Effect of latitude on flavonoid biosynthesis in ... - Wiley Online Library

Plant, Cell and Environment (2010) 33, 1239–1247

doi: 10.1111/j.1365-3040.2010.02154.x

Effect of latitude on flavonoid biosynthesis in plants

pce_2154

1239..1247

LAURA JAAKOLA & ANJA HOHTOLA

Department of Biology, University of Oulu, FIN-90014 Oulu, Finland

ABSTRACT The growth conditions in different latitudes vary markedly with season, day length, light quality and temperature. Many plant species have adapted well to the distinct environments through different strategies, one of which is the production of additional secondary metabolites. Flavonoids are a widely spread group of plant secondary metabolites that are involved in many crucial functions of plants. Our understanding of the biosynthesis, occurrence and function of flavonoids has increased rapidly in recent decades. Numerous studies have been published on the influence of environmental factors on the biosynthesis of flavonoids. However, extensive long-term studies that examine the effect of the characteristics of northern climates on flavonoid biosynthesis are still scarce. This review focuses on the current knowledge about the effect of light intensity, photoperiod and temperature on the gene–environment interaction related to flavonoid biosynthesis in plants. Key-words: light intensity; light quality; temperature.

INTRODUCTION Light intensity, photoperiod and temperature have been reported to influence the biosynthesis of many secondary metabolites in a number of plant species. The long days and cool night temperatures in the northern latitudes appear, for example, to increase the production of aromatic compounds compared with the same plant species in the south (Davik et al. 2006). A similar pattern is considered to occur in other compounds that are connected with taste and colour in berries and herbs. However, the scientific evidence for the assumption about increased biosynthesis of secondary metabolites in plants growing in the northern climates is based on only a small number of scientific studies. One of the few is an intra-Nordic project which proved that, in addition to a higher carotene, ascorbic acid and sugar content, the flavour and aroma of certain vegetables were more pronounced in the northern latitudes compared with plants grown in the southern parts (Hårdh, Persson & Ottoson 1977). A Finnish study showed that the red colour (i.e. anthocyanin content) of strawberries is more intense in the north than in the south (Hårdh & Hårdh 1977). Similarly, a recent study showed that northern bilberry (Vaccinium myrtillus) populations have a higher Correspondence: L. Jaakola. Fax: +358 8 553 1061; e-mail: [email protected] © 2010 Blackwell Publishing Ltd

anthocyanin content than their southern counterparts (Lätti, Riihinen & Kainulainen 2008). Flavonoids are a large group of phenolic secondary metabolites that are widespread among plants. They have various functional roles, for instance, as attractants that enhance pollination and seed dispersal and as a part of the plant defence mechanism (Koes, Verweij & Quattrocchio 2005; Grotewold 2006). Flavonoids are also of interest in human nutrition as they have been reported to possess multiple biological properties that are beneficial for health. As colourful compounds, anthocyanins have been a popular research target in plant science. Although numerous studies have been carried out on the occurrence, distribution, biosynthesis, metabolism and function of flavonoid compounds in plants, detailed long-term studies focusing on the effect of northern growth conditions – long, cool days – on flavonoid contents in plants are scarce. The aim of this review was to compile knowledge about the effects of latitude on the gene–environment interaction of flavonoid biosynthesis in plants.The reports on this topic are examined with a focus to determine if there are quantitative and/or qualitative characteristics in flavonoid composition that are related to the latitudinal origin, growth site or growth conditions of the plants. The focus of the review is on the Northern Hemisphere because of the number of studies made in these areas. The most latitudinal studies that have been carried out so far and that are reviewed in the current paper concern the North-European climate conditions, which differ from those of the same latitudes in NorthAmerica orAsia.However,the literature has been reviewed with a special focus on regional differences in the south–north axis.Moreover,because solely latitudinal studies are scarce, the effect of light and temperature conditions on flavonoid biosynthesis in plants has been analysed also in a broader sense.

