Antonio Heredia

Biophysical and biochemical characteristics of cutin, a plant barrier biopolymer.

Cutin is a support biopolyester involved in waterproofing the leaves and fruits of higher plants, regulating the flow of nutrients among various plant cells and organs, and minimizing the deleterious impact of pathogens. Despite the complexity and intractable nature of this biopolymer, significant progress in chemical composition, molecular architecture and, more recently, biosynthesis have been made in the past 10 years. This review is focused in the description of these advances and their physiological impacts to improve our knowledge on plant cutin, an unusual topic in most plant physiology and biochemistry books and reviews.

Pollen sporopollenin: degradation and structural elucidation

Abstract We report the isolation of purified sporopollenin from pollen grains of different species and its complete solubilization. Exine from Pinus pinaster, Betula alba, Ambrosia elatior and Capsicum annuum was extracted by treatment with hydrogen fluoride in pyridine. These exines were purified from their aromatic moieties and from fatty acids linked by ester bonds using acidolysis and saponification treatments. The biopolymer obtained retains almost completely the shape of the original pollen grain. Fourier-transform infrared spectroscopy analysis of the isolated sporopollenin showed the absence of polysaccharide and phenolic material and the presence of carboxylic acid groups joined to unsaturations and ether linkages. Sporopollenin samples were successfully degraded by exhaustive 24-h ozonolysis at room temperature. Gentle ozonolysis (3 h at 0°C) did not completely degrade the biopolymer. The compounds obtained after exhaustive ozonolysis were analysed by gas chromatography-mass spectrometry. Dicarboxylic acids with a low number of carbon atoms were identified as major components of sporopollenin from P. pinaster, A. elatior and C. annuum, representing 28.8%, 63.2% and 88.5%, respectively, of the total compounds obtained. Fatty acids and n-alkanes also were identified in P. pinaster, A. elatior and B. alba sporopollenin. From the data obtained, an hypothesis about the chemical nature and structural arrangement of the sporopollenin is proposed.

Improved germination under osmotic stress of tobacco plants overexpressing a cell wall peroxidase

The cell wall is a fundamental component in the response of plants to environmental changes. To directly assess the role of the cell wall we have increased the expression and activity of a cell wall associated peroxidase (TPX2), an enzyme involved in modifying cell wall architecture. Overexpression of TPX2 had no effect on wild‐type development, but greatly increased the germination rate under high salt or osmotic stress. Differential scanning calorimetry showed that transgenic seeds were able to retain more water available for germination than wild‐type seeds. Thermoporometry calculations indicated that this could be due to a lower mean pore size in the walls of transgenic seeds. Therefore, the higher capacity of transgenic seeds in retaining water could result in higher germination rates in conditions where the availability of water is restricted.

An overview on plant cuticle biomechanics

Plant biomechanics combines the principles of physics, chemistry and engineering to answer questions about plant growth, development and interaction with the environment. The epidermal-growth-control theory, postulated in 1867 and verified in 2007, states that epidermal cells determine the rate of organ elongation since they are under tension, while inner tissues are under compression. The lipid cuticle layer is deposited on the surface of outer epidermal cell walls and modifies the chemical and mechanical nature of these cell walls. Thus, the plant cuticle plays a key role in plant interaction with the environment and in controlling organ expansion. Rheological analyses indicate that the cuticle is a mostly viscoelastic and strain-hardening material that stiffens the comparatively more elastic epidermal cell walls. Cuticle stiffness can be attributed to polysaccharides and flavonoids present in the cuticle whereas a cutin matrix is mainly responsible for its extensibility. Environmental conditions such as temperature and relative humidity have a plasticizing effect on the mechanical properties of cuticle since they lower cuticle stiffness and strength. The external appearance of agricultural commodities, especially fruits, is of great economic value. Mechanical properties of the cuticle can have a positive or negative effect on disorders like fruit cracking, fungal pathogen penetration and pest infestation. Cuticle rheology has significant variability within a species and thus can be subjected to selection in order to breed cultivars resistant to pests, infestation and disorders.

Characterization and biosynthesis of non-degradable polymers in plant cuticles

Abstract. The structure and monomeric composition of the highly aliphatic and non-saponifiable fraction of cutans isolated from the leaf cuticles of Agave americana L. and Clivia miniata Reg. have been elucidated. Spectroscopic Fourier transform infrared and 13C-nuclear magnetic resonance, calorimetric and X-ray diffraction studies, together with biopolymer analysis after exhaustive ozonolysis, showed that the cutan fraction consists of an amorphous three-dimensional network linked by ether bonds containing double bonds and free carboxylic acid functions. Data obtained from fatty acid sorption indicated that the new biopolymer investigated here has a highly hydrophobic character constituting an additional barrier biopolymer in those cuticles where it is present. Labelled [14C]linoleic acid was preferentially incorporated into the non-ester part of C. miniata leaf disks in comparison with the cutin fraction of the cuticular membrane. This indicates that the cis-pentadiene system of polyunsaturated fatty acids is involved in the formation of intramolecular linkages, mainly ether bonds, of the aliphatic biopolymer.

Infrared and Raman spectroscopic features of plant cuticles: a review

The cuticle is one of the most important plant barriers. It is an external and continuous lipid membrane that covers the surface of epidermal cells and whose main function is to prevent the massive loss of water. The spectroscopic characterization of the plant cuticle and its components (cutin, cutan, waxes, polysaccharides and phenolics) by infrared and Raman spectroscopies has provided significant advances in the knowledge of the functional groups present in the cuticular matrix and on their structural role, interaction and macromolecular arrangement. Additionally, these spectroscopies have been used in the study of cuticle interaction with exogenous molecules, degradation, distribution of components within the cuticle matrix, changes during growth and development and characterization of fossil plants.

