Survival strategies of plants during secondary growth: barrier properties of phellems and lenticels towards water, oxygen, and carbon dioxide

Ever since plants began to conquer the terrestrial environment, a simple but effective evolutionary strategy has been employed to cope with the combined necessities of preventing an excessive loss of water via the aerial surface while also supporting the vital exchange of CO(2) and O(2) for photosynthesis and respiration. Large areas of the primary above-ground surface of plants are covered by a hydrophobic, non-cellular cuticle which effectively minimizes evaporation and very strongly reduces exchange of CO(2) and O(2). Hence, gas exchange is controlled by regulating stomatal apertures. Upon wounding or entering into secondary growth, however, the epidermis, cuticle, and stomata are replaced by a phellem (cork), which is produced by a lateral cambium, the phellogen. Former stomata are replaced by lenticels, which are multicellular structures and functionally analogous to stomata. In the secondary plant body, phellems effectively prevent the loss of water from the cortex of the stem while lenticels support the exchange of vital gases such as CO(2), O(2), and water vapour. The permeance of these gases via the lenticels reaches a maximum during July and is minimal during autumn and winter. In contrast to stomatal control, gas exchange through phellems is regulated by long-term structural changes. The permeances of cuticles, phellems, and lenticels are compared and discussed.     

Cuticular water permeability and its physiological significance

Cuticles act as solution-diffusion membranes for water transport.Diffusion in pores does not contribute to cuticular transpiration.An extensive literature survey of cuticular permeances (P) andminimum leaf conductances (g_min) to water is presented. Thetwo variables cannot be distinguished with most experimentaltechniques. Results from different experiments are in good agreementwith each other for some species, for example, Fagus sytvaticaL., but not for others, such as Picea abies (L.) Karst. In adata set of 313 values of P or g_min from 200 species, distributionsof results obtained with different techniques were found todiffer significantly. Likely reasons include water loss fromincompletely closed or incompletely sealed stomata, and thedependence of P on moisture content of the cuticle and on storagetime of isolated cuticles. Contrasting evidence for an interactionbetween cuticular transpiration and stomatal sensitivity toair humidity is presented. The occurrence of unusually highg_min in trees growing at the alpine treeline and its physiologicalsignificance are discussed. It is shown that g_min is of littlevalue as a predictor for drought resistance of crops, with thepossible exception of Sorghum bicolor L. Moench. Possible wateruptake from fog or dew across cuticles is considered briefly. Key words: Epidermal conductance, VPD-response, water absorption, waxes, winter desiccation     

Cuticular permeability of the three tree species Prunus Iaurocerasus L., Ginkgo biloba L. and Juglans regia L.: comparative investigation of the transport properties of intact leaves, isolated cuticles and reconstituted cuticular waxes

Cuticular transport properties of intact leaves, isolated cuticularmembranes and reconstituted cuticular waxes of the three treespecies Prunus laurocerasus L., Ginkgo biloba L. and Juglansregia L. were measured using six different ¹⁴C-labelled compounds,benzoic acid, salicylic acid, 2,4-dichlorophenoxy acid, metribuzin,4-nitrophenol, and atrazine. For the same compound and the samespecies, the permeance of the intact leaf and the isolated cuticlewas equal. This provides strong evidence demonstrating thattransport properties of cuticles are not altered during isolation.Additionally, diffusion coefficients of the ¹⁴C-labelled compoundsin isolated and subsequently reconstituted cuticular wax ofthe three tree species were measured. Permeances of intact leavesand isolated cuticles could be predicted from diffusion coefficients,wax/water partition coefficients and the thickness of the transport-limitingwax layer with a mean deviation of about 1.7. This providesevidence that transport properties of recrystallized cuticularwaxes do indeed reflect barrier properties of isolated cuticularmembranes and intact leaves with in situ waxes. Thus, it canbe concluded that the investigation of cuticular permeabilityusing the three independent experimental systems of differentcomplexity give comparable results. Finally, it was observedthat permeances and diffusion coefficients measured with P.laurocerasus were always significantly lower than those measuredwith G. biloba and J. regia. This is interpreted as an ecologicaladaptation of the respective species. The evergreen speciesP. laurocerasus must be more adapted to environmental stresssuch as drought and frost injury compared to the two deciduousspecies G. biloba and J. regia. Key words: Cuticular permeability, diffusion coefficient, leaf surface, permeance, plant cuticle, transport     

