Molecular genetic and biochemical evidence for adaptive evolution of leaf abaxial epicuticular wax crystals in the genus Lithocarpus (Fagaceae).

BACKGROUND: Leaf epicuticular wax is an important functional trait for physiological regulation and pathogen defense. This study tests how selective pressure may have forced the trait of leaf abaxial epicuticular wax crystals (LAEWC) and whether the presence/absence of LAEWC is associated with other ecophysiological traits. Scanning Electron Microscopy was conducted to check for LAEWC in different Lithocarpus species. Four wax biosynthesis related genes, including two wax backbone genes ECERIFERUM 1 (CER1) and CER3, one regulatory gene CER7 and one transport gene CER5, were cloned and sequenced. Ecophysiological measurements of secondary metabolites, photosynthesis, water usage efficiency, and nutrition indices were also determined. Evolutionary hypotheses of leaf wax character transition associated with the evolution of those ecophysiological traits as well as species evolution were tested by maximum likelihood. RESULTS: Eight of 14 studied Lithocarpus species have obvious LAEWC appearing with various types of trichomes. Measurements of ecophysiological traits show no direct correlations with the presence/absence of LAEWC. However, the content of phenolic acids is significantly associated with the gene evolution of the wax biosynthetic backbone gene CER1, which was detected to be positively selected when LAEWC was gained during the late-Miocene-to-Pliocene period. CONCLUSIONS: Changes of landmass and vegetation type accelerated the diversification of tropical and subtropical forest trees and certain herbivores during the late Miocene. As phenolic acids were long thought to be associated with defense against herbivories, co-occurrence of LAEWC and phenolic acids may suggest that LAEWC might be an adaptive defensive mechanism in Lithocarpus.

Chemical composition and functional properties of native chestnut starch (Castanea sativa Mill).

Starch isolation methods can change their physico-chemical and functional characteristics hindering the establishment of a starch-food functionality relation. A simple high yield and soft isolation method was applied for chestnut (Castanea sativa Mill) starch consisting in steeping and fruit disintegration in a 25 mM sodium bisulfite solution and purification by sedimentation. Starch integrity, physico-chemical composition, morphology and functional properties were determined, being observed significant differences from previous described methods for chestnut starch isolation. The X-ray pattern was of B-type, with a degree of crystallinity ranging from 51% to 9%, dependent on the starch moisture content. The onset, peak, and conclusion gelatinization temperatures were 57.1°C, 61.9°C and 67.9°C, respectively. Total amylose content was 26.6%, and there was not found any evidence for lipid complexed amylose. Swelling power at 90°C was 19 g/g starch, and the amount of leached amylose was 78% of the total amylose content. Native chestnut starch presents a type B pasting profile similar to corn starch but with a lower gelatinization (56.1°C) and peak viscosity (79.5°C) temperatures, making native chestnut starch a potential technological alternative to corn starch, especially in application where lower processing temperatures are needed.

Programmed cell death is responsible for replaceable bud senescence in chestnut (Castanea mollissima BL.).

UNLABELLED: In the chestnut "replaceable bud" cultivar 'Tima zhenzhu', the auxiliary bud formed on the fruiting branch dies after fruiting, giving rise to a morphology more suitable than the wild type's for intensive cultivation and heightened production. Here, we show that many of the hallmarks of programmed cell death (PCD) occur during the senescence of the replaceable bud, including DNA degradation, a high ratio of PCD cells and the breakdown of cell ultrastructure. The time course of the senescence was followed by sampling the developing bud from 20 to 40 days after flowering. In cv. 'Tima zhenzhu', DNA degradation was detectable prior to any visible sign of bud senescence, while it did not occur in the wild type (cv. 'Dabanhong'). The ratio of PCD cells (as determined by flow cytometry) rose over the sampling period and was consistently higher in cv. 'Tima zhenzhu' than in cv. 'Dabanhong'. After staining the bud cell nuclei with propidium iodide, it was clear that both their chromatin content and overall size fell over the sampling period in cv. 'Tima zhenzhu' while in cv. 'Dabanhong', no such decrease occurred. Other characteristics of PCD were noted in cv. 'Tima zhenzhu''s bud cells, including chromatin condensation, tonoplast invagination and DNA cleavage. We conclude that the replaceable bud senescence phenomenon is driven by PCD. The manipulation of this trait may have potential for remodeling the pattern of development of the fruit-bearing branches of chestnut. KEY MESSAGE: This paper first reported the occurrence of programmed cell death during the senescence of vegetative buds in a woody species, and the results extend the range of knowledge of PCD.

