Michel A. Haring

Rapid phosphatidic acid accumulation in response to low temperature stress in Arabidopsis is generated through diacylglycerol kinase

Phosphatidic acid (PtdOH) is emerging as an important signaling lipid in abiotic stress responses in plants. The effect of cold stress was monitored using 32P-labeled seedlings and leaf discs of Arabidopsis thaliana. Low, non-freezing temperatures were found to trigger a very rapid 32P-PtdOH increase, peaking within 2 and 5 min, respectively. In principle, PtdOH can be generated through three different pathways, i.e., (1) via de novo phospholipid biosynthesis (through acylation of lyso-PtdOH), (2) via phospholipase D hydrolysis of structural phospholipids, or (3) via phosphorylation of diacylglycerol (DAG) by DAG kinase (DGK). Using a differential 32P-labeling protocol and a PLD-transphosphatidylation assay, evidence is provided that the rapid 32P-PtdOH response was primarily generated through DGK. A simultaneous decrease in the levels of 32P-PtdInsP, correlating in time, temperature dependency, and magnitude with the increase in 32P-PtdOH, suggested that a PtdInsP-hydrolyzing PLC generated the DAG in this reaction. Testing T-DNA insertion lines available for the seven DGK genes, revealed no clear changes in 32P-PtdOH responses, suggesting functional redundancy. Similarly, known cold-stress mutants were analyzed to investigate whether the PtdOH response acted downstream of the respective gene products. The hos1, los1, and fry1 mutants were found to exhibit normal PtdOH responses. Slight changes were found for ice1, snow1, and the overexpression line Super-ICE1, however, this was not cold-specific and likely due to pleiotropic effects. A tentative model illustrating direct cold effects on phospholipid metabolism is presented.

Dissection of the fusarium I2 gene cluster in tomato reveals six homologs and one active gene copy.

The I2 locus in tomato confers resistance to race 2 of the soil-borne fungus Fusarium oxysporum f sp lycopersici. The selective restriction fragment amplification (AFLP) positional cloning strategy was used to identify I2 in the tomato genome. A yeast artificial chromosome (YAC) clone covering ~750 kb encompassing the I2 locus was isolated, and the AFLP technique was used to derive tightly linked AFLP markers from this YAC clone. Genetic complementation analysis in transgenic R1 plants using a set of overlapping cosmids covering the I2 locus revealed three cosmids giving full resistance to F. o. lycopersici race 2. These cosmids shared a 7-kb DNA fragment containing an open reading frame encoding a protein with similarity to the nucleotide binding site leucine-rich repeat family of resistance genes. At the I2 locus, we identified six additional homologs that included the recently identified I2C-1 and I2C-2 genes. However, cosmids containing the I2C-1 or I2C-2 gene could not confer resistance to plants, indicating that these members are not the functional resistance genes. Alignments between the various members of the I2 gene family revealed two significant variable regions within the leucine-rich repeat region. They consisted of deletions or duplications of one or more leucine-rich repeats. We propose that one or both of these leucine-rich repeats are involved in Fusarium wilt resistance with I2 specificity.

Differential Timing of Spider Mite-Induced Direct and Indirect Defenses in Tomato Plants

Through a combined metabolomics and transcriptomics approach we analyzed the events that took place during the first 5 d of infesting intact tomato (Lycopersicon esculentum) plants with spider mites (Tetranychus urticae). Although the spider mites had caused little visible damage to the leaves after 1 d, they had already induced direct defense responses. For example, proteinase inhibitor activity had doubled and the transcription of genes involved in jasmonate-, salicylate-, and ethylene-regulated defenses had been activated. On day four, proteinase inhibitor activity and particularly transcript levels of salicylate-regulated genes were still maintained. In addition, genes involved in phospholipid metabolism were up-regulated on day one and those in the secondary metabolism on day four. Although transcriptional up-regulation of the enzymes involved in the biosynthesis of monoterpenes and diterpenes already occurred on day one, a significant increase in the emission of volatile terpenoids was delayed until day four. This increase in volatile production coincided with the increased olfactory preference of predatory mites (Phytoseiulus persimilis) for infested plants. Our results indicate that tomato activates its indirect defenses (volatile production) to complement the direct defense response against spider mites.

Jasmonic acid is a key regulator of spider mite-induced volatile terpenoid and methyl salicylate emission in tomato.

