Publications - Cell and Metabolic Biology

BackgroundAdventitious roots (ARs) are often necessary for plant survival, and essential for successful micropropagation. In Arabidopsis thaliana dark-grown seedlings AR-formation occurs from the hypocotyl and is enhanced by application of indole-3-butyric acid (IBA) combined with kinetin (Kin). The same IBA + Kin-treatment induces AR-formation in thin cell layers (TCLs). Auxin is the main inducer of AR-formation and xylogenesis in numerous species and experimental systems. Xylogenesis is competitive to AR-formation in Arabidopsis hypocotyls and TCLs. Jasmonates (JAs) negatively affect AR-formation in de-etiolated Arabidopsis seedlings, but positively affect both AR-formation and xylogenesis in tobacco dark-grown IBA + Kin TCLs. In Arabidopsis the interplay between JAs and auxin in AR-formation vs xylogenesis needs investigation. In de-etiolated Arabidopsis seedlings, the Auxin Response Factors ARF6 and ARF8 positively regulate AR-formation and ARF17 negatively affects the process, but their role in xylogenesis is unknown. The cross-talk between auxin and ethylene (ET) is also important for AR-formation and xylogenesis, occurring through EIN3/EIL1 signalling pathway. EIN3/EIL1 is the direct link for JA and ET-signalling. The research investigated JA role on AR-formation and xylogenesis in Arabidopsis dark-grown seedlings and TCLs, and the relationship with ET and auxin. The JA-donor methyl-jasmonate (MeJA), and/or the ET precursor 1-aminocyclopropane-1-carboxylic acid were applied, and the response of mutants in JA-synthesis and -signalling, and ET-signalling investigated. Endogenous levels of auxin, JA and JA-related compounds, and ARF6, ARF8 and ARF17 expression were monitored.ResultsMeJA, at 0.01 μM, enhances AR-formation, when combined with IBA + Kin, and the response of the early-JA-biosynthesis mutant dde2–2 and the JA-signalling mutant coi1–16 confirmed this result. JA levels early change during TCL-culture, and JA/JA-Ile is immunolocalized in AR-tips and xylogenic cells. The high AR-response of the late JA-biosynthesis mutant opr3 suggests a positive action also of 12-oxophytodienoic acid on AR-formation. The crosstalk between JA and ET-signalling by EIN3/EIL1 is critical for AR-formation, and involves a competitive modulation of xylogenesis. Xylogenesis is enhanced by a MeJA concentration repressing AR-formation, and is positively related to ARF17 expression.ConclusionsThe JA concentration-dependent role on AR-formation and xylogenesis, and the interaction with ET opens the way to applications in the micropropagation of recalcitrant species.

BackgroundType VI glandular trichomes represent the most abundant trichome type on leaves and stems of tomato plants and significantly contribute to herbivore resistance, particularly in the wild species. Despite this, their development has been poorly studied so far. The goal of this study is to fill this gap. Using a variety of cell imaging techniques, a detailed record of the anatomy and developmental stages of type VI trichomes in the cultivated tomato (Solanum lycopersicum) and in a related wild species (S. habrochaites) is provided.ResultsIn both species, the development of these structures follows a highly reproducible cell division pattern. The two species differ in the shape of the trichome head which is round in S. habrochaites and like a four-leaf clover in S. lycopersicum, correlating with the presence of a large intercellular cavity in S. habrochaites where the produced metabolites accumulate. In both species, the junction between the intermediate cell and the four glandular cells constitute a breaking point facilitating the decapitation of the trichome and thereby the quick release of the metabolites. A strongly auto-fluorescent compound transiently accumulates in the early stages of development suggesting a potential role in the differentiation process. Finally, immuno-labelling with antibodies recognizing specific cell wall components indicate a key role of pectin and arabinogalactan components in the differentiation of type VI trichomes.ConclusionsOur observations explain the adaptive morphologies of type VI trichomes for metabolite storage and release and provide a framework for further studies of these important metabolic cellular factories. This is required to better exploit their potential, in particular for the breeding of pest resistance in tomato.

BackgroundPetunia is a model to study the process of adventitious root (AR) formation on leafy cuttings. Excision of cuttings leads to a transient increase in jasmonates, which is regarded as an early, transient and critical event for rooting. Here, the role of jasmonates in AR formation on petunia cuttings has been studied by a reverse genetic approach.ResultsTo reduce the endogenous levels of jasmonates, transgenic plants were generated expressing a Petunia hybrida ALLENE OXIDE CYCLASE (PhAOC)-RNAi construct. The transgenic plants exhibited strongly reduced PhAOC transcript and protein levels as well as diminished accumulation of cis-12-oxo-phytodienoic acid, jasmonic acid and jasmonoyl-isoleucine after wounding in comparison to wild type and empty vector expressing plants. Reduced levels of endogenous jasmonates resulted in formation of lower numbers of ARs. However, this effect was not accompanied by altered levels of auxin and aminocyclopropane carboxylate (ACC, precursor of ethylene) or by impaired auxin and ethylene-induced gene expression. Neither activity of cell-wall invertases nor accumulation of soluble sugars was altered by jasmonate deficiency.ConclusionsDiminished numbers of AR in JA-deficient cuttings suggest that jasmonates act as positive regulators of AR formation in petunia wild type. However, wound-induced rise in jasmonate levels in petunia wild type cuttings seems not to be causal for increased auxin and ethylene levels and for sink establishment.

