Changes in leaf trichomes and epicuticular flavonoids during leaf development in three birch taxa

BACKGROUND AND AIMS: Changes in number of trichomes and in composition and concentrations of their exudates throughout leaf development may have important consequences for plant adaptation to abiotic and biotic factors. In the present study, seasonal changes in leaf trichomes and epicuticular flavonoid aglycones in three Finnish birch taxa (Betula pendula, B. pubescens ssp. pubescens, and B. pubescens ssp. czerepanovii) were followed. METHODS: Trichome number and ultrastructure were studied by means of light, scanning and transmission electron microscopy, while flavonoid aglycones in ethanolic leaf surface extracts were analysed by high-pressure liquid chromatography. KEY RESULTS: Density of both glandular and non-glandular trichomes decreased drastically with leaf expansion while the total number of trichomes per leaf remained constant, indicating that the final number of trichomes is established early in leaf development. Cells of glandular trichomes differentiate before those of the epidermis and produce secreted material only during the relatively short period (around 1-2 weeks) of leaf unfolding and expansion. In fully expanded leaves, glandular trichomes appeared to be at the post-secretory phase and function mainly as storage organs; they contained lipid droplets and osmiophilic material (probably phenolics). Concentrations (mg g(-1) d. wt) of surface flavonoids decreased with leaf age in all taxa. However, the changes in total amount ( microg per leaf) of flavonoids during leaf development were taxon-specific: no changes in B. pubescens ssp. czerepanovii, increase in B. pendula and in B. pubescens ssp. pubescens followed by the decline in the latter taxon. Concentrations of most of the individual leaf surface flavonoids correlated positively with the density of glandular trichomes within species, suggesting the participation of glandular trichomes in production of surface flavonoids. CONCLUSIONS: Rapid decline in the density of leaf trichomes and in the concentrations of flavonoid aglycones with leaf age suggests that the functional role of trichomes is likely to be most important at the early stages of birch leaf development.     

Comparative analysis of leaf trichome structure and composition of epicuticular flavonoids in Finnish birch species

The morphology, ultrastructure, density and distribution of trichomes on leaves of Betula pendula, B. pubescens ssp. pubescens, B. pubescens ssp. czerepanovii and B. nana were examined by means of light, scanning and transmission electron microscopy. The composition of flavonoids in ethanolic leaf surface extracts was analysed by high pressure liquid chromatography. All taxa examined contained both glandular and non-glandular trichomes (short and/or long hairs) but differed from each other in trichome ultrastructure, density and location on the leaf. Leaves of B. pubescens were more hairy than those of B. pendula, but the latter species had a higher density of glandular trichomes. Of the two subspecies of B. pubescens, leaves of ssp. pubescens had more short hairs on the leaf surface and four times the density of glandular trichomes of leaves of ssp. czerepanovii, whereas, in the latter subspecies, short hairs occurred largely on leaf veins, as in B. nana. The glandular trichomes were peltate glands, consisting of medullar and cortical cells, which differed structurally. Cortical cells possessed numerous small, poorly developed plastids and small vacuoles, whereas medullar cells had several large plastids with well-developed thylakoid systems and fewer vacuoles. In B. pubescens subspecies, vacuoles of the glandular cells contained osmiophilic deposits, which were probably phenolic, whereas in B. pendula, vacuoles of glandular trichomes were characterized by the presence of numerous myelin-like membranes. The composition of epicuticular flavonoids also differed among species. The two subspecies of B. pubescens and B. nana shared the same 12 compounds, but five of these occurred only in trace amounts in B. nana. Leaf surface extracts of B. pendula contained just six flavonoids, three of which occurred only in this species. In summary, the structure, density and distribution of leaf trichomes and the composition of epicuticular flavonoids represent good taxonomic markers for Finnish birch species.     