The tomato (Lycopersicon esculentum) mutant def-1, which is deficient in induced jasmonic acid (JA) accumulation upon wounding or herbivory, was used to study the role of JA in the direct and indirect defense responses to phytophagous mites (Tetranychus urticae). In contrast to earlier reports, spider mites laid as many eggs and caused as much damage on def-1 as on wild-type plants, even though def-1 lacked induction of proteinase inhibitor activity. However, the hatching-rate of eggs on def-1 was significantly higher, suggesting that JA-dependent direct defenses enhanced egg mortality or increased the time needed for embryonic development. As to gene expression, def-1 had lower levels of JA-related transcripts but higher levels of salicylic acid (SA) related transcripts after 1 d of spider mite infestation. Furthermore, the indirect defense response was absent in def-1, since the five typical spider mite-induced tomato-volatiles (methyl salicylate [MeSA], 4,8,12-trimethyltrideca-1,3,7,11-tetraene [TMTT], linalool, trans-nerolidol, and trans-β-ocimene) were not induced and the predatory mite Phytoseiulus persimilis did not discriminate between infested and uninfested def-1 tomatoes as it did with wild-type tomatoes. Similarly, the expression of the MeSA biosynthetic gene salicylic acid methyltransferase (SAMT) was induced by spider mites in wild type but not in def-1. Exogenous application of JA to def-1 induced the accumulation of SAMT and putative geranylgeranyl diphosphate synthase transcripts and restored MeSA- and TMTT-emission upon herbivory. JA is therefore necessary to induce the enzymatic conversion of SA into MeSA. We conclude that JA is essential for establishing the spider mite-induced indirect defense response in tomato.

ODORANT1 Regulates Fragrance Biosynthesis in Petunia Flowers

Floral scent is important to plant reproduction because it attracts pollinators to the sexual organs. Therefore, volatile emission is usually tuned to the foraging activity of the pollinators. In Petunia hybrida, volatile benzenoids determine the floral aroma. Although the pathways for benzenoid biosynthesis have been characterized, the enzymes involved are less well understood. How production and emission are regulated is unknown. By targeted transcriptome analyses, we identified ODORANT1 (ODO1), a member of the R2R3-type MYB family, as a candidate for the regulation of volatile benzenoids in Petunia hybrida cv W115 (Mitchell) flowers. These flowers are only fragrant in the evening and at night. Transcript levels of ODO1 increased before the onset of volatile emission and decreased when volatile emission declined. Downregulation of ODO1 in transgenic P. hybrida Mitchell plants strongly reduced volatile benzenoid levels through decreased synthesis of precursors from the shikimate pathway. The transcript levels of several genes in this pathway were reduced by suppression of ODO1 expression. Moreover, ODO1 could activate the promoter of the 5-enol-pyruvylshikimate-3-phosphate synthase gene. Flower pigmentation, which is furnished from the same shikimate precursors, was not influenced because color and scent biosynthesis occur at different developmental stages. Our studies identify ODO1 as a key regulator of floral scent biosynthesis.

Regulation of floral scent production in petunia revealed by targeted metabolomics

Petunia hybrida line W115 (Mitchell) has large white flowers that produce a pleasant fragrance. By applying solid phase micro extraction (SPME) techniques coupled to GC-MS analysis, volatile emission was monitored in vivo using a targeted metabolomics approach. Mature flowers released predominantly benzenoid compounds of which benzaldehyde, phenylacetaldehyde, methylbenzoate, phenylethylalcohol, iso-eugenol and benzylbenzoate were most abundant. This emission had a circadian rhythm reaching its maximum at dusk. During petal limb expansion two sesquiterpenes were emitted by the petunia flowers, tentatively identified as germacrene D and cadina-3,9-diene. In vitro analysis showed that the petal limbs and stigma were the main producers of the benzenoids and sesquiterpenes, respectively. Moreover, comparison of in vivo and in vitro analysis indicated that volatiles were not stored during periods of low emission but rather were synthesized de novo. DNA-microarray analysis revealed that genes of the pathways leading to the production of volatile benzenoids were upregulated late during the day, preceding the increase of volatile emission. RNA-gel blot analyses confirmed that the levels of phenylalanine ammonia lyase (PAL) and S-adenosyl methionine (SAM) synthase transcripts increased towards the evening. Our results suggest that the circadian production of volatile benzenoids in petunia W115 is, at least partly, regulated at the transcript level.