Characterization of hydrophilic and lipophilic pathways of Hedera helix L. cuticular membranes: permeation of water and uncharged organic compounds

The permeability of astomatous leaf cuticular membranes of Hedera helix L. was measured for uncharged hydrophilic (octanol/water partition coefficient log K(O/W) < or =0) and lipophilic compounds (log K(O/W) >0). The set of compounds included lipophilic plant protection agents, hydrophilic carbohydrates, and the volatile compounds water and ethanol. Plotting the mobility of the model compounds versus the molar volume resulted in a clear differentiation between a lipophilic and a hydrophilic pathway. The size selectivity of the lipophilic pathway was described by the free volume theory. The pronounced tortuosity of the diffusional path was caused by cuticular waxes, leading to an increase in permeance for the lipophilic compounds after wax extraction. The size selectivity of the hydrophilic pathway was described by hindered diffusion in narrow pores of molecular dimensions. A distinct increase in size selectivity was observed for hydrophilic compounds with a molar volume higher than 110 cm3 mol(-1). Correspondingly, the size distribution of passable hydrophilic pathways was interpreted as a normal distribution with a mean pore radius of 0.3 nm and a standard deviation of 0.02 nm. The increased permeance of the hydrophilic compounds by the removal of cuticular waxes was attributed to an increase in the porosity, a decrease in the tortuosity, and a widening of the pore size distribution. Cuticular transpiration resulted from the permeation of water across the hydrophilic pathway. The far-reaching implications of two parallel pathways for the establishment of correlations between cuticular structure, chemistry, and function are discussed.     

Isolation of cuticular membranes from various conifer needles

Summary A method of isolating intact needle cuticles is presented. Cuticles were separated enzymatically from needles of Abies alba Mill., Picea abies (L.) Karst., Picea pungens Engelm., Pinus mugo Turra, and Taxus baccata L. Cuticle separation depended on the enzyme concentration, the developmental stage of the needles and the duration of incubation in the hydrolytic pectinase/cellulase solution. Cuticles could not be removed from needles older than 2 years. Scanning electron micrographs of enzymatically isolated cuticles are presented. The permeance coefficients for water and oxygen transport across the isolated cuticular membranes indicate their functional intactness. But permeance coefficients also show that isolation of cuticular membranes with chromic acid is an unacceptable method, since they are lo longer structurally or functionally intact following isolation by this method.     

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.     

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.     

Co-permeability of 3H-labelled water and 14C-labelled organic acids across isolated Prunus laurocerasus cuticles: effect of temperature on cuticular paths of diffusion

Co‐permeability of ³H‐labelled water and ¹⁴C‐labelled benzoic acid or 2,4‐dichlorophenoxyacetic acid across isolated cuticular membranes of Prunus laurocerasus L. was measured at temperatures ranging from 15 to 50 °C. The water and benzoic acid permeances were highly correlated over the whole temperature range investigated, whereas water and 2,4‐dichlorophenoxyacetic acid permeances were only correlated between 15 and 30 °C. The activation energies of cuticular permeability calculated from Arrhenius plots were 40 kJ mol^?1 for water and benzoic acid and 115 kJ mol^?1 for 2,4‐dichlorophenoxyacetic acid. The slopes of the Arrhenius plots of 2,4‐dichlorophenoxyacetic acid were linear between 15 and 50 °C, whereas pronounced phase transitions around 30 °C were observed for water and benzoic acid permeability. However, with isolated polymer matrix membranes, where cuticular waxes forming the transport‐limiting barrier of cuticles have been extracted, phase transitions were not observed for water and benzoic acid. It is concluded that temperatures above 30 °C caused structural changes in the transport‐limiting barrier of the cuticles leading to additional paths of diffusion for water and benzoic acid but not for 2,4‐dichlorophenoxyacetic acid.     