Extensive sharing of chloroplast haplotypes among European birches indicates hybridization among Betula pendula, B. pubescens and B. nana

Extensive sharing of chloroplast haplotypes among the silver birch, Betula pendula Roth., the downy birch, B. pubescens Ehrh., and the dwarf birch, B. nana L., was discovered using polymerase chain reaction-restriction fragment length polymporphism markers. The geographical component of the genetic variation was stronger than the species component: the species were not significantly different while 11% of the variation could be attributed to differentiation between the two main regions studied, Scandinavia and western Russia. All haplotypes occurring in more than 2% of the individuals were shared among the species and the introgression ratios were quite large: 0.79 between B. pubescens and B. pendula and 0.67 between B. pubescens and B. nana. The data also indicate that B. pendula individuals are more similar to sympatric B. pubescens than to B. pendula individuals from nearby forests. However, this trend is not as pronounced when B. pubescens is considered, suggesting that introgression is not symmetrical. The haplotype sharing among the three Betula species is most likely caused by hybridization and subsequent cytoplasmic introgression.     

Quantitative analysis of polymeric proanthocyanidins in birch leaves with normal-phase HPLC

The proanthocyanidin composition and content in the leaves of nine birch species (Betula albosinensis, B. ermanii B. maximowicziana, B. nana, B. papyrifera, B. pendula, B. platyphylla, B. pubescens, and B. pubescens ssp. czerepanovii) were studied with different methods including colorimetric assay, HPLC coupled with PAD or ESI/MS and NMR. Total proanthocyanidin content was determined using the acid butanol assay. A normal phase-HPLC method was applied for the analysis of polymeric proanthocyanidins. The content of polymeric proanthocyanidins was estimated from a late eluting peak in the chromatogram. With this HPLC method, quantitative analysis of polymeric proanthocyanidins could be performed directly from leaf extracts: no additional purification or preparation steps were required. It was shown that birch leaves contained mainly polymeric proanthocyanidins with a degree of polymerisation greater than 10. Total proanthocyanidin content (expressed as dry weight) was found to vary from 44mg/g (B. papyrifera) to 145mg/g (B. nana), and polymeric proanthocyanidin content from 39mg/g (B. pendula) to 119 mg/g (B. nana).     

Reproductive patterns of Betula pendula and B. pubescens coll. along a regional altitudinal gradient in northern Sweden

The Scandinavian birches have different distribution patterns Betula pendula reaches its upper tree limit at the bottom of the valleys in the Scandes, while B pubescens ssp pubescens extends above the B pendula limit, and merges into B pubescens ssp tortuosa at higher elevations In a 6 yr study I quantified the spatial and temporal variation in the reproduction of these species along a regional altitudinal gradient Betula pendula showed a steeper altitudinal decrease of seed germinability and seed germination rate than B pubescens coll Seed weight was strongly and positively correlated with altitude for B pubescens , but not for B pendula The proportion of seeds infected by gall midges Semudobia coll, was much higher in B pendula than in B pubescens coll especially in the mountain area All birch taxa showed large annual variations in quantity and quality of seeds The sapling density was higher in B pubescens ssp pubescens stands than in B pendula and B pubescens ssp tortuosa stands in the mountains The vegetative reproduction was about three times higher for B pubescens ssp tortuosa , than for the other two birches These results indicate that the altitudinal variation in reproduction among the birch taxa mirror their present distribution patterns     

Distribution of hydrolysable tannins in the foliage of Finnish birch species

On the basis of qualitative and quantitative analysis with liquid chromatography--electrospray ionisation mass spectrometry, the foliage of dwarf birch (Betula nana L.), silver birch (B. pendula Roth) and mountain birch (B. pubescens ssp. czerepanovii (Orlova) H?met-Ahti) were found, for the first time, to contain the same individual HTs that were described earlier for white birch (B. pubescens Ehrh.). In addition, one previously unidentified ellagitannin was preliminarily identified from the leaves of white and mountain birches, being totally absent from the foliage of the other two species. There were large variations in the contents of HTs between species. Seasonal variation affected significantly the contents of some individual HTs within species, and these changes were mainly in accordance with the biosynthetic pathway of HTs. All species converted galloylglucoses (GGs) into ellagitannins (ETs), dwarf birch being the only one that's efficient ET synthesis resulted in seasonally increased contents of ETs and thereof total HTs as well. The presence of insoluble ETs as well as the absence of insoluble GGs was confirmed in all four birch species for the first time. Furthermore, the amounts of insoluble ETs per one birch leaf were found to accumulate during the growing season. These findings complemented our knowledge of the biosynthetic pathway of birch leaf HTs: from soluble GGs via soluble ETs into insoluble ETs. The possible role of HTs in the herbivore defence of these species is discussed.     