FLAVONOIDS The flavonoid family is divided into a number of subgroups: the six main classes are flavonols, flavones, flavan-3-ols, isoflavones, flavanones and anthocyanidins. Flavonoids usually accumulate in the plant vacuoles as glycosides (Bohm 1998). Flavonols and flavones are synthesized from a branch of the phenylpropanoid pathway. Flavonols and flavones are conjugated to sugars, primarily glucose, rhamnose and rutinose. Flavonols, particularly kaempferol and quercetin, are widespread in different plant species and organs, and they have, for instance, been shown to be essential for pollen germination and pollen tube formation in 1239

1240 L. Jaakola & A. Hohtola

(a)

(b)

R1

R1 OH HO

O

A

B

+

HO

R2

C

OH O

A

O

Flavonol aglycons: Kaempferol R1 = R2 = H Quercetin R1 = OH, R2 = H Myrisetin R1 = R2 = OH Isorhamnetin R1 OCH3 = R2 = H

R2

C OH

OH OH

B

OH

Anthocyanidins: Pelargonidin R1 = R2 = H Cyanidin R1 = OH, R2 = H Delphinidin R1 = R2 = OH Peonidin R1 OCH3 = R2 = H Petudinin R1 OCH3 = R2 = OH Malvidin R1 = R2 = OCH3

some plants. The chemical structures of the most common flavonols are presented (Fig. 1a). Some flavonoids have more restricted distribution than others. Isoflavones are produced almost exclusively by the members of Fabaceae/Leguminosae family, and flavanones, such as hesperidin and naringenin, are specific to citrus fruits. In isoflavones, the position of one phenyl group differs compared with flavones (Bohm 1998). Proanthocyanidins are colourless oligomers and polymers of flavan-3-ol units [e.g. (+)-catechin and (-)epicatechin]. Proanthocyanidins accumulate in different plant organs and tissues to provide protection against predation. Only quite recently the study of proanthocyanidins has intensified and the understanding of proanthocyanidin biosynthesis has expanded, but many open questions still remain (Xie & Dixon 2005). Anthocyanins belong to a class of flavonoids and are important pigments in flowers, fruits and leaves, serving as visual signals for pollinators and seed dispersers (Koes et al. 2005). The anthocyanin pigments are responsible for the majority of the red, purple and blue colours of flowers. In addition to reproductive organs, anthocyanins commonly accumulate in green vegetative tissues as a consequence of different environmental stimuli (Steyn et al. 2002). Anthocyanin pigments are predominantly present in glycosylated forms of their aglycon (Fig. 1b), which contribute to the increased stability of anthocyanins and aqueous solubility in vacuoles (Ogata et al. 2005). The flavonoid biosynthetic enzymes are localized in the cytosol (Koes et al. 2005). After biosynthesis, flavonoids are transported to vacuoles or cell walls. In addition to the intracellular transport of flavonoids, there are some indications of long-distance transport in relation to flavonoid biosynthesis. In recent years, plenty of work has been directed at elucidating flavonoid biosynthesis from a molecular point of view (see, e.g. Koes et al. 2005; Grotewold 2006). Both the structural genes that encode the key enzymes of the flavonoid pathway (see Fig. 2) and regulatory genes that are

Figure 1. (a) Chemical structures of the most common flavonol aglycons and (b) anthocyanidins.

required for flavonoid biosynthesis have been characterized in a variety of plant species. The regulation of flavonoid biosynthesis occurs via co-ordinated transcriptional control of the structural genes, by the interaction of DNA binding R2R3 MYB transcription factors, MYC-like basic helix loop helix (bHLH) and WD40 proteins (Koes et al. 2005; Grotewold 2006). However, a general view on how the signals needed on regulation of flavonoid biosynthesis are mediated in the different tissues during developmental processes in response to environmental factors is still missing.

PHOTOPERIOD AND DAY LENGTH Photoperiod influences the growth and development of plants. The biosynthesis of secondary metabolites can be regulated by the same photoperiodic conditions that control developmental phenomena. The biosynthesis of anthocyanins in some flowers has been found to be controlled by photoperiodic conditions (Taylor 1965). Earlier studies on various plants have shown that a longer photoperiod in general increases the content of phenolic compounds (Taylor 1965). However, the increase in secondary metabolites in long-day conditions can be a result of an increase in incident light energies. In 26 d photoperiodic treatments (20, 16, 15 or 8 h light), a reduction of 40% was detected in the content of phenolic compounds between 20 and 8 h light treatments with Xanthium pensylvanicum (Taylor 1965). Similarly, Carvalho et al. (2010) detected a massive induction in transcription of flavonoid biosynthesis genes and accumulation of anthocyanins, flavonols and catechins in sweet potato (Ipomoea batatas) leaves growing under long (16 h) compared with short (8 h) day conditions for a 30 d research period. Anthocyanin contents especially have been found to be affected by day length, whereas data from other flavonoids are variable. Camm et al. (1993) investigated the accumulation of anthocyanins, proanthocyanidins and flavan-3-ols in Pinus contorta seedlings in two different day lengths in

© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1239–1247

Effect of latitude on flavonoid biosynthesis 1241 Phenylalanine PAL

Cinnamic acid C4H

p-Coumaric acid

Hydroxycinnamic acid conjugates

4CL General phenylpropanoid pathway

4-Coumaroyl-CoA

Flavonoid pathway

CHS

Malonyl-Co-A

Naringenin chalcone CHI

Naringenin (flavanone) F3H FLS

Kaempferol

Dihydrokaempferol

F3’H

FLS

Dihydroquercetin

F3’5’H

DFR

Quercetin LCR

F3’5’H

Leucocyanidin

DFR

DFR

Leucopelargonidin

Figure 2. A schematic presentation

Myricetin

Leucodelphinidin ANS

ANS

ANS

Procyanidin

Dihydromyricetin FLS

LCR

Prodelphinidin

ANR

Cyanidin UFGT

Cyanidin-3-glucoside MT

Delphinidin

Pelargonidin UFGT

Pelargonidin3-glucoside

Peonidin-3-glucoside

UFGT

Delphinidin-3-glucoside MT

Petudinin-3-glucoside

MT

Malvidin-3-glucoside

(Cyanidin-3-galactoside, or -arabinoside)

Vancouver, Canada (49°15′49′N). The seedlings were grown for 4 weeks under short-day (10 h) or long-day (15.5–14 h) conditions. Afterwards, all seedlings were maintained in natural day length for 15 weeks, from the end of August to late December, followed by cold storage at +3 °C in the dark for 2 months. As a result, the concentration of anthocyanins was notably lower in seedlings growing under short-day treatment, whereas the concentration of proanthocyanins and flavan-3-ols varied less. A study conducted on purple and red-flesh potatoes (Reyes, Miller & CisnerosZevallos 2004) also showed that in longer days (14–15 h), the anthocyanin content in potatoes was higher compared with shorter days (12–14 h), but the content of total phenolics was not affected by the treatments. It is worth noticing that tuber tissues do not grow under direct light exposure, which suggests an indirect effect of light on the anthocyanin production. However, in the long-day treatment also, the temperature was 6–9 °C lower compared with shorter-day conditions. Graglia et al. (2001) showed site-specific differences in flavonoids in Betula nana leaves from dwarf shrub tundra in Abisko, northern Sweden (68°21′N, 18°49′E), and from tussock tundra at Toolik Lake, Alaska (68°38′N, 149°34′E).

of the flavonoid biosynthesis pathway. Enzyme abbreviations: PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumaroyl:CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′ hydroxylase; F3′5′H, flavonoid 3′5′ hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol 4-reductase; LCR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; ANS, anthocyanidin synthase; UFGT, UDP glucose-flavonoid 3-o-glucosyl transferase; RT, rhamnosyl transferase; MT, methyltransferase.

The content of myricetin derivatives was significantly higher in Abisko than at Toolik Lake; by contrast, the content of quercetin derivatives, another group of flavonols, was higher at Toolik. The authors conclude that the Toolik Lake population has much higher flexibility in its carbon allocation and that this is caused by differences in genetic control, different environmental conditions or a differential evolutionary response to stress and especially herbivore pressure. Hence, although day length and light conditions can explain part of the variation in flavonoid composition, site-specific variation occurs in different growth sites at the same latitude. Cues on the light-mediated regulation of latitudinal genetic adaptation in plants have been found recently. Tessadori et al. (2009) examined the variation in chromatin compaction in 21 Arabidopsis thaliana phenotypes from different geographical origins and habitats. A positive correlation between the latitude of origin and chromatin compaction was detected. The level of compaction appeared to be dependent on light intensity. Moreover, the photoreceptor PHYTOCHROME-B (PHYB) and the histone modifier HISTONE DEACETYLASE-6 (HDA6) were found to act as positive regulators of light-controlled


1242 L. Jaakola & A. Hohtola chromatin compaction. The authors conclude that chromatin plasticity, controlled by light intensity, could be associated with acclimation of Arabidopsis to each latitudinal environment.