Water permeability of isolated cuticular membranes: The effect of cuticular waxes on diffusion of water

Summary The water permeability of astomatous cuticular membranes isolated from Citrus aurantium L. leaves, pear (Pyrus communis L.) leaves and onion (Allium cepa L.) bulb scales was determined before and after extraction of cuticular waxes with lipid solvents. In pear, the permeability coefficients for diffusion of tritiated water across cuticular membranes (CM) prior to extraction [P _d(CM)] decreased by a factor of four during leaf expansion. In all three species investigated P _d(CM) values of cuticular membranes from fully expanded leaves varied between 1 to 2×10^-7 cm^-3 s^-1·P _d(CM) values were not affected by pH. Extraction of cuticular waxes from the membranes increased their water permeability by a factor of 300 to 500. Permeability coefficients for diffusion of THO across the cutin matrix (MX) after extraction [P _d(MX)] increased with increasing pH. P _dvalues were not inversely proportional to the thickness of cuticular membranes. By treating the cutin matrix and cuticular waxes as two resistances acting in series it was shown that the water permeability of cuticles is completely determined by the waxes. The lack of the P _d(CM) values to respond to pH appeared to be due to structural effects of waxes in the cutin matrix. Cuticular membranes from the submerse leaves of the aquatic plant Potamogeton lucens L. were three orders of magnitude more permeable to water than the cuticular membranes of the terrestrial species investigated.Abbreviations CM cuticular membrane - MX cutin matrix - WAX waxes This study was supported by a grant from the Deutsche Forschungsgemeinschaft.     

Particulate Matter on Foliage of 13 Woody Species: Deposition on Surfaces and Phytostabilisation in Waxes – a 3-Year Study

Particulate matter (PM) as an air pollutant can be harmful for human health through allergic, mutagenic and carcinogenic effects. Although the main focus is on decreasing air pollution, after PM has been emitted to the atmosphere, one of the realistic options to decrease it's concentrations in urbanized area will be phytoremediation. This study compared the capacity to capture PM from air of seven tree species commonly cultivated in Poland (Catalpa bignonioides Walter, Corylus colurna L., Fraxinus pennsylvanica Marsh., Ginkgo biloba L., Platanus × hispanica Mill. ex Muenchh., Quercus rubra L., Tilia tomentosa Moench ‘Brabant’) and six shrub species (Acer tataricum subsp. ginnala (Maxim.) Wesm., Sambucus nigra L., Sorbaria sorbifolia (L.) A.Br., Spiraea japonica L.f., Syringa meyeri C.K. Schneid. ‘Palibin’, Viburnum lantana L.). Significant differences were found between species in mass of total PM accumulation for two PM categories and three size fractions determined and in amount of waxes. A positive correlation was found between in-wax PM of diameter 2.5–10 μm and amount of waxes, but not between amount of waxes and amount of total PM or of any size fraction.     

两个油菜种对水分胁迫的适应方式

本文报道了2个油菜种对水分胁迫的适应方式。研究表明:芥菜型油菜对水分胁迫的适应性强于甘蓝型。其主要原因是由于形态方面的抗旱性,包括发育良好的根系,较厚的蜡质关闭了气孔。同时芥菜型油菜能将其余部分的水分调用到生长区而免遭旱害。甘蓝型油菜叶水势下降快,在相同水势下其相对电导值低于芥菜型。同时还观察了2个种在干旱条件下叶绿体与其基粒的变化。总之,芥菜型具典型的高水势耐旱特性,而甘蓝型具低水势耐旱特性。     

Effect of humidity on cuticular water permeability of isolated cuticular membranes and leaf disks