Reproductive variability and pollen limitation in three Betula taxa in northern Sweden

This 3-yr study documents large variation in pollination and seed quality in birches (Betula) in northern Sweden between taxa, high and low altitude populations, and among years Seed quality was positively correlated with pollination degree and improved by pollen supplementation in B pubescens ssp pubescens in the mountain area, indicating pollen limitation Betula pendula showed the same tendency, but less pronounced Reduced sexual reproduction of B pendula in the mountain area might more often be due to limited resources for seed maturation and defense of the seeds against predispersal seed predation In contrast, coastal populations of both species display relatively high pollination rates and supplemental pollination had no effect on seed quality, suggesting that pollen limitation is rare or absent here The mountain birch Betula pubescens ssp tortuosa showed generally lower pollination and seed quality compared with B pubescens ssp pubescens , but not compared with mountain populations of B pendula It is concluded that pollen limitation may occur in Betula , but to what extent varies with taxon, altitude and year The seed quality is also strongly affected by other factors such as infections of gall midges (Semudobia spp, Diptera Cecidomyndae) and incomplete seed maturation     

Foraging by moose on two species of birch when these occur in different proportions

The two tree-like birch species Betula pendula and B. pubescens are of medium preference to moose during winter in northern Sweden. Because these birches are abundant in many biotopes, they form a major part of the moose diet. The two birches are very similar in appearance and often occur in mixed stands. Twenty-one birch stands where B. pendula formed from 2 to 97% of the birches were investigated to determine how the relative composition of the stands affected browsing of the two species by moose. In stands composed of 60% or less of B. pendula , there was a preference for this species when regarding 1.5–3.0 m high birches, which carry the largest amount of available browse per tree. At higher proportions B. pendula was still preferred, but use was closer to availability. However, in all stands there were significantly more moose bites per tree of B. pendula than of B. pubescens . In four birch stands browsing on 1.0–1.5 m high birches was also investigated. For this height class there was still a preference for B. pendula , but the difference between the two species was less pronounced than for the 1.5–3.0 m height class.     

Foliar phenolic composition of European white birch during bud unfolding and leaf development

We studied the between-tree and within-tree variation in the composition and content of foliar low-molecular-weight phenolics (LMWP) of European white birch ( Betula pendula Roth) during the unfolding of vegetative buds and during early leaf development. In buds, the major groups of phenolic compounds were hydrolysable tannins and flavonoid aglycones, whereas, later during leaf development, the flavonoid glycosides accounted for most of the total LMWP. The content of total LMWP, as well as individual compounds, varied largely among individual trees, while variation within an individual tree was low. The biosynthetic origin of individual compounds or compound groups is discussed in order to explain the main patterns in leaf chemistry during bud unfolding and early leaf development.     

Content and Composition of Fatty Acids in Total Lipids from the Buds of Betula pubescens,B. pendula,and B. pendula var. carelica

Russian Journal of Plant Physiology - Absolute content and composition of fatty acids (FAs) in total lipids from the buds of white birch (Betula pubescens Ehrh.), silver birch (B. pendula Roth),...     

