Jana Leide

The Developmental Pattern of Tomato Fruit Wax Accumulation and Its Impact on Cuticular Transpiration Barrier Properties: Effects of a Deficiency in a β-Ketoacyl-Coenzyme A Synthase (LeCER6)

Cuticular waxes play a pivotal role in limiting transpirational water loss across the primary plant surface. The astomatous fruits of the tomato (Lycopersicon esculentum) ‘MicroTom’ and its lecer6 mutant, defective in a β-ketoacyl-coenzyme A synthase, which is involved in very-long-chain fatty acid elongation, were analyzed with respect to cuticular wax load and composition. The developmental course of fruit ripening was followed. Both the ‘MicroTom’ wild type and lecer6 mutant showed similar patterns of quantitative wax accumulation, although exhibiting considerably different water permeances. With the exception of immature green fruits, the lecer6 mutant exhibited about 3- to 8-fold increased water loss per unit time and fruit surface area when compared to the wild type. This was not the case with immature green fruits. The differences in final cuticular barrier properties of tomato fruits in both lines were fully developed already in the mature green to early breaker stage of fruit development. When the qualitative chemical composition of fruit cuticular waxes during fruit ripening was investigated, the deficiency in a β-ketoacyl-coenzyme A synthase in the lecer6 mutant became discernible in the stage of mature green fruits mainly by a distinct decrease in the proportion of n-alkanes of chain lengths > C28 and a concomitant increase in cyclic triterpenoids. This shift in cuticular wax biosynthesis of the lecer6 mutant appears to be responsible for the simultaneously occurring increase of water permeance. Changes in cutin composition were also investigated as a function of developmental stage. This integrative functional approach demonstrates a direct relationship between cuticular transpiration barrier properties and distinct chemical modifications in cuticular wax composition during the course of tomato fruit development.

The positional sterile (ps) mutation affects cuticular transpiration and wax biosynthesis of tomato fruits.

Cuticular waxes are known to play a pivotal role in limiting transpirational water loss across primary plant surfaces. The astomatous tomato fruit is an ideal model system that permits the functional characterization of intact cuticular membranes and therefore allows direct correlation of their permeance for water with their qualitative and quantitative composition. The recessive positional sterile (ps) mutation, which occurred spontaneously in tomato (Solanum lycopersicum L.), is characterized by floral organ fusion and positional sterility. Because of a striking phenotypical similarity with the lecer6 wax mutant of tomato, which is defective in very-long-chain fatty acid elongation, ps mutant fruits were analyzed for their cuticular wax and cutin composition. We also examined their cuticular permeance for water following the developmental course of fruit ripening. Wild type and ps mutant fruits showed considerable differences in their cuticular permeance for water, while exhibiting similar quantitative wax accumulation. The ps mutant fruits showed a five- to eightfold increase in water loss per unit time and surface area when compared to the corresponding wild type fruits. The cuticular waxes of ps mutant fruits were characterized by an almost complete absence of n-alkanes and aldehydes, with a concomitant increase in triterpenoids and sterol derivatives. We also noted the occurrence of alkyl esters not present in the wild type. Quantitative and qualitative cutin monomer composition remained largely unaffected. The significant differences in the cuticular wax composition of ps mutant fruits induced a distinct increase of cuticular water permeance. The fruit wax compositional phenotype indicates the ps mutation is responsible for effectively blocking the decarbonylation pathway of wax biosynthesis in epidermal cells of tomato fruits.

Distribution of cutin and suberin biomarkers under forest trees with different root systems

Background and aimsDifferences in chemical composition of root compounds and root systems among tree species may affect organic matter (OM) distribution, source and composition in forest soils. The objective of this study was to elucidate the contribution of species specific cutin and suberin biomarkers as proxies for shoot- and root-derived organic carbon (OC) to soil OM at different depths with increasing distance to the stems of four different tree species.MethodsThe contribution of cutin- and suberin-derived lipids to OM in a Cutanic Alisol was analyzed with increasing soil depth and distance to the stems of Fagus sylvatica L., Picea abies (L.) Karst., Quercus robur L. and Pseudotsuga menziesii (Mirb.) Franco. Cutin and suberin monomers of plants and soils were analyzed by alkaline hydrolysis and subsequent gas chromatography–mass spectrometry.ResultsThe amount and distribution of suberin-derived lipids in soil clearly reflected the specific root system of the different tree species. The amount of cutin-derived lipids decreased strongly with soil depth, indicating that the input of leaf/needle material is restricted to the topsoil. In contrast to the suberin-derived lipids, the spatial pattern of cutin monomer contribution to soil OM did not depend on tree species.ConclusionsOur results document the importance of tree species as a main factor controlling the composition and distribution of OM in forest soils. They reveal the impact of tree species on root-derived OM distribution and the necessity to distinguish among different zones when studying soil OM storage in forests.

