of CK biosynthesis-, signaling- and degradation-gene expression. In addition, 35S::SlIPT3 tomato plants displayed improved tolerance to salinity consecutive to photosynthetic pigments and K(+)/Na(+) ratio retention. Involvement of SlIPT3 and SlIPT4 in salt stress response was also observed in Arabidopsis ipt3 knock-out complemented plants, through maintenance of CK homeostasis. CONCLUSIONS: SlIPT3 and SlIPT4 are functional IPTs encoded by differently expressed genes, distinctively taking part in the salinity response. The substantial participation of SlIPT3 in CK metabolism during salt stress has been determined in 35S::SlIPT3 tomato transformants, where enhancement of CKs accumulation significantly improved plant tolerance to salinity, underlining the importance of this phytohormone in stress response.

The Solanum lycopersicum Zinc Finger2 cysteine-2/histidine-2 repressor-like transcription factor regulates development and tolerance to salinity in tomato and Arabidopsis.

The zinc finger superfamily includes transcription factors that regulate multiple aspects of plant development and were recently shown to regulate abiotic stress tolerance. Cultivated tomato (Solanum lycopersicum Zinc Finger2 [SIZF2]) is a cysteine-2/histidine-2-type zinc finger transcription factor bearing an ERF-associated amphiphilic repression domain and binding to the ACGTCAGTG sequence containing two AGT core motifs. SlZF2 is ubiquitously expressed during plant development, and is rapidly induced by sodium chloride, drought, and potassium chloride treatments. Its ectopic expression in Arabidopsis (Arabidopsis thaliana) and tomato impaired development and influenced leaf and flower shape, while causing a general stress visible by anthocyanin and malonyldialdehyde accumulation. SlZF2 enhanced salt sensitivity in Arabidopsis, whereas SlZF2 delayed senescence and improved tomato salt tolerance, particularly by maintaining photosynthesis and increasing polyamine biosynthesis, in salt-treated hydroponic cultures (125 mm sodium chloride, 20 d). SlZF2 may be involved in abscisic acid (ABA) biosynthesis/signaling, because SlZF2 is rapidly induced by ABA treatment and 35S::SlZF2 tomatoes accumulate more ABA than wild-type plants. Transcriptome analysis of 35S::SlZF2 revealed that SlZF2 both increased and reduced expression of a comparable number of genes involved in various physiological processes such as photosynthesis, polyamine biosynthesis, and hormone (notably ABA) biosynthesis/signaling. Involvement of these different metabolic pathways in salt stress tolerance is discussed.

Subcellular homeostasis of phytohormone auxin is mediated by the ER-localized PIN5 transporter.

The plant signalling molecule auxin provides positional information in a variety of developmental processes by means of its differential distribution (gradients) within plant tissues. Thus, cellular auxin levels often determine the developmental output of auxin signalling. Conceptually, transmembrane transport and metabolic processes regulate the steady-state levels of auxin in any given cell. In particular, PIN auxin-efflux-carrier-mediated, directional transport between cells is crucial for generating auxin gradients. Here we show that Arabidopsis thaliana PIN5, an atypical member of the PIN gene family, encodes a functional auxin transporter that is required for auxin-mediated development. PIN5 does not have a direct role in cell-to-cell transport but regulates intracellular auxin homeostasis and metabolism. PIN5 localizes, unlike other characterized plasma membrane PIN proteins, to endoplasmic reticulum (ER), presumably mediating auxin flow from the cytosol to the lumen of the ER. The ER localization of other PIN5-like transporters (including the moss PIN) indicates that the diversification of PIN protein functions in mediating auxin homeostasis at the ER, and cell-to-cell auxin transport at the plasma membrane, represent an ancient event during the evolution of land plants.

Kaempferol 3-O-rhamnoside-7-O-rhamnoside is an endogenous flavonol inhibitor of polar auxin transport in Arabidopsis shoots.

Polar auxin transport (PAT) plays key roles in the regulation of plant growth and development. Flavonoids have been implicated in the inhibition of PAT. However, the active flavonoid derivative(s) involved in this process in vivo has not yet been identified. Here, we provide evidence that a specific flavonol bis-glycoside is correlated with shorter plant stature and reduced PAT. Specific flavonoid-biosynthetic or flavonoid-glycosylating steps were genetically blocked in Arabidopsis thaliana. The differential flavonol patterns established were analyzed by high-performance liquid chromatography (HPLC) and related to altered plant stature. PAT was monitored in stem segments using a radioactive [(3)H]-indole-3-acetic acid tracer. The flavonoid 3-O-glucosyltransferase mutant ugt78d2 exhibited a dwarf stature in addition to its altered flavonol glycoside pattern. This was accompanied by reduced PAT in ugt78d2 shoots. The ugt78d2-dependent growth defects were flavonoid dependent, as they were rescued by genetic blocking of flavonoid biosynthesis. Phenotypic and metabolic analyses of a series of mutants defective at various steps of flavonoid formation narrowed down the potentially active moiety to kaempferol 3-O-rhamnoside-7-O-rhamnoside. Moreover, the level of this compound was negatively correlated with basipetal auxin transport. These results indicate that kaempferol 3-O-rhamnoside-7-O-rhamnoside acts as an endogenous PAT inhibitor in Arabidopsis shoots.