Metabolic modifications of birch leaf phenolics by an herbivorous insect: detoxification of flavonoid aglycones via glycosylation

The metabolic modifications of birch (Betula pubescens Ehrh.) leaf phenolics in the digestive tract of its major defoliator, larvae of the autumnal moth Epirrita autumnata, were studied. The main phenolic acids of birch, i.e. chlorogenic and p-coumaroylquinic acids, were isomerised in the alkaline digestive tract. Moreover, only 16 to 92% of the ingested amounts of chlorogenic acid were found in the faeces of individual larvae; the missing portion is possibly being used in the formation of reactive o-quinones. Water-soluble flavonoid glycosides were mostly excreted unaltered. In contrast, lipophilic flavonoid aglycones were not excreted as such, but as glycosides after being detoxified by E. autumnata via glycosylation. When the larvae were fed with leaf-painted acacetin and kaempferide, i.e. two naturally occurring birch leaf flavonoid aglycones, acacetin-7-O-glucoside and kaempferide-3-O-glucoside appeared in larval faeces as major metabolites. However, the efficiency of aglycone glycosylation varied-, ranging from 17 to 33%, depending on the aglycone and its dietary level. There was also large variation in the efficiency of glycosylation--from 2 to 57%--among individual larvae. These results demonstrate a compound-specific metabolism of phenolic compounds, leading to different phenolic profiles in the insect gut compared to its leaf diet.     

Comparative phylogeography and population structure of European Betula species, with particular focus on B. pendula and B. pubescens

Aim  To compare the population genetic structures of the haplotype-sharing species Betula pendula and B. pubescens and to draw phylogeographic inferences using chloroplast DNA markers. In particular, we tested whether B. pendula and B. pubescens exhibited the same or different phylogeographic structures.
Location  Western Europe and Russia.
Methods  In this study we used both chloroplast DNA polymerase chain reaction-restriction fragment length polymorphism and microsatellites to genotype B. pendula , B. pubescens and, to a limited extent, B. nana , in 53 populations across Eurasia. A spatial amova ( samova ) was used to identify major clusters within each species.
Results  The low level of phylogeographic structure previously observed in B. pendula was confirmed, and the samova analysis retrieved only two major clusters. In contrast, seven clusters were observed in B. pubescens , although the overall level of population differentiation was similar to that of B. pendula .
Main conclusions  We detected a difference in the population genetic structure between the two species, despite extensive haplotype sharing. It is difficult to ascribe this finding to a single factor, but divergence in ecology between the two species may provide part of the explanation. For both species, the contribution of southern western populations to the recolonization after the Last Glacial Maximum seems to have been limited, and eastern and western European populations apparently had different histories.     

Leaf surface traits: overlooked determinants of birch resistance to herbivores and foliar micro-fungi?

Previous studies on the mechanisms of birch resistance to herbivores and foliar micro-fungi (both pathogenic and endophytic) have focused mainly on the role of internal leaf chemistry. In the present study, we examined genetic correlations between leaf surface traits (glandular trichome density and total concentrations of surface flavonoid aglycones) and occurrence of three species of foliar micro-fungi, one pathogenic rust (Melampsoridium betulinum) and two endophytic fungi (Fusicladium sp. and Melanconium sp.), and performance of autumnal moth larvae (Epirrita autumnata) in two birch species, Betula pubescens ssp. czerepanovii and B. pendula. The performance of autumnal moth larvae on B. pubescens ssp. czerepanovii was negatively correlated with density of glandular trichomes (RGR: r=–0.855; pupal mass: r=–0.709). In addition, rust infection was negatively correlated with trichome density in B. pendula (r=–0.675) and with epicuticular flavonoid aglycones in B. pubescens ssp. czerepanovii (r=–0.855). The frequency of the endophytic fungus Fusicladium sp., was related to epicuticular flavonoid aglycones (r=–0.782), while another endophytic fungus, Melanconium sp., showed no associations with any of the studied variables in B. pubescens ssp. czerepanovii. Our results indicate that leaf surface traits may be at least as important determinants of herbivore performance and micro-fungi abundance in birch as leaf internal chemistry.     