The effects of humidity on water permeability of astomatous, isolated cuticular membranes and leaf disks of Citrus aurantium L., Vinca major L., Prunus laurocerasus L., Hedera helix L. and Forsythia intermedia (Thunb.) Vahl. were investigated by a new method using 3H2O. With isolated cuticular membranes of P. laurocerasus the isotope method resulted in values similar to those obtained by a well-established gravimetric method. Cuticular water permeability significantly increased by factors of 2 to 3 when air humidities increased from 2 to 100%. Plots of permeances vs. air humidity were non-linear and the slope increased with increasing air humidity. Permeances of intact leaf disks showed a response to increasing humidity similar to those of isolated cuticular membranes. When cuticular water permeability was measured using wax-free, isolated polymer matrix membranes that had been methylated, the effect of air humidity was significantly suppressed compared to non-methylated polymer matrix membranes. From this observation it is concluded that non-esterified, free carboxyl groups present in the cutin polymer matrix significantly contribute to the effect of humidity on cuticular water permeability. These and other polar groups sorb water, which in turn increases the water permeability of polar domains of the cuticle. This humidity-sensitive, polar path of cuticular water permeability is arranged in parallel with the major, dominating and humidity-independent, non-polar path of cuticular water permeability formed by the lipophilic wax components of the cuticle. This conclusion is supported by the fact that cuticular transpiration can be increased by orders of magnitude upon (i) wax extraction, (ii) increase in temperature or (iii) the action of plasticizers, none of which influenced or only marginally influenced the permeability of inorganic ions penetrating plant cuticles across humidity-sensitive polar pores.     

Stem hypoxia and root respiration of flooded maple and birch seedlings

Some researchers have attributed flood tolerance of woody plants to air entering the shoot through stems, leaves, or lenticels and diffusing to the roots to sustain aerobie respiration. The purpose of this study was to determine if internal aeration of roots by lower stems, changes in gross morphology of lower stems, or both, contribute to flood tolerance of certain tree species. Greenhouse-grown seedlings of red maple ( Acer rubrum L.) and river birch ( Betula nigra L.) tolerated at least 30 days of flooding, where as sugar maple ( Acer saccharum Marsh) and European white birch (also called silver birch, Betula pendula Roth) were intolerant. Flood treatment induced lentieel intumescences and adventitious root formation on red maple stems, but only adventitious roots formed on river birch stems. Stem morphology of sugar maple and European birch was unchanged by flooding. Flood stress decreased oxygen consumption capacity of excised roots from both tolerant and intolerant species. Exclusion of oxygen from the lower stems of flooded red maple and river birch prevented lenticel intumescence and adventitious root formation, but flood tolerance and root respiration capacity were unchanged. Neither internal aeration nor changes in stem morphology appear to account for flood tolerance of red maple and river birch.     

Histological Changes of the Peanut (Arachis hypogaea) Gynophore and Fruit Surface During Development, and their Potential Significance for Nutrient Uptake

Histological changes in gynophores and fruits of Arachis hypogaeaL.cv. White Spanish were examined, utilizing scanning electronmicroscopy as well as light microscopy. The epidermis of theabove ground parts of gynophores is characterized by the presenceof stomata, lenticels and multicellular trichomes. Below groundportions of the same plant organ exhibit unicellular root-hair-likestructures. These protuberances of the epidermal cells can reacha very high density and length (up to 0.75mm) . Identical structurescan be found on the developing pod and are most prominent atthe reproductive stages R5-R6. In later developmental stagesthe hairs degenerate and the presence of large lenticels becomesthe obvious external feature of the pod. It is suggested thatthe substantial increase in surface area due to the hairs maywell be an anatomical adaptation for nutrient and water uptake. Arachis hypogaea, peanut fruit development, nutrient uptake     

Wax Layers on Cosmos bipinnatus Petals Contribute Unequally to Total Petal Water Resistance