Deficiency in a Very-Long-Chain Fatty Acid β-Ketoacyl-Coenzyme A Synthase of Tomato Impairs Microgametogenesis and Causes Floral Organ Fusion

Previously, it was shown that β-ketoacyl-coenzyme A synthase ECERIFERUM6 (CER6) is necessary for the biosynthesis of very-long-chain fatty acids with chain lengths beyond C28 in tomato (Solanum lycopersicum) fruits and C26 in Arabidopsis (Arabidopsis thaliana) leaves and the pollen coat. CER6 loss of function in Arabidopsis resulted in conditional male sterility, since pollen coat lipids are responsible for contact-mediated pollen hydration. In tomato, on the contrary, pollen hydration does not rely on pollen coat lipids. Nevertheless, mutation in SlCER6 impairs fertility and floral morphology. Here, the contribution of SlCER6 to the sexual reproduction and flower development of tomato was addressed. Cytological analysis and cross-pollination experiments revealed that the slcer6 mutant has male sterility caused by (1) hampered pollen dispersal and (2) abnormal tapetum development. SlCER6 loss of function provokes a decrease of n- and iso-alkanes with chain lengths of C27 or greater and of anteiso-alkanes with chain lengths of C28 or greater in flower cuticular waxes, but it has no impact on flower cuticle ultrastructure and cutin content. Expression analysis confirmed high transcription levels of SlCER6 in the anther and the petal, preferentially in sites subject to epidermal fusion. Hence, wax deficiency was proposed to be the primary reason for the flower fusion phenomenon in tomato. The SlCER6 substrate specificity was revisited. It might be involved in elongation of not only linear but also branched very-long-chain fatty acids, leading to production of the corresponding alkanes. SlCER6 implements a function in the sexual reproduction of tomato that is different from the one in Arabidopsis: SlCER6 is essential for the regulation of timely tapetum degradation and, consequently, microgametogenesis.

Light promotes an increase of cytokinin oxidase/dehydrogenase activity during senescence of barley leaf segments

Following a study of the relationship between cytokinin oxidase/dehydrogenase (CKX) and senescence in darkened barley leaf segments, we have now investigated the influence of light on the in vitro activity of CKX. Seedlings of Hordeum vulgare L. were grown for 8d under a light/dark regime of 18h white light and 6h darkness. Then apical parts of 7cm length were cut from the first foliage leaves and their bases were placed in water. In segments kept in the dark, the CKX activity measured by cleavage of N(6)-(Δ(2)-isopentenyl)adenine rose from 0.1pkat (gFW)(-1) to 0.8pkat (g initial FW)(-1) within the first 4d of incubation. In contrast, in segments kept under the light/dark regime it reached a value of 8.6pkat (g initial FW)(-1) over the same time period. The chlorophyll a content declined slightly slower during light/dark cycling than in darkness. In contrast to segments and isolated laminae, corresponding attached laminae exhibited less CKX activity after 2d under light/dark conditions than after 2d in the dark. The activity in attached laminae of first foliage leaves of plants growing in light/dark cycling increased strongly only when the plants were older than 4 weeks. In line with this, the CKX activity in attached laminae of flag leaves of barley growing in fields increased in a late developmental state. The senescence of darkened isolated laminae of Zea mays L. and Phragmites australis (Cav.) Trin. ex Steudel was associated with an enhancement of CKX activity too. Because in most cases a positive correlation between CKX activity and senescence was found, it is likely that the enzyme promotes senescence by destroying cytokinins, which help to keep Poaceae leaves green. Light may promote not only cytokinin degradation but also the formation of bioactive cytokinins in leaf segments.

Abscisic acid mediates the formation of a suberized stem scar tissue in tomato fruits

During harvest, fleshy berry tomato fruits (Solanum lycopersicum) were wounded at their stem scar. Within 3 d, this wound was rapidly sealed by a process covering the wound site with a membranous layer which effectively protects the tomato fruit from excessive water loss, nutrient elution and the entry of pathogens. Chemical analysis of the de novo synthesized stem scar tissue revealed the presence of aromatic and aliphatic components characteristic of the biopolyester suberin. Gene expression patterns associated with suberization were identified at the stem scar region. Changes in the relative abundance of different transcripts suggested a potential involvement of the plant hormone abscisic acid (ABA) in the wound-healing processes. The amount of ABA present in the stem scar tissue showed a significantly increased level during wound healing, whereas ABA-deficient mutants notabilis, flacca and sitiens were largely devoid of this rise in ABA levels. The mutant fruits showed a retarded and less efficient suberization response at the stem scar wound, whereas the rate and strength of this response were positively correlated with ABA content. These results clearly indicate in vivo the involvement of ABA in the suberization-based wound-healing processes at the stem scar tissue of tomato fruits.