Temporal variation in epidermal flavonoids due to altered solar UV radiation is moderated by the leaf position in Betula pendula

The physiological mechanisms controlling plant responses to dynamic changes in ambient solar ultraviolet (UV) radiation are not fully understood: this information is important to further comprehend plant adaptation to their natural habitats. We used the fluorimeter Dualex to estimate in vivo the epidermal flavonoid contents by measuring epidermal UV absorbance (A(375) ) in Betula pendula Roth (silver birch) leaves of different ages under altered UV. Seedlings were grown in a greenhouse for 15 days without UV and transferred outdoors under three UV treatments (UV-0, UV-A and UV-A+B) created by three types of plastic film. After 7 and 13 days, Dualex measurements were taken at adaxial and abaxial epidermis of the first three leaves (L1, L2 and L3) of the seedlings. After 14 days, some of the seedlings were reciprocally swapped amongst the treatments to study the accumulation of epidermal flavonoids in the youngest unfolded leaves (L3) during leaf expansion under changing solar UV environments. A(375) of the leaves responded differently to the UV treatment depending on their position. UV-B increased the A(375) in the leaves independently of leaf position. L3 quickly adjusted A(375) in their epidermis according to the UV they received and these adjustments were affected by previous UV exposure. The initial absence of UV-A+B or UV-A, followed by exposure to UV-A+B, particularly enhanced leaf A(375) . Silver birch leaves modulate their protective pigments in response to changes in the UV environment during their expansion, and their previous UV exposure history affects the epidermal-absorbance achieved during later UV exposure.     

Oncopsis flavicollis (L.) associated with tree birches (Betula): a complex of biological species or a host plant utilization polymorphism?

The closely related species of leafhoppers, Oncopsis flavicollis (L.) and 0. subangulata (Sahl.), are restricted to birches, Betula pendula Roth and B. pubescens Ehrh., as host plants. Morphometric discriminant analyses of adult insects from S Wales showed O. flavicollis populations from the two Betula species to be significantly different. The best discrimination was provided by characters of the male dorsal abdominal apodemes. Analyses of such apodeme morphology in populations more widely from S Britain showed three distinct types: in western localities type 1 dominantly on B. pubescens , type 2 dominantly on B. pendula and type 3 absent; in eastern localities type 3 only on B. pendula , type 2 on both species of Betula and type 1 only on B. pubescens. Acoustic calling and courtship signals of males showed clear differences between the three apodeme types of 0. flavicollis. It is concluded that the three 0. flavicollis types, together with 0. subangulata , are distinct but very closely related biological species. Contrary to earlier suggestions, this example provides no evidence for host plant utilization polymorphisms.     

Fragmented environment affects birch leaf endophytes

The effect of environmental fragmentation on the species distribution and frequency of horizontally transmitted endophytic fungi in Betula pubescens and Betula pendula leaves was studied in an archipelago in southwestern Finland. The study system consisted of 14 islands, ranging in size and distance to the mainland, and five mainland sites. Endophytic fungi were grown out from surface-sterilized leaves. The frequency of endophytic fungi mainly depended on the size of the island, explaining 32-35% of the variation, and the distance to the mainland explaining 29-35% of the variation. The birch trees on the largest islands near the mainland had the highest endophyte frequencies. Fusicladium betulae, Gnomonia setacea and Melanconium betulinum were the most commonly isolated fungi. Foliar endophytes of birch trees are able to disperse to fairly fragmented areas, but their frequencies seem to depend on environmental isolation and size of the island.     

Exkret-Flavonoide bei Höheren Pflanzen arider Gebiete

Flavonoids generally occur in higher plants as water-soluble glycosides. However, lipophilic flavonoid aglycones can be excreted by glandular trichomes or extruded through the cuticle. They are accumulated on the plant surface as constituents of leaf resins or thin epicuticular layers. External flavonoid aglycones are found in various families throughout the higher plants, but appear to be most abundant in theAsteraceae. They occur relatively frequently in plants of (semi-)arid habitats. It is advantageous for chemotaxonomic studies that the structural diversity of exudate flavonoids is normally greater than that of the tissue glycosides in these plants.

Vorgetragen auf der Tagung der Deutschen Botanischen Gesellschaft, Wien, September 1984.     