LIGHT QUALITY The quality and duration of radiation are different in different areas of the globe. Light quality is not stable but varies because of many factors even in the same area. Light quality (i.e. the spectral distribution of light) is influenced by the solar angle, which depends on the latitude and the time of day. In northern areas (above 69°N latitude), the sun remains continuously above the horizon in the summer, and the evening represents a relatively long ‘end-of-day’ period, which lasts for up to 20% of the daily hours. During this time, the ratio of red to far-red light, the R : FR ratio, is reduced and has an effect on growth (Sarala et al. 2007). Pecot et al. (2005) found that R : FR decreases with increasing solar angle from maximum zenith for the study site under blue skies, is greater under overcast skies and decreases under canopies. The ratio of R : FR regulates important aspects of plant development, including stem extension, specific leaf area and seed germination. The R : FR ratio also has an effect on the biosynthesis of secondary metabolites. Northern A. thaliana populations were found to be more responsive to red and far-red light than southern populations (Stenøien et al. 2002). Northern populations of downy birch (Betula pubescens) and Norway spruce (Picea abies) also show a requirement for FR light in the light period to maintain growth, and this demand increases along with increasing northern latitude of origin (Clapham et al. 1998). Alokam, Chinnappa & Reid (2002) studied differences in anthocyanin accumulation between alpine and prairie plants of Stellaria longipes in the same environmental conditions but under different R : FR ratios. The levels of anthocyanins in prairie plants were significantly higher under high R : FR than their alpine counterparts. Under low R : FR, both alpine and prairie plants showed almost the same levels of anthocyanins. Tegelberg, Julkunen-Tiitto & Aphalo (2004) reported that silver birch (Betula pendula Roth) leaves that were exposed to supplemental FR related to R contained higher levels of phenolic acids. The UV-Binduced production of the flavonols kaempferol and quercetin or chlorogenic acid was not modified by the R : FRratio. Hence, it appears that the long ‘end-of-day’ period along with a reduced R : FR ratio in high latitudes does have an impact on the growth and metabolism of northern plants. However, its impact on production of the flavonoids is still not clear. Excess solar radiation has a clear effect on flavonoid composition. Tattini et al. (2000) observed that the content, but not the composition, of leaf tissue phenylpropanoids varied significantly between sun and shade leaves of Phillyrea latifolia with a notable increase in flavonoid glycosides in sun leaves. Furthermore, the localization of flavonoids showed differences between sun and shade leaves.

Similarly, an increase in the content of all flavonoids, except proanthocyanidins, and the activation of flavonoid biosynthetic genes were detected in bilberry leaves growing under direct sun exposure (Jaakola et al. 2004). Anthocyanins were detected only in the bilberry leaves growing under direct solar radiation. However, even though anthocyanins commonly accumulate in excess light, their possible role in photoprotection is not clear (Steyn et al. 2002).

UV-LIGHT Because of the depletion of the ozone layer, the level of UV radiation has increased in the polar areas, including the Northern Hemisphere. Exposure to high amounts of UV radiation can cause damage to macromolecules, such as DNA. It has been found that UV-B (280–315 nm) stimulates protection mechanisms and some photomorphogenetic responses that modify plant resistance to UV-B and other types of abiotic stress (Ballare 2003). An increasing number of reports deal with the influence of UV radiation on secondary metabolism, especially flavonoids (e.g. Kootstra 1994; Casati & Walbot 2003; Stracke et al. 2010). It has been shown that light-absorbing flavonoid compounds, flavonols and anthocyanins, in particular, accumulate in epidermal cells to protect the internal tissues of the leaves and stem from the damaging effects of UV-B radiation (Nybakken, Aubert & Bilger 2004; Treutter 2006). Flavonoids are also potent scavengers of reactive oxygen species, and thus prevent peroxidation of lipids in plant tissues. In many studies, flavonols, especially moieties of quercetin or kaempferol, have been reported to accumulate in response to increased UV-B radiation (Treutter 2006). Specifically, UV-B- or UV-A-induced accumulation of anthocyanins has been observed in various species (Zhou et al. 2007 and references therein). UVB-induced accumulation of anthocyanins and other phenols, mediated by PAL, may confer freezing tolerance, as shown by Teklemariam & Blake (2004). A 20 h exposure to UVB increased freezing (-15 °C) tolerance in jack pine (Pinus banksiana) seedlings when PAL inhibitor was not used. In PAL-inhibited seedlings, the UVB pretreatment increased freezing injury by 48%. Similarly, Nozzolillo et al. (2002) reported that anthocyanins in P. banksiana seedlings were produced from the time of the first exposure to freezing temperatures. Altitude also has an effect on the contents of secondary metabolites in higher plants. In addition to incurring many climatic differences, altitude influences the quality of radiation. Especially, UV-B radiation is high in alpine sites compared with lower habitats (Barnes, Flint & Caldwell 1987). The higher solar radiation at higher altitudes has often been implicated as having an impact on secondary metabolite profiles. For example, an increase in phenolic compounds with altitude as a response to increasing UV radiation has been demonstrated. A study by Turunen & Latola (2005) showed that the alpine timberline plants are generally adapted to UV-B, but that, on the other hand, alpine timberline plants of northern latitudes may be less protected


Effect of latitude on flavonoid biosynthesis 1243 against increasing UV-B radiation than plants from more southern latitudes and higher elevations.