Cuticular waxes coat all primary aboveground plant organs as a crucial adaptation to life on land. Accordingly, the properties of waxes have been studied in much detail, albeit with a strong focus on leaf and fruit waxes. Flowers have life histories and functions largely different from those of other organs, and it remains to be seen whether flower waxes have compositions and physiological properties differing from those on other organs. This work provides a detailed characterization of the petal waxes, using Cosmos bipinnatus as a model, and compares them with leaf and stem waxes. The abaxial petal surface is relatively flat, whereas the adaxial side consists of conical epidermis cells, rendering it approximately 3.8 times larger than the projected petal area. The petal wax was found to contain unusually high concentrations of C₂₂ and C₂₄ fatty acids and primary alcohols, much shorter than those in leaf and stem waxes. Detailed analyses revealed distinct differences between waxes on the adaxial and abaxial petal sides and between epicuticular and intracuticular waxes. Transpiration resistances equaled 3 × 10⁴ and 1.5 × 10⁴ s m⁻¹ for the adaxial and abaxial surfaces, respectively. Petal surfaces of C. bipinnatus thus impose relatively weak water transport barriers compared with typical leaf cuticles. Approximately two-thirds of the abaxial surface water barrier was found to reside in the epicuticular wax layer of the petal and only one-third in the intracuticular wax. Altogether, the flower waxes of this species had properties greatly differing from those on vegetative organs.The flowers of many plants are especially adapted to ensure reproductive success by attracting, orienting, and assisting pollinators. Petals must also resist unfavorable environmental conditions such as a desiccating atmosphere. Some characteristics that increase reproductive success, including their high surface areas and surface permeability to small scent molecules, may also make petals more vulnerable to drying out (Goodwin et al., 2003; Bergougnoux et al., 2007). Thus, despite their ephemeral nature, petals may need to compromise between competing physiological and ecological functions. This raises questions: How effective are petal skins at blocking water? Do petal skin compositions differ from those on other plant parts in order to balance multiple functions?To answer these questions, both the chemical composition and the transpiration barrier properties of petal skins must be determined. It is well established that petals are covered by cuticles comparable to those on vegetative organs (Whitney et al., 2011). The waxes coating all primary parts of shoots consist of very-long-chain compounds, including alkanes, aldehydes, primary and secondary alcohols, fatty acids, esters, and ketones ranging in chain length from 20 to 70 carbons (Jetter et al., 2007). The ratio between these derivatives varies temporally and spatially between organs and layers within the cuticle (Jenks et al., 1995, 1996; Jetter and Schäffer, 2001). As well, wax may contain cyclic compounds such as pentacyclic triterpenoids (Buschhaus and Jetter, 2011). Even though it has long been known that the waxes, rather than the accompanying cutin polymer, are essential for the cuticular transpiration barrier (Schönherr, 1976), it is currently not clear how individual wax components contribute to this physiological function.In contrast to other organs, relatively few studies so far have addressed the chemical composition of petal waxes. Noteworthy exceptions are detailed analyses of petal waxes for Crataegus monogyna and three cultivars of Rubus idaeus (Griffiths et al., 2000), Antirrhinum majus (Goodwin et al., 2003), Vicia faba (Griffiths et al., 1999), Cistus albidus (Hennig et al., 1988), Petunia hybrida (King et al., 2007), Arabidopsis (Arabidopsis thaliana; Shi et al., 2011), and Rosa damascena (Stoianova-Ivanova et al., 1971). Selected compound classes have been investigated for some more species, including selected Ericaceae (Salasoo, 1989), Rosaceae (Wollrab, 1969a, 1969b), and Asteraceae (Akihisa et al., 1998) species. Some major plant families, such as the Asteraceae, have not been investigated in much detail.Along with chemical analyses, the physiological properties of waxes on fruits and leaves of diverse plant species also have been investigated in the past. The effectiveness of a water barrier may be characterized by quantifying the permeance for water (P; m s⁻¹) or, inversely, the transpiration resistance (s m⁻¹; Riederer and Schreiber, 1995). These characteristics may, in turn, be determined by measuring the water flux (J; kg m⁻² s⁻¹) across the cuticle under controlled conditions according to the equation P = J/Δc (where Δc is the water concentration gradient driving the diffusion across the barrier). Because both permeance and resistance are physiological characteristics independent of water concentration, their values enable comparisons between water barriers of different plant species and organs. Water permeance values and the corresponding barrier effectiveness vary widely between plant species and organs, with a range of 0.36 to 200 × 10⁻⁶ m s⁻¹ (Kerstiens, 1996; Schreiber and Riederer, 1996). The mean and median leaf permeances (1.42 × 10⁻⁵ and 0.58 × 10⁻⁵ m s⁻¹, respectively) were lower than those of fruit (9.93 × 10⁻⁵ and 9.46 × 10⁻⁵ m s⁻¹), leading to the conclusion that leaves typically produce a better barrier against water movement than does fruit (Kerstiens, 1996). This difference in the physiological performance of waxes on different organs raises the question of how effective the transpiration barrier of cuticular waxes on petals may be. However, to date, water permeance values for petals have not been published and thus cannot be compared with those for other organs.To fill important gaps in our understanding of cuticle function and composition, we initiated a detailed analysis of petal waxes using Cosmos bipinnatus as a first model. We recently reported the identification of novel compounds from the C. bipinnatus petal waxes (Buschhaus et al., 2013) but not the overall wax composition of the petal waxes. Therefore, the ray flowers of this species were examined here to determine (1) the wax composition on the adaxial and abaxial petal surfaces in comparison with the stem and leaf wax and (2) the corresponding petal water permeances.     