BackgroundIdentification of genes with invariant levels of gene expression is a prerequisite for validating transcriptomic changes accompanying development. Ideally expression of these genes should be independent of the morphogenetic process or environmental condition tested as well as the methods used for RNA purification and analysis.ResultsIn an effort to identify endogenous genes meeting these criteria nine reference genes (RG) were tested in two Petunia lines (Mitchell and V30). Growth conditions differed in Mitchell and V30, and different methods were used for RNA isolation and analysis. Four different software tools were employed to analyze the data. We merged the four outputs by means of a non-weighted unsupervised rank aggregation method. The genes identified as optimal for transcriptomic analysis of Mitchell and V30 were EF1α in Mitchell and CYP in V30, whereas the least suitable gene was GAPDH in both lines.ConclusionsThe least adequate gene turned out to be GAPDH indicating that it should be rejected as reference gene in Petunia. The absence of correspondence of the best-suited genes suggests that assessing reference gene stability is needed when performing normalization of data from transcriptomic analysis of flower and leaf development.

Diadenosine 5′,5′”‐P1,P4‐tetraphosphate (Ap4A) cleaving enzymes are assumed to regulate intracellular levels of Ap4A, a compound known to affect cell proliferation and stress responses. From plants an Ap4A hydrolase was recently purified using tomato cells grown in suspension. It was partially sequenced and a peptide antibody was prepared (Feussner et al., 1996). Using this polyclonal monospecific antibody, an abundant nuclear location of Ap4A hydrolase in 4‐day‐old cells of atomato cell suspension culture is demonstrated here by means of immunocytochemical techniques using FITC (fluorescein‐5‐isothiocyanate) labeled secondary antibodies. The microscopic analysis of the occurrence of Ap4A hydrolase performed for different stages of the cell cycle visualized by parallel DAPI (4,6‐diamidino‐2‐phenylindole) staining revealed that the protein accumulates within nuclei of cells in the interphase, but is absent in the nucleus as well as cytoplasm during all stages of mitosis. This first intracellular localization of an Ap4A degrading enzyme within the nucleus and its pattern of appearance during the cell cycle is discussed in relation to the suggested role of Ap4A in triggering DNA synthesis and cell proliferation.

Changes in lipoxygenase (LOX) protein pattern and/or activity were investigated in relation to acquired resistance of cucumber (Cucumis sativus L.) leaves against two powdery mildews, Sphaerotheca fuliginea (Schlecht) Salmon and Erysiphe cichoracearum DC et Merat. Acquired resistance was established by spraying leaves with salicylic acid (SA) or 2,6‐dichloroisonicotinic acid (INA) and estimated in whole plants by infested leaf area compared to control plants. SA was more effective than INA. According to Western blots, untreated cucumber leaves contained a 97 kDa LOX form, which remained unchanged for up to 48 h after pathogen inoculation. Upon treatment with SA alone for 24 h or with INA plus pathogen, an additional 95 kDa LOX form appeared which had an isoelectric point in the alkaline range. For the induction of this form, a threshold concentration of 1 mM SA was required, higher SA concentrations did not change LOX‐95 expression which remained similar between 24 h and 96 h but further increased upon mildew inoculation. Phloem exudates contained only the LOX‐97 form, in intercellular washing fluid no LOX was detected. dichloroisonicotinic localization revealed LOX protein in the cytosol of the mesophyll cells without differences between the forms.

The plant growth substance jasmonic acid and its methyl ester (JA‐Me) induce a set of proteins (jasmonate‐induced proteins, JIPs) when applied to leaf segments of barley (Hordeum vulgare L. cv. Salome). Most of these JIPs could be localized within different cell compartments by using a combination of biochemical and histochemical methods. Isolation and purification of various cell organelles of barley mesophyll cells, the separation of their proteins by one‐dimensional polyacrylamide gel electrophoresis and the identification of the major abundant JIPs by Western blot analysis, as well as the immuno‐gold labelling of JIPs in ultrathin sections were performed to localize JIPs intracellularly. JIP‐23 was found to be in vacuoles, peroxisomes, and in the granular parts of the nucleus as well as within the cytoplasm; JIP‐37 was detected in vacuoles and in the nucleoplasm; JIP‐66 is a cytosolic protein. Some less abundant JIPs were also localized within different cell compartments: JIP‐100 was found within the stromal fraction of chloroplasts; JIP‐70 is present in the peroxisome and the nucleus; JIP‐50 and JIP‐6 accumulate in vacuoles. The location of JIP‐66 and JIP‐6 confirms their possible physiological role deduced from molecular analysis of their cDNA.