Multifunctional Contribution of the Inflated Fruiting Calyx: Implication for Cuticular Barrier Profiles of the Solanaceous Genera Physalis, Alkekengi, and Nicandra.

Pivotal barrier properties of the hydrophobic plant cuticle covering aerial plant surfaces depend on its physicochemical composition. Among plant species and organs, compounds of this boundary layer between the plant interior and the environment vary considerably but cuticle-related studies comparing different organs from the same plant species are still scarce. Thus, this study focused on the cuticle profiles of Physalis peruviana, Physalis ixocarpa, Alkekengi officinarum, and Nicandra physalodes species. Inflated fruiting calyces enveloping fruits make Physalis, Alkekengi, and Nicandra highly recognizable genera among the Solanoideae subfamily. Although the inflation of fruiting calyces is well discussed in the literature still little is known about their post-floral functionalities. Cuticular composition, surface structure, and barrier function were examined and compared in fully expanded amphistomatous leaves, ripe astomatous fruits, and fully inflated hypostomatous fruiting calyces. Species- and organ-specific abundances of non-glandular and glandular trichomes revealed high structural diversity, covering not only abaxial and adaxial leaf surfaces but also fruiting calyx surfaces, whereas fruits were glabrous. Cuticular waxes, which limit non-stomatal transpiration, ranged from <1 µg cm-2 on P. peruviana fruiting calyces and N. physalodes fruits to 22 µg cm-2 on P. peruviana fruits. Very-long-chain aliphatic compounds, notably n-alkanes, iso-, and anteiso-branched alkanes, alkanols, alkanoic acids, and alkyl esters, dominated the cuticular wax coverages (≥86%). Diversity of cuticular wax patterns rose from leaves to fruiting calyces and peaked in fruits. The polymeric cutin matrix providing the structural framework for cuticular waxes was determined to range from 81 µg cm-2 for N. physalodes to 571 µg cm-2 for A. officinarum fruits. Cuticular transpiration barriers were highly efficient, with water permeabilities being ≤5 × 10-5 m s-1. Only the cuticular water permeability of N. physalodes fruits was 10 × 10-5 m s-1 leading to their early desiccation and fruits that easily split, whereas P. peruviana, P. ixocarpa, and A. officinarum bore fleshy fruits for extended periods after maturation. Regarding the functional significance, fruiting calyces establish a physicochemical shield that reduces water loss and enables fruit maturation within a protective microclimate, and promotes different seed dispersal strategies among plant species investigated.

Optogenetic control of plant growth by a microbial rhodopsin.

While rhodopsin-based optogenetics has revolutionized neuroscience1,2, poor expression of opsins and the absence of the essential cofactor all-trans-retinal has complicated the application of rhodopsins in plants. Here, we demonstrate retinal production in plants and improved rhodopsin targeting for green light manipulation of plant cells using the Guillardia theta light-gated anion channelrhodopsin GtACR13. Green light induces a massive increase in anion permeability and pronounced membrane potential changes when GtACR1 is expressed, enabling non-invasive manipulation of plant growth and leaf development. Using light-driven anion loss, we could mimic drought conditions and bring about leaf wilting despite sufficient water supply. Expressed in pollen tubes, global GtACR1 activation triggers membrane potential depolarizations due to large anion currents. While global illumination was associated with a reversible growth arrest, local GtACR1 activation at the flanks of the apical dome steers growth direction away from the side with increased anion conductance. These results suggest a crucial role of anion permeability for the guidance of pollen tube tip growth. This plant optogenetic approach could be expanded to create an entire pallet of rhodopsin-based tools4, greatly facilitating dissection of plant ion-signalling pathways.

Compositional, structural and functional cuticle analysis of Prunus laurocerasus L. sheds light on cuticular barrier plasticity.

Barrier properties of the hydrophobic plant cuticle depend on its physicochemical composition. The cuticular compounds vary considerably among plant species but also among organs and tissues of the same plant and throughout developmental stages. As yet, these intraspecific modifications at the cuticular wax and cutin level are only rarely examined. Attempting to further elucidate cuticle profiles, we analysed the adaxial and abaxial surfaces of the sclerophyllous leaf and three developmental stages of the drupe fruit of Prunus laurocerasus, an evergreen model plant native to temperate regions. According to gas chromatographic analyses, the cuticular waxes contained primarily pentacyclic triterpenoids dominated by ursolic acid, whereas the cutin biopolyester mainly consisted of 9/10,ω-dihydroxy hexadecanoic acid. Distinct organ- and side-specific patterns were found for cuticular lipid loads, compositions and carbon chain length distributions. Compositional variations led to different structural and functional barrier properties of the plant cuticle, which were investigated further microscopically, infrared spectroscopically and gravimetrically. The minimum water conductance was highlighted at 1 × 10-5 m s-1 for the perennial, hypostomatous P. laurocerasus leaf and at 8 × 10-5 m s-1 for the few-month-living, stomatous fruit suggesting organ-specific cuticular barrier demands.