Gap-junctional single-channel permeability for fluorescent tracers in mammalian cell cultures

We have developed a simple dye transfer method that allows quantification of the gap-junction permeability of small cultured cells. Fluorescent dyes (calcein and Lucifer yellow) were perfused into one cell of an isolated cell pair using a patch-type micropipette in the tight-seal whole cell configuration. Dye spreading into the neighboring cells was monitored using a low-light charge-coupled device camera. Permeation rates for calcein and Lucifer yellow were then estimated by fitting the time course of the fluorescence intensities in both cells. For curve fitting, we used a set of model equations derived from a compartment model of dye distribution. The permeation rates were correlated to the total ionic conductance of the gap junction measured immediately after the perfusion experiment. Assuming that dye permeation is through a unit-conductance channel, we were then able to calculate the single-channel permeance for each tracer dye. We have applied this technique to HeLa cells stably transfected with rat-Cx46 and Cx43, and to BICR/M1R(k) cells, a rat mammary tumor cell line that has very high dye coupling through endogenous Cx43 channels. Scatter plots of permeation rates versus junctional conductance did not show a strictly linear correlation of ionic versus dye permeance, as would have been expected for a simple pore. Instead, we found that the data scatter within a wide range of different single-channel permeances. In BICR/M1R(k) cells, the lower limiting single-channel permeance is 2.2 +/- 2.0 x 10(-12) mm3/s and the upper limit is 50 x 10(-12) mm3/s for calcein and 6.8 +/- 2.8 x 10(-12) mm3/s and 150 x 10(-12) mm3/s for Lucifer yellow, respectively. In HeLa-Cx43 transfectants we found 2.0 +/- 2.4 x 10(-12) mm3/s and 95 x 10(-12) mm3/s for calcein and 2.1 +/- 6.8 x 10(-12) mm3/s and 80 x 10(-12) mm3/s for Lucifer yellow, and in HeLa-Cx46 transfectants 1.7 +/- 0.3 x 10(-12) mm3/s and 120 x 10(-12) mm3/s for calcein and 1.3 +/- 1.1 x 10(-12) mm3/s and 34 x 10(-12) mm3/s for Lucifer yellow, respectively. This variability is most likely due to a yet unknown mechanism that differentially regulates single-channel permeability for larger molecules and for small inorganic ions.     

Transpiration from Tomato Fruit Occurs Primarily via Trichome-Associated Transcuticular Polar Pores

Nonstomatal water loss by transpiration through the hydrophobic cuticle is ubiquitous in land plants, but the pathways along which this occurs have not been identified. Tomato (Solanum lycopersicum) provides an excellent system in which to study this phenomenon, as its fruit are astomatous and a major target for desiccation resistance to enhance shelf life. We screened a tomato core collection of 398 accessions from around the world and selected seven cultivars that collectively exhibited the lowest and highest degrees of transpirational water loss for a more detailed study. The transpirational differences between these lines reflected the permeances of their isolated cuticles, but this did not correlate with various measures of cuticle abundance or composition. Rather, we found that fruit cuticle permeance has a strong dependence on the abundance of microscopic polar pores. We further observed that these transcuticular pores are associated with trichomes and are exposed when the trichomes are dislodged, revealing a previously unreported link between fruit trichome density and transpirational water loss. During postharvest storage, limited self-sealing of the pores was detected for certain cultivars, in contrast with the stem scar, which healed relatively rapidly. The abundance of trichome-associated pores, together with their self-sealing capacity, presents a promising target for breeding or engineering efforts to reduce fruit transpirational water loss.