Putting the insect into the birch–insect interaction

Leaf maturation in mountain birch (Betula pubescens ssp. czerepanovii) is characterized by rapid shifts in the types of dominant phenolics: from carbon-economic flavonoids aglycons in flushing leaves, via hydrolysable tannins and flavonoid glycosides, to carbon-rich proanthocyanidins (condensed tannins) in mature foliage. This shift accords with the suggested trade-offs between carbon allocation to plant defense and growth, but may also relate to the simultaneous decline in nutritive leaf traits, such as water, proteins and sugars, which potentially limit insect growth. To elucidate how birch leaf quality translates into insect growth, I introduce a simple model that takes into account defensive compounds but also acknowledges insect demand for nutritive compounds. The effects of defensive compounds on insect growth depend strongly on background variation in nutritive leaf traits: compensatory feeding on low nutritive diets increases the intake of defensive compounds, and the availability of growth-limiting nutritive compounds may modify the effects of defenses. The ratio of consumption to larval growth (both in dry mass) increases very rapidly with leaf maturation: from 2.9 to 9.8 over 2 weeks in June-July, and to 15 by August. High concentrations in mature birch leaves of "quantitative" defenses, such as proanthocyanidins (15-20% of dry mass), presumably prevent further consumption. If the same compounds had also protected half-grown leaves (which supported the same larval growth with only one third of the dry matter consumption of older leaves), the same intake of proanthocyanidins would have demanded improbably high concentrations (close to 50%) in young leaves. The model thus suggests an adaptive explanation for the high levels of "quantitative" defenses, such as proanthocyanidins, in low-nutritive but not in high-nutritive leaves because of the behavioral responses of insect feeding to leaf nutritive levels.     

Flavonoids as chemotaxonomic markers for Erythroxylum australe

Methanolic leaf extracts of Erythroxylum australe F. Muell. produced eight O-conjugated flavonoids. Six of the flavonoid aglycones were dihydroisoflavones (all dihydro-orobol derivatives), one a flavanone, eriodictyol, and one a flavonol, quercetin. The major glycosides of the flavonoids included mono-glucosyl-rhamnosyls and dirhamnosyl-glucosides with either 3, 5, 7 or 3', 4' linkage or a combination thereof The array of flavonoids present in E. australe suggests kinship to E. ulei and linkage to the four cultivated alkaloid-bearing Erythroxylum, especially the ancestral E. coca var. coca. Because of the uniqueness of the flavonoids present in leaf tissue of E. australe they are unambiguously useful as chemotaxonomic markers for the taxon.     

Interspecific variation in resistance of Asian, European, and North American birches (Betula spp.) to bronze birch borer (Coleoptera: Buprestidae)

Bronze birch borer (Agrilus anxius Gory) is the key pest of birches (Betula spp.) in North America, several of which have been recommended for ornamental landscapes based on anecdotal reports of borer resistance that had not been confirmed experimentally. In a 20-yr common garden experiment initiated in 1979 in Ohio, North American birch species, including paper birch (Betula papyrifera Marshall), 'Whitespire' gray birch (Betula populifolia Marshall), and river birch (Betula nigra L.), were much more resistant to bronze birch borer than species indigenous to Europe and Asia, including European white birch (Betula pendula Roth), downy birch (Betula pubescens Ehrh.), monarch birch (Betula maximowicziana Regel), and Szechuan white birch (Betula szechuanica Jansson). Within 8 yr of planting, every European white, downy, and Szechuan birch had been colonized and killed, although 100% of monarch birch had been colonized and 88% of these plants were killed after nine years. Conversely, 97% of river birch, 76% of paper birch, and 73% Whitespire gray birch were alive 20 yr after planting, and river birch showed no evidence of colonization. This pattern is consistent with biogeographic theory of plant defense: North American birch species that share a coevolutionary history with bronze birch borer were much more resistant than na?ve hosts endemic to Europe and Asia, possibly by virtue of evolution of targeted defenses. This information suggests that if bronze birch borer were introduced to Europe or Asia, it could threaten its hosts there on a continental scale. This study also exposed limitations of anecdotal observation as evidence of host plant resistance.