Mutations in the NB-ARC Domain of I-2 That Impair ATP Hydrolysis Cause Autoactivation

Resistance (R) proteins in plants confer specificity to the innate immune system. Most R proteins have a centrally located NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) domain. For two tomato (Lycopersicon esculentum) R proteins, I-2 and Mi-1, we have previously shown that this domain acts as an ATPase module that can hydrolyze ATP in vitro. To investigate the role of nucleotide binding and hydrolysis for the function of I-2 in planta, specific mutations were introduced in conserved motifs of the NB-ARC domain. Two mutations resulted in autoactivating proteins that induce a pathogen-independent hypersensitive response upon expression in planta. These mutant forms of I-2 were found to be impaired in ATP hydrolysis, but not in ATP binding, suggesting that the ATP- rather than the ADP-bound state of I-2 is the active form that triggers defense signaling. In addition, upon ADP binding, the protein displayed an increased affinity for ADP suggestive of a change of conformation. Based on these data, we propose that the NB-ARC domain of I-2, and likely of related R proteins, functions as a molecular switch whose state (on/off) depends on the nucleotide bound (ATP/ADP).

Tomato linalool synthase is induced in trichomes by jasmonic acid

Tomato (Lycopersicon esculentum) plants emit a blend of volatile organic compounds, which mainly consists of terpenes. Upon herbivory or wounding, the emission of several terpenes increases. We have identified and characterized the first two tomato monoterpene synthases, LeMTS1 and LeMTS2. Although these proteins were highly homologous, recombinant LeMTS1 protein produced (R)-linalool from geranyl diphosphate (GPP) and (E)-nerolidol from farnesyl diphosphate (FPP), while recombinant LeMTS2 produced β-phellandrene, β-myrcene, and sabinene from GPP. In addition, these genes were expressed in different tissues: LeMTS1 was expressed in flowers, young leaves, stems, and petioles, while LeMTS2 was strongest expressed in stems and roots. LeMTS1 expression in leaves was induced by spider mite-infestation, wounding and jasmonic acid (JA)-treatment, while LeMTS2 did not respond to these stimuli. The expression of LeMTS1 in stems and petioles was predominantly detected in trichomes and could be induced by JA. Because JA treatment strongly induced emission of linalool and overexpression of LeMTS1 in tomato resulted in increased production of linalool, we propose that LeMTS1 is a genuine linalool synthase. Our results underline the importance of trichomes in JA-induced terpene emission in tomato.

The Role of Specific Tomato Volatiles in Tomato-Whitefly Interaction

Bemisia tabaci (whitefly) infestations and the subsequent transfer of viruses are the cause of severe losses in crop production and horticultural practice. To improve biological control of B. tabaci, we investigated repellent properties of plant-produced semiochemicals. The mix of headspace volatiles, collected from naturally repellent wild tomato accessions, influenced B. tabaci initial choice behavior, indicating a role for plant semiochemicals in locating host plants. A collection of wild tomato accessions and introgression lines (Solanum pennellii LA716 × Solanum lycopersicum ‘Moneyberg’) were extensively screened for attractiveness to B. tabaci, and their headspace profiles were determined by means of gas chromatography-mass spectrometry. Correlation analysis revealed that several terpenoids were putatively involved in tomato-whitefly interactions. Several of these candidate compounds conferred repellence to otherwise attractive tomato plants when applied to the plants branches on paper cards. The sesquiterpenes zingiberene and curcumene and the monoterpenes p-cymene, α-terpinene, and α-phellandrene had the strongest effects in free-choice bioassays. These terpenes also elicited a response of receptors on the insects antennae as determined by electroantennography. Conversely, the monoterpene β-myrcene showed no activity in both assays. B. tabaci apparently uses, besides visual cues, specific plant volatile cues for the initial selection of a host. Altering whitefly choice behavior by manipulation of the terpenoid composition of the host headspace may therefore be feasible.

Multiple PLDs Required for High Salinity and Water Deficit Tolerance in Plants

High salinity and drought have received much attention because they severely affect crop production worldwide. Analysis and comprehension of the plants response to excessive salt and dehydration will aid in the development of stress-tolerant crop varieties. Signal transduction lies at the basis of the response to these stresses, and numerous signaling pathways have been implicated. Here, we provide further evidence for the involvement of phospholipase D (PLD) in the plants response to high salinity and dehydration. A tomato (Lycopersicon esculentum) α-class PLD, LePLDα1, is transcriptionally up-regulated and activated in cell suspension cultures treated with salt. Gene silencing revealed that this PLD is indeed involved in the salt-induced phosphatidic acid production, but not exclusively. Genetically modified tomato plants with reduced LePLDα1 protein levels did not reveal altered salt tolerance. In Arabidopsis (Arabidopsis thaliana), both AtPLDα1 and AtPLDδ were found to be activated in response to salt stress. Moreover, pldα1 and pldδ single and double knock-out mutants exhibited enhanced sensitivity to high salinity stress in a plate assay. Furthermore, we show that both PLDs are activated upon dehydration and the knock-out mutants are hypersensitive to hyperosmotic stress, displaying strongly reduced growth.