In later-diverging land plants, water is lost primarily by transpiration through stomata. However, when tissues begin to dry, the stomata close, restricting transpiration through this low-resistance route (Saliendra et al., 1995; Brodribb and Holbrook, 2003). Indeed, under such conditions, stomata contribute very little to the overall rate of water loss (Burghardt and Riederer, 2003; Santrůcek et al., 2004) and transpiration mostly occurs directly from the apoplast of epidermal cells. The hydrophobic cuticle that coats the epidermis of aerial organs provides the key barrier against this flux; however, despite extensive research, the key factors that determine the permeances of cuticles, which vary by over 500-fold across plant species (Riederer and Schreiber, 2001), have not been fully resolved.Answering this question is complicated by the compositional and structural complexity of cuticles, which consist of a lipidic polyester, termed cutin, along with polysaccharides and various soluble compounds referred to collectively as waxes (Riederer and Muller, 2006; Schreiber, 2010; Yeats and Rose, 2013). Furthermore, each of these constituents is compositionally diverse, with the types and amounts varying between species and even between organs of a single individual (Riederer and Muller, 2006; Schreiber, 2010; Yeats and Rose, 2013). However, cuticular waxes are believed to be critical for restricting transpiration, as various studies have found that extraction of the wax component increases permeance by 100- to 2,000-fold (Schreiber and Schönherr, 2009; Schreiber, 2010). In contrast, the >90% reduction in cutin levels in fruit of the cutin deficient2 (cd2) and cd3 mutants of tomato (Solanum lycopersicum) were reported to have a minimal effect on water loss (Isaacson et al., 2009). Despite the substantial effect of waxes, there does not appear to be a correlation between total wax levels and cuticular permeance across, or within, species (Jordan et al., 1984; Schreiber and Riederer, 1996; Riederer and Schreiber, 2001; Parsons et al., 2013). Indeed, a single monolayer of wax could theoretically account for the entire resistance of a cuticle to water diffusion (Schreiber and Schönherr, 2009). The wax compositional profile, rather than amount, is likely to be the key determinant of permeance.Triterpenoids, a common class of cyclic waxes, do not appear to contribute to transpirational resistance (Grncarevic and Radler, 1967), and several studies have found that their abundance relative to aliphatic wax components correlates with increased permeability (Vogg et al., 2004; Leide et al., 2011; Buschhaus and Jetter, 2012; Parsons et al., 2013; Jetter and Riederer, 2016). Thus, the ratio of aliphatic compounds to triterpenoids has been proposed as a key determinant of differences in permeability of up to 8-fold in the most extreme case (Leide et al., 2011). However, importantly, this only accounts for a small amount of the total variation in permeance observed between cuticles from different sources. An alternative hypothesis is that there exist polar pores, possibly created by the presence of polysaccharides that span the cuticle and provide a lower resistance route for water movement and largely dictate water flux (Schreiber, 2005). Diffusion experiments with isolated cuticles have offered support for this idea (Schönherr, 1976, 2006; Popp et al., 2005), but the presence of such pores has yet to be demonstrated. If they indeed exist, variation in their abundance or size might result in substantial differences in cuticular transpiration rates.Tomato fruit represents a useful model system in which to study the relationships between cuticle structure, composition, and properties, as they are astomatous and can be easily isolated, allowing direct measurement of permeance and biomechanical characteristics (Martin and Rose, 2014). Notably, while there is considerable variability in the amount and composition of waxes among tomato cultivars (Bauer et al., 2004), studies to date of tomato fruit cuticles have mainly focused on specific genetic variants or mutants, with little utilization of varietal diversity. A recent study has shown that even among just three varieties, there is significant variation in fruit transpiration rate and cuticle permeability (Romero and Rose, 2019). In this study, we used a germplasm diversity panel of tomato accessions, similar to the collection described by Lin et al. (2014), and assessed the range of fruit cuticular permeance. Specifically, we investigated water loss rates from harvested mature green (MG)-stage fruits and asked whether variation in cuticular permeability can be explained by wax composition or whether other cuticle properties or features play an important role.     