EFFECT OF LIGHT ON FLAVONOIDS IN FLOWERS AND FRUITS Some studies have indicated that fruits growing in northern latitudes have higher contents of phenolic compounds compared with their southern counterparts (Hårdh & Hårdh 1977; Lätti et al. 2008, 2010). The northern light conditions are speculated to have the main role in the differences between northern and southern plant populations in the Northern Hemisphere. Light is one of the most important regulating factors during flower pigmentation, as shown in many studies (Dong et al. 1998; Farzad, Griesbach & Weiss 2002; Meng et al. 2004). Blocking UV or natural light (dark treatment) before flower bud break can reduce the gene expression involved in anthocyanin biosynthesis, resulting in either pink (UV block treatment) or pure white (dark treatment) apple flowers (Dong et al. 1998). Interestingly, it was found that the pure white flowers were unable to resynthesize anthocyanins even if they were re-exposed to light, which suggests developmental control of anthocyanin biosynthesis. An effect of light on grape berry phenolics has been reported in many papers. Pereira et al. (2006) found that light exposure increases the flavonol content of skin and pulp. The skin phenolics pattern was so strongly dependent on light exposure that the researchers were able to visually distinguish sun berries from shade berries. Exposure to sunlight increases anthocyanin concentrations in grape skin regardless of ambient temperature (Spayd et al. 2002). Shading of the grape berries during berry development has been found to reduce the accumulation of flavonoids, especially flavonols and proanthocyanidins, and to inhibit the transcription of the corresponding flavonoid pathway genes (Fujita et al. 2006, 2007). Bakhshi & Arakawa (2006) showed that the content of phenolic acids, anthocyanin and flavonols increased rapidly by irradiation, whereas flavanols, procyanidins and dihydrochalcones did not change in either mature or in ripe apple fruits.

TEMPERATURE Plants growing in cold climates can maintain higher photosynthetic rates at lower temperatures than plants growing in warmer areas, and they can thus increase the amount of fixed carbon available for secondary metabolites. Several studies show the effect of temperature, high or low, on the composition or concentrations of flavonoids. Low temperature has been shown to induce anthocyanin synthesis in various species (Chalker-Scott 1999; Choi et al. 2009). However, the accumulation of anthocyanins in cold temperatures is light dependent: in the absence of light, low temperatures prevent anthocyanin biosynthesis. The regulation of cold induction of anthocyanins and the role of light are not yet well understood. Choi et al. (2009) identified an enhanced level of anthocyanin 1 (ela1) mutant of