Effectiveness of cuticular transpiration barriers in a desert plant at controlling water loss at high temperatures

Maintaining the integrity of the cuticular transpiration barrier even at elevated temperatures is of vital importance especially for hot-desert plants. Currently, the temperature dependence of the leaf cuticular water permeability and its relationship with the chemistry of the cuticles are not known for a single desert plant. This study investigates whether (i) the cuticular permeability of a desert plant is lower than that of species from non-desert habitats, (ii) the temperature-dependent increase of permeability is less pronounced than in those species and (iii) whether the susceptibility of the cuticular permeability barrier to high temperatures is related to the amounts or properties of the cutin or the cuticular waxes. We test these questions with Rhazya stricta using the minimum leaf water vapour conductance (gmin) as a proxy for cuticular water permeability. gmin of R. stricta (5.41 × 10−5 m s−1 at 25 °C) is in the upper range of all existing data for woody species from various non-desert habitats. At the same time, in R. stricta, the effect of temperature (15–50 °C) on gmin (2.4-fold) is lower than in all other species (up to 12-fold). Rhazya stricta is also special since the temperature dependence of gmin does not become steeper above a certain transition temperature. For identifying the chemical and physical foundation of this phenomenon, the amounts and the compositions of cuticular waxes and cutin were determined. The leaf cuticular wax (251.4 μg cm−2) is mainly composed of pentacyclic triterpenoids (85.2% of total wax) while long-chain aliphatics contribute only 3.4%. In comparison with many other species, the triterpenoid-to-cutin ratio of R. stricta (0.63) is high. We propose that the triterpenoids deposited within the cutin matrix restrict the thermal expansion of the polymer and, thus, prevent thermal damage to the highly ordered aliphatic wax barrier even at high temperatures.

The Nonspecific Lipid Transfer Protein AtLtpI-4 Is Involved in Suberin Formation of Arabidopsis thaliana Crown Galls

AtLtpI-4 involvement in suberin formation is essential for crown gall growth, while ectopic expression in epidermal cells provides evidence for AtLp-I-4 functioning in extracellular lipid deposition. Nonspecific lipid transfer proteins reversibly bind different types of lipid molecules in a hydrophobic cavity. They facilitate phospholipid transfer between membranes in vitro, play a role in cuticle and possibly in suberin formation, and might be involved in plant pathogen defense signaling. This study focuses on the role of the lipid transfer protein AtLTPI-4 in crown gall development. Arabidopsis (Arabidopsis thaliana) crown gall tumors, which develop upon infection with the virulent Agrobacterium tumefaciens strain C58, highly expressed AtLTPI-4. Crown galls of the atltpI-4 loss-of-function mutant were much smaller compared with those of wild-type plants. The gene expression pattern and localization of the protein to the plasma membrane pointed to a function of AtLTPI-4 in cell wall suberization. Since Arabidopsis crown galls are covered by a suberin-containing periderm instead of a cuticle, we analyzed the suberin composition of crown galls and found a reduction in the amounts of long-chain fatty acids (C18:0) in the atltpI-4 mutant. To demonstrate the impact of AtLtpI-4 on extracellular lipid composition, we expressed the protein in Arabidopsis epidermis cells. This led to a significant increase in the very-long-chain fatty acids C24 and C26 in the cuticular wax fraction. Homology modeling and lipid-protein-overlay assays showed that AtLtpI-4 protein can bind these very-long-chain fatty acids. Thus, AtLtpI-4 protein may facilitate the transfer of long-chain as well as very-long-chain fatty acids into the apoplast, depending on the cell type in which it is expressed. In crown galls, which endogenously express AtLtpI-4, it is involved in suberin formation.

Chemical Composition and Water Permeability of Fruit and Leaf Cuticles of Olea europaea L.

The plant cuticle, protecting against uncontrolled water loss, covers olive (Olea europaea) fruits and leaves. The present study describes the organ-specific chemical composition of the cuticular waxes and the cutin and compares three developmental stages of fruits (green, turning, and black) with the leaf surface. Numerous organ-specific differences, such as the total coverage of cutin monomeric components (1034.4 μg cm-2 and 630.5 μg cm-2) and the cuticular waxes (201.6 μg cm-2 and 320.4 μg cm-2) among all three fruit stages and leaves, respectively, were detected. Water permeability as the main cuticular function was 5-fold lower in adaxial leaf cuticles (2.1 × 10-5 m s-1) in comparison to all three fruit stages (9.5 × 10-5 m s-1). The three fruit developmental stages have the same cuticular water permeability. It is hypothesized that a higher weighted average chain length of the acyclic cuticular components leads to a considerably lower permeability of the leaf as compared to the fruit cuticle.