Diffusive and convective transport through hollow fiber membranes for liver cell culture

For an efficient membrane bioreactor design, transport phenomena determining the overall mass flux of metabolites, catabolites, cell regulatory factors, and immune-related soluble factors, need to be clarified both experimentally and theoretically. In this work, experiments and calculations aimed at discerning the simultaneous influence of both diffusive and convective mechanisms to the transport of metabolites. In particular, the transmembrane mass flux of glucose, bovine serum albumin (BSA), APO-transferrin, immunoglobulin G, and ammonia was experimentally measured, under pressure and concentration gradients, through high-flux microporous hydrophilic poly-ether-sulphone (PES-HFMs) and poly-sulphone hollow fiber membranes (PS-HFMs). These data were analyzed by means of a model based on the mechanism of capillary pore diffusion, assuming that solute spherical molecules pass through an array of solvent-filled cylindrical pores with a diffusive permeation corrected for friction and steric hindrances. Additionally, resistances to the mass transfer were taken into account. Convective permeation data were discussed in terms of morphological properties of the polymeric membranes, molecular Stokes radius, and solute-membrane interactions according to information given by contact angle measurements. The observed steady-state hydraulic permeance of PS-HFMs was 0.972 L/m2hmbar, about 15.6-fold lower than that measured for PES-HFMs (15.2 L/m2h); in general, PS-HFMs provided a significant hindrance to the transport of target species. Diffusion coefficients of metabolites were found to be similar to the corresponding values in water through PES-HFMs, but significantly reduced through PS-HFMs (D(Glucose)(Membrane)=2.8x10(-6)+/-0.6x10(-6)cm2/s, D(BSA)(Membrane)=6.4 x 10(-7)+/-1 x 10(-7)cm(/s, D(Apotransferrin)(Membrane)=2.3 x 10(-7)+/-0.25 x 10(-7)cm2/s).     

Water permeability of plant cuticles: permeance,diffusion and partition coefficients

Summary Using isolated cuticular membranes from ten woody and herbaceous plant species, permeance and diffusion coefficients for water were measured, and partition coefficients were calculated. The cuticular membranes of fruit had much higher permeance and diffusion coefficients than leaf cuticular membranes from either trees or herbs. Both diffusion and partition coefficients increased with increasing membrane thickness. Thin cuticles, therefore, tend to be better and more efficient water barriers than thick cuticles. We compared the diffusion coefficients and the water content of cuticles as calculated from transport measurements with those obtained from water vapor sorption. There is good to fair agreement for cuticular membranes with a low water content, but large discrepancies appear for polymer matrix membranes with high permeance. This is probably due to the fact that diffusion coefficients obtained from transport measurements on membranes with high permeance and water content are underestimated. Water permeabilities of polyethylene and polypropylene membranes are similar to those of leaf cuticular membranes. However, leaf cuticles have much lower diffusion coefficients and a much greater water content than these synthetic polymers. This suggests that cuticles are primarily mobility barriers as far as water transport is concerned.     

Artichoke Leaf Morphology and Surface Features in Different Micropropagation Stages

Artichoke (Cynara scolymus L.) leaf size and shape, glandular and covering trichomes, stomatal density, stomata shape, pore area and epicuticular waxes during micropropagation stages were studied by scanning electron microscopy (SEM) and morphometric analysis with the aim to improve the survival rate after transfer to greenhouse conditions. Leaves from in vitro shoots at the proliferation stage showed a spatular shape, ring-shaped stomata, a large number of glandular trichomes and juvenile covering hairs, but failed to show any epicuticular waxes. Leaves from in vitro plants at the root elongation stage showed a lanceolated elliptic shape with a serrated border, elliptical stomata, decreased pore area percentage, stomatal density, and mature covering trichomes. One week after transfer to ex vitro conditions, epicuticular waxes appeared on the leaf surface and stomata and pore area were smaller as compared to in vitro plants. Artichoke acclimatization may be improved by hormonal stimulation of root development, since useful morphological changes on leaves occurred during root elongation.