The Pseudomonas type III effector HopQ1 activates cytokinin signaling and interferes with plant innate immunity.

We characterized the molecular function of the Pseudomonas syringae pv. tomato DC3000 (Pto) effector HopQ1. In silico studies suggest that HopQ1 might possess nucleoside hydrolase activity based on the presence of a characteristic aspartate motif. Transgenic Arabidopsis lines expressing HopQ1 or HopQ1 aspartate mutant variants were characterized with respect to flagellin triggered immunity, phenotype and changes in phytohormone content by high-performance liquid chromatography-MS (HPLC-MS). We found that HopQ1, but not its aspartate mutants, suppressed all tested immunity marker assays. Suppression of immunity was the result of a lack of the flagellin receptor FLS2, whose gene expression was abolished by HopQ1 in a promoter-dependent manner. Furthermore, HopQ1 induced cytokinin signaling in Arabidopsis and the elevation in cytokinin signaling appears to be responsible for the attenuation of FLS2 expression. We conclude that HopQ1 can activate cytokinin signaling and that moderate activation of cytokinin signaling leads to suppression of FLS2 accumulation and thus defense signaling.

Constitutive salicylic acid accumulation in pi4kIIIß1ß2 Arabidopsis plants stunts rosette but not root growth.

Phospholipids have recently been found to be integral elements of hormone signalling pathways. An Arabidopsis thaliana double mutant in two type III phosphatidylinositol-4-kinases (PI4Ks), pi4kIIIß1ß2, displays a stunted rosette growth. The causal link between PI4K activity and growth is unknown. Using microarray analysis, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and multiple phytohormone analysis by LC-MS we investigated the mechanism responsible for the pi4kIIIß1ß2 phenotype. The pi4kIIIß1ß2 mutant accumulated a high concentration of salicylic acid (SA), constitutively expressed SA marker genes including PR-1, and was more resistant to Pseudomonas syringae. pi4kIIIß1ß2 was crossed with SA signalling mutants eds1 and npr1 and SA biosynthesis mutant sid2 and NahG. The dwarf phenotype of pi4kIIIß1ß2 rosettes was suppressed in all four triple mutants. Whereas eds1 pi4kIIIß1ß2, sid2 pi4kIIIß1ß2 and NahG pi4kIIIß1ß2 had similar amounts of SA as the wild-type (WT), npr1pi4kIIIß1ß2 had more SA than pi4kIIIß1ß2 despite being less dwarfed. This indicates that PI4KIIIß1 and PI4KIIIß2 are genetically upstream of EDS1 and need functional SA biosynthesis and perception through NPR1 to express the dwarf phenotype. The slow root growth phenotype of pi4kIIIß1ß2 was not suppressed in any of the triple mutants. The pi4kIIIß1ß2 mutations together cause constitutive activation of SA signalling that is responsible for the dwarf rosette phenotype but not for the short root phenotype.

Transcriptional and hormonal regulation of petal and stamen development by STAMENLESS, the tomato (Solanum lycopersicum L.) orthologue to the B-class APETALA3 gene.

Four B-class MADS box genes specify petal and stamen organ identities in tomato. Several homeotic mutants affected in petal and stamen development were described in this model species, although the causal mutations have not been identified for most of them. In this study we characterized a strong stamenless mutant in the tomato Primabel cultivar (sl-Pr), which exhibited homeotic conversion of petals into sepals and stamens into carpels and we compared it with the stamenless mutant in the LA0269 accession (sl-LA0269). Genetic complementation analysis proved that both sl mutants were allelic. Sequencing revealed point mutations in the coding sequence of the Tomato APETALA3 (TAP3) gene of the sl-Pr genome, which lead to a truncated protein, whereas a chromosomal rearrangement in the TAP3 promoter was detected in the sl-LA0269 allele. Moreover, the floral phenotype of TAP3 antisense plants exhibited identical homeotic changes to sl mutants. These results demonstrate that SL is the tomato AP3 orthologue and that the mutant phenotype correlated to the SL silencing level. Expression analyses showed that the sl-Pr mutation does not affect the expression of other tomato B-class genes, although SL may repress the A-class gene MACROCALYX. A partial reversion of the sl phenotype by gibberellins, gene expression analysis, and hormone quantification in sl flowers revealed a role of phytohormones in flower development downstream of the SL gene. Together, our results indicated that petal and stamen identity in tomato depends on gene-hormone interactions, as mediated by the SL gene.