Identification of a homolog of Arabidopsis DSP4 (SEX4) in chestnut: its induction and accumulation in stem amyloplasts during winter or in response to the cold.

Oligosaccharide synthesis is an important cryoprotection strategy used by woody plants during winter dormancy. At the onset of autumn, starch stored in the stem and buds is broken down in response to the shorter days and lower temperatures resulting in the buildup of oligosaccharides. Given that the enzyme DSP4 is necessary for diurnal starch degradation in Arabidopsis leaves, this study was designed to address the role of DSP4 in this seasonal process in Castanea sativa Mill. The expression pattern of the CsDSP4 gene in cells of the chestnut stem was found to parallel starch catabolism. In this organ, DSP4 protein levels started to rise at the start of autumn and elevated levels persisted until the onset of spring. In addition, exposure of chestnut plantlets to 4 °C induced the expression of the CsDSP4 gene. In dormant trees or cold-stressed plantlets, the CsDSP4 protein was immunolocalized both in the amyloplast stroma and nucleus of stem cells, whereas in the conditions of vegetative growth, immunofluorescence was only detected in the nucleus. The studies indicate a potential role for DSP4 in starch degradation and cold acclimation following low temperature exposure during activity-dormancy transition.

Involvement of reactive oxygen species during early stages of ectomycorrhiza establishment between Castanea sativa and Pisolithus tinctorius.

Evidence for the participation of reactive oxygen species (ROS) and antioxidant systems in ectomycorrhizal (ECM) establishment is lacking. In this paper, we evaluated ROS production and the activities of superoxide dismutase (SOD) and catalase (CAT) during the early contact of the ECM fungus Pisolithus tinctorius with the roots of Castanea sativa (chestnut tree). Roots were placed in contact with P. tinctorius mycelia, and ROS production was evaluated by determining the levels of H(2)O(2) and O(2) (.-) during the early stages of fungal contact. Three peaks of H(2)O(2) production were detected, the first two coinciding with O(2) (.-) bursts. The first H(2)O(2) production peak coincided with an increase in SOD activity, whereas CAT activity seemed to be implicated in H(2)O(2) scavenging. P. tinctorius growth was evaluated in the presence of P. tinctorius-elicited C. sativa crude extracts prepared during the early stages of fungal contact. Differential hyphal growth that matched the H(2)O(2) production profile with a delay was detected. The result suggests that during the early stages of ECM establishment, H(2)O(2) results from an inhibition of ROS-scavenging enzymes and plays a role in signalling during symbiotic establishment.

The potential role of orbicules as a vector of allergens.

BACKGROUND: In the locules of anthers of flowering plants, tiny (1.5-2 microm) granules of sporopollenin may occur next to the pollen grains. Those granules, called orbicules, mostly occur on the radial and innermost tangential wall of secretory tapetum cells. METHODS: We have investigated the presence of orbicules in 15 important European allergenic species with scanning electron microscopy (SEM). RESULTS: Orbicules were present in all species investigated of the families Betulaceae, Chenopodiaceae, Fagaceae, Poaceae, Polygonaceae, and Urticaceae. However, in the Asteraceae and Oleaceae species studied, orbicules were lacking. In all Chenopodiaceae, Poaceae, and Urticaceae species, orbicules were attached to the pollen exine. These observations indicate the possibility of the dispersal of orbicules into the atmosphere during anthesis. CONCLUSIONS: The hypothesis of the potential role of orbicules as possible important vectors of allergens is put forward, based on the comparison of our results with recent literature about the evidence of allergenic activity in the smaller micronic atmospheric aerosol fraction. Our results provide evidence that an in-depth investigation of the sites of allergens across the whole anther is required. We suggest that allergen researchers apply immunoelectron microscopy on whole anthers to determine whether orbicules possess allergens.