Leaf cuticle analyses: implications for the existence of cutan/non-ester cutin and its biosynthetic origin.

BACKGROUND AND AIMS: The cuticle of a limited number of plant species contains cutan, a chemically highly resistant biopolymer. As yet, the biosynthesis of cutan is not fully understood. Attempting to further unravel the origin of cutan, we analysed the chemical composition of enzymatically isolated cuticular membranes of Agave americana leaves. METHODS: Cuticular waxes were extracted with organic solvents. Subsequently, the dewaxed cuticular membrane was depolymerized by acid-catalysed transesterification yielding cutin monomers and cutan, a non-hydrolysable, cuticular membrane residue. The cutan matrix was analysed by thermal extraction, flash pyrolysis and thermally assisted hydrolysis and methylation to elucidate the monomeric composition and deduce a putative biosynthetic origin. KEY RESULTS: According to gas chromatography-mass spectrometry analyses, the cuticular waxes of A. americana contained primarily very-long-chain alkanoic acids and primary alkanols dominated by C32, whereas the cutin biopolyester of A. americana mainly consisted of 9,10-epoxy ω-hydroxy and 9,10,ω-trihydroxy C18 alkanoic acids. The main aliphatic cutan monomers were alkanoic acids, primary alkanols, ω-hydroxy alkanoic acids and alkane-α,ω-diols ranging predominantly from C28 to C34 and maximizing at C32. Minor contributions of benzene-1,3,5-triol and derivatives suggested that these aromatic moieties form the polymeric core of cutan, to which the aliphatic moieties are linked via ester and possibly ether bonds. CONCLUSIONS: High similarity of aliphatic moieties in the cutan and the cuticular wax component indicated a common biosynthetic origin. In order to exclude species-specific peculiarities of A. americana and to place our results in a broader context, cuticular waxes, cutin and cutan of Clivia miniata, Ficus elastica and Prunus laurocerasus leaves were also investigated. A detailed comparison showed compositional and structural differences, indicated that cutan was only found in leaves of perennial evergreen A. americana and C. miniata, and made clear that the phenomenon of cutan is possibly less present in plant species than suggested in the literature.

Cuticular wax coverage and its transpiration barrier properties in Quercus coccifera L. leaves: does the environment matter?

Plants prevent uncontrolled water loss by synthesizing, depositing and maintaining a hydrophobic layer over their primary aerial organs-the plant cuticle. Quercus coccifera L. can plastically respond to environmental conditions at the cuticular level. When exposed to hot summer conditions with high vapour-pressure deficit (VPD) and intense solar radiation (Mediterranean atmospheric conditions; MED), this plant species accumulates leaf cuticular waxes even over the stomata, thereby decreasing transpirational water loss. However, under mild summer conditions with moderate VPD and regular solar radiation (temperate atmospheric conditions; TEM), this effect is sharply reduced. Despite the ecophysiological importance of the cuticular waxes of Q. coccifera, the wax composition and its contribution to avoiding uncontrolled dehydration remain unknown. Thus, we determined several leaf traits for plants exposed to both MED and TEM conditions. Further, we qualitatively and quantitatively investigated the cuticular lipid composition by gas chromatography. Finally, we measured the minimum leaf conductance (gmin) as an indicator of the efficacy of the cuticular transpiration barrier. The MED leaves were smaller, stiffer and contained a higher load of cuticular lipids than TEM leaves. The amounts of leaf cutin and cuticular waxes of MED plants were 1.4 times and 2.6 times higher than that found for TEM plants, respectively. In detail, MED plants produced higher amounts of all compound classes of cuticular waxes, except for the equivalence of alkanoic acids. Although MED leaves contained higher cutin and cuticular wax loads, the gmin was not different between the two habitats. Our findings suggest that the qualitative accumulation of equivalent cuticular waxes might compensate for the higher wax amount of MED plants, thereby contributing equally to the efficacy of the cuticular transpirational barrier of Q. coccifera. In conclusion, we showed that atmospheric conditions profoundly affect the cuticular lipid composition of Q. coccifera leaves, but do not alter its transpiration barrier properties.