Relative humidity and temperature modify the mechanical properties of isolated tomato fruit cuticles

The mechanical properties of enzymatically isolated cuticular membrane (CM) from ripe tomato fruits were investigated at 10 to 45°C and relative humidity (RH) of 40 to wet. CM samples were stressed by uniaxial tension loads to determine their tensile modulus, E, breaking stress (strength), σ(max), and maximum elongation, ε(max). The CM stress-strain curves revealed a biphasic behavior when tested at RH values below wet conditions. In the first phase, CM responded to the loads by instantaneous extension with no further extension recorded until a further load was added: defined as pure elastic strain (E(e)). In the second phase, CM responded by instantaneous extension and by some additional time-dependent extension, defined as viscoelastic strain (E(v)). When CMs were submerged in aqueous solution (wet), the stress-strain curves were monophasic, with both elastic and viscoelastic strain. E(e) depended on RH and was higher than E(v), which was independent of RH. Temperature decreased E(e) and σ(max) of tomato fruit CM. Temperature response was not linear but consisted of two temperature-independent phases separated by a transition temperature. This transition zone has been related previously to the presence of a secondary phase transition in the cutin matrix of the tomato fruit CM.

New Insights into the Properties of Pubescent Surfaces: Peach Fruit as a Model

The surface of peach (Prunus persica ‘Calrico’) is covered by a dense indumentum, which may serve various protective purposes. With the aim of relating structure to function, the chemical composition, morphology, and hydrophobicity of the peach skin was assessed as a model for a pubescent plant surface. Distinct physicochemical features were observed for trichomes versus isolated cuticles. Peach cuticles were composed of 53% cutan, 27% waxes, 23% cutin, and 1% hydroxycinnamic acid derivatives (mainly ferulic and p-coumaric acids). Trichomes were covered by a thin cuticular layer containing 15% waxes and 19% cutin and were filled by polysaccharide material (63%) containing hydroxycinnamic acid derivatives and flavonoids. The surface free energy, polarity, and work of adhesion of intact and shaved peach surfaces were calculated from contact angle measurements of water, glycerol, and diiodomethane. The removal of the trichomes from the surface increased polarity from 3.8% (intact surface) to 23.6% and decreased the total surface free energy chiefly due to a decrease on its nonpolar component. The extraction of waxes and the removal of trichomes led to higher fruit dehydration rates. However, trichomes were found to have a higher water sorption capacity as compared with isolated cuticles. The results show that the peach surface is composed of two different materials that establish a polarity gradient: the trichome network, which has a higher surface free energy and a higher dispersive component, and the cuticle underneath, which has a lower surface free energy and higher surface polarity. The significance of the data concerning water-plant surface interactions is discussed within a physiological context.

Leaf structural changes associated with iron deficiency chlorosis in field-grown pear and peach: physiological implications

Plants grown in calcareous, high pH soils develop Fe deficiency chlorosis. While the physiological parameters of Fe-deficient leaves have been often investigated, there is a lack of information regarding structural leaf changes associated with such abiotic stress. Iron-sufficient and Fe-deficient pear and peach leaves have been studied, and differences concerning leaf epidermal and internal structure were found. Iron deficiency caused differences in the aspect of the leaf surface, which appeared less smooth in Fe-deficient than in Fe-sufficient leaves. Iron deficiency reduced the amount of soluble cuticular lipids in peach leaves, whereas it reduced the weight of the abaxial cuticle in pear leaves. In both plant species, epidermal cells were enlarged as compared to healthy leaves, whereas the size of guard cells was reduced. In chlorotic leaves, bundle sheaths were enlarged and appeared disorganized, while the mesophyll was more compacted and less porous than in green leaves. In contrast to healthy leaves, chlorotic leaves of both species showed a significant transient opening of stomata after leaf abscission (Iwanoff effect), which can be ascribed to changes found in epidermal and guard cells. Results indicate that Fe-deficiency may alter the barrier properties of the leaf surface, which can significantly affect leaf water relations, solute permeability and pest and disease resistance.

Wettability, Polarity, and Water Absorption of Holm Oak Leaves: Effect of Leaf Side and Age

The highly pubescent abaxial side of holm oak leaves is unwettable and water repellent, while the adaxial side is wettable and can take up water, which may be an adaptation to growing under Mediterranean conditions. Plant trichomes play important protective functions and may have a major influence on leaf surface wettability. With the aim of gaining insight into trichome structure, composition, and function in relation to water-plant surface interactions, we analyzed the adaxial and abaxial leaf surface of holm oak (Quercus ilex) as a model. By measuring the leaf water potential 24 h after the deposition of water drops onto abaxial and adaxial surfaces, evidence for water penetration through the upper leaf side was gained in young and mature leaves. The structure and chemical composition of the abaxial (always present) and adaxial (occurring only in young leaves) trichomes were analyzed by various microscopic and analytical procedures. The adaxial surfaces were wettable and had a high degree of water drop adhesion in contrast to the highly unwettable and water-repellent abaxial holm oak leaf sides. The surface free energy and solubility parameter decreased with leaf age, with higher values determined for the adaxial sides. All holm oak leaf trichomes were covered with a cuticle. The abaxial trichomes were composed of 8% soluble waxes, 49% cutin, and 43% polysaccharides. For the adaxial side, it is concluded that trichomes and the scars after trichome shedding contribute to water uptake, while the abaxial leaf side is highly hydrophobic due to its high degree of pubescence and different trichome structure, composition, and density. Results are interpreted in terms of water-plant surface interactions, plant surface physical chemistry, and plant ecophysiology.