Cuticular wax coverage and its transpiration barrier properties in Quercus coccifera L. leaves: does the environment matter?

Plants prevent uncontrolled water loss by synthesizing, depositing and maintaining a hydrophobic layer over their primary aerial organs-the plant cuticle. Quercus coccifera L. can plastically respond to environmental conditions at the cuticular level. When exposed to hot summer conditions with high vapour-pressure deficit (VPD) and intense solar radiation (Mediterranean atmospheric conditions; MED), this plant species accumulates leaf cuticular waxes even over the stomata, thereby decreasing transpirational water loss. However, under mild summer conditions with moderate VPD and regular solar radiation (temperate atmospheric conditions; TEM), this effect is sharply reduced. Despite the ecophysiological importance of the cuticular waxes of Q. coccifera, the wax composition and its contribution to avoiding uncontrolled dehydration remain unknown. Thus, we determined several leaf traits for plants exposed to both MED and TEM conditions. Further, we qualitatively and quantitatively investigated the cuticular lipid composition by gas chromatography. Finally, we measured the minimum leaf conductance (gmin) as an indicator of the efficacy of the cuticular transpiration barrier. The MED leaves were smaller, stiffer and contained a higher load of cuticular lipids than TEM leaves. The amounts of leaf cutin and cuticular waxes of MED plants were 1.4 times and 2.6 times higher than that found for TEM plants, respectively. In detail, MED plants produced higher amounts of all compound classes of cuticular waxes, except for the equivalence of alkanoic acids. Although MED leaves contained higher cutin and cuticular wax loads, the gmin was not different between the two habitats. Our findings suggest that the qualitative accumulation of equivalent cuticular waxes might compensate for the higher wax amount of MED plants, thereby contributing equally to the efficacy of the cuticular transpirational barrier of Q. coccifera. In conclusion, we showed that atmospheric conditions profoundly affect the cuticular lipid composition of Q. coccifera leaves, but do not alter its transpiration barrier properties.

Effects of temperature on the cuticular transpiration barrier of two desert plants with water-spender and water-saver strategies.

The efficacy of the cuticular transpiration barrier and its resistance to elevated temperatures are significantly higher in a typical water-saver than in a water-spender plant growing in hot desert.

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.

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

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.

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.

Deficiency in a very-long-chain fatty acid ß-ketoacyl-coenzyme a synthase of tomato impairs microgametogenesis and causes floral organ fusion.

Previously, it was shown that ß-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.

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.

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.

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.

The developmental pattern of tomato fruit wax accumulation and its impact on cuticular transpiration barrier properties: effects of a deficiency in a beta-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 beta-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 beta-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 > C(28) 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.

Tomato fruit cuticular waxes and their effects on transpiration barrier properties: functional characterization of a mutant deficient in a very-long-chain fatty acid beta-ketoacyl-CoA synthase.

Cuticular waxes play a pivotal role in limiting transpirational water loss across the plant surface. The correlation between the chemical composition of the cuticular waxes and their function as a transpiration barrier is still unclear. In the present study, intact tomato fruits (Lycopersicon esculentum) are used, due to their astomatous surface, as a novel integrative approach to investigate this composition- function relationship: wax amounts and compositions of tomato were manipulated before measuring unbiased cuticular transpiration. First, successive mechanical and extractive wax-removal steps allowed the selective modification of epi- and intracuticular wax layers. The epicuticular film consisted exclusively of very-long-chain aliphatics, while the intracuticular compartment contained large quantities of pentacyclic triterpenoids as well. Second, applying reverse genetic techniques, a loss-of-function mutation with a transposon insertion in a very-long-chain fatty acid elongase beta-ketoacyl-CoA synthase was isolated and characterized. Mutant leaf and fruit waxes were deficient in n-alkanes and aldehydes with chain lengths beyond C30, while shorter chains and branched hydrocarbons were not affected. The mutant fruit wax also showed a significant increase in intracuticular triterpenoids. Removal of the epicuticular wax layer, accounting for one-third of the total wax coverage on wild-type fruits, had only moderate effects on transpiration. By contrast, reduction of the intracuticular aliphatics in the mutant to approximately 50% caused a 4-fold increase in permeability. Hence, the main portion of the transpiration barrier is located in the intracuticular wax layer, largely determined by the aliphatic constituents, but modified by the presence of triterpenoids, whereas epicuticular aliphatics play a minor role.