Arabidopsis, which has elevated levels of flavonoids and also exhibits UV and cold stress tolerance in a temperature of 4 °C. The authors suggest that in the future, ela1 will serve as a good model in studies on the regulation of cold stress-related anthocyanin accumulation. Many studies have shown that a poor flower colouration develops under high temperature (30–40 °C) conditions. The decrease in anthocyanin content is caused by a decline in gene expression and enzyme activity in the anthocyanin biosynthesis pathway, as has been shown in rose (Dela et al. 2003), chrysanthemum (Nozaki, Takamura & Fukai 2006) and petunia (Shvarts, Borochov & Weiss 1997). Moreover, Plantago lanceolata produced darker flowers at cooler ambient temperatures (Stiles et al. 2007). Most of the 17 anthocyanins, derived from both cyanidin and delphinidin branches of the anthocyanin biosynthetic pathway, significantly increased when P. lanceolata was grown under cool-temperature conditions (15 °C 16 h day/10 °C 8 h night) compared with high-temperature ones (27 °C 16 h day/22 °C 8 h night). Significant differences were also detected in the anthocyanin levels and in the sensitivity to temperature changes between the genotypes. The authors conclude that the temperature regulation of the anthocyanin biosynthetic pathway occurs both upstream and downstream of the divergence of the cyanidin and delphinidin branches. Boo, Chon & Lee (2006) showed that anthocyanin biosynthesis in chicory (Cichorium intybus) was highest under 15/10 °C (day/night) temperatures, decreased with higher day and night temperatures, and was almost totally inhibited at 30/25 °C. In contrast to these results, Wahid (2007) showed that the levels of anthocyanins in 1-month-old sugarcane plants sharply increased within 24 h of exposure to heat stress (40 °C). Wang & Zheng (2001) found that strawberries grown at 18/12 °C generally had the lowest anthocyanin, flavonol (quercetin 3-glucoside and quercetin 3-glucoronide) and phenolic acid contents, whereas a temperature of 30/22 °C yielded the highest content. Several studies show the effect of temperature on the colouration of berry or fruit skin. The optimum temperature for the biosynthesis of phenolic acids, anthocyanins and flavonols in apple fruit was 24 °C of the tested range (10, 17, 24 and 30 °C), and was not dependent on maturity stage or variety (Bakhshi & Arakawa 2006). Anthocyanin accumulation in the grape berry skins was significantly higher at 20 °C than at 30 °C, and the most sensitive stage for the temperature treatment was 1–3 weeks after colouring began (Yamane et al. 2006). A high temperature (max 35 °C) also reduced the total anthocyanin content of grape skin to less than half of that in the control berries growing in max 25 °C (Mori et al. 2007). The analyses of transcript abundance of the flavonoid pathway genes and the stable isotope-labelled tracer experiments on anthocyanin degradation suggested that the loss of anthocyanins under a high temperature is caused by both anthocyanin degradation and inhibition of mRNA transcription (Mori et al. 2007). A decline in temperature has been reported to cause qualitative changes in flavonoid composition. Albert et al.


1244 L. Jaakola & A. Hohtola (2009) noticed a pronounced increase in the quercetin : kaempferol ratio in the flowering heads of Arnica montana along with a 5 °C decrease of temperature (from 25 °C day/7.5 °C night to 20 °C day/12.5 °C night) in the alpine climate regime. Recently, Schmidt et al. (2010) studied the climatic and genotypic influences on the concentration and composition of flavonoids in eight kale (Brassica oleracea) cultivars growing in cool (0.3–9.6 °C) temperatures. The samples were collected at 4 week intervals, during a 4 month period (October–January). The main flavonoid aglycon in kale was flavonol kaempferol, followed by quercetin and isorhamnetin. According to the results, the total flavonoid contents were not markedly affected, but an increased quercetin : kaempferol ratio along with the decline in temperature was detected. The reported studies show that plants can react to elevated and low temperatures by altering flavonoid synthesis in a species-specific way. In general, too high a temperature can inhibit biosynthesis and cause degradation of flavonoids. A low temperature can increase flavonoid production, although the accumulation of flavonoids in cold temperatures is light dependent. There appears to be some evidence that cooler temperatures favour the production of flavonoids with a higher hydroxylation level.

GENE ¥ ENVIRONMENT INTERACTION Long-term studies that focus on the gene–environment interaction regarding flavonoid biosynthesis have been scarce. One of the few that show the significance of plant origin and growth site to the profile of secondary metabolites was carried out by Oleszek et al. (2002). They analysed the flavonoid composition and concentration in the needles of Pinus sylvestris that originated in five areas in Eastern Europe and Siberia, and that had grown in Poland for over 80 years. The aim of the study was to determine whether a long-lasting influence of the environment can modify flavonoid profiles and their total concentration. The northernmost P. sylvestris origin was from the latitude 63°50′N and the southernmost from 40°30′N, whereas the experimental sites were in latitudes 51°37′N and 52°15′N. The authors were able to show the relationship between the flavonoid level and the latitude of the population origin. Interestingly, the needles of the trees that originated from the higher latitude had the lowest levels of taxifolin 3′-O-glucoside, quercetin, taxifolin and total flavonoids. This common garden experiment shows that there are local adaptations based on genetic differences in the regulation of flavonoid biosynthesis between populations of P. sylvestris. Unfortunately, there are no data on the flavonoid contents of the P. sylvestris needles growing in the original growth site. Thus, many open questions remain; for example, do the needles of the northern P. sylvestris populations have lower capacity to produce flavonoids in a shorter day? Would the result be the same if the trees had grown in the northern growth site? Few publications report changes in flavonoid contents along with increasing latitude. In juniper (Juniperus communis) needles, a positive correlation was detected with

increasing latitude (59°N–69°N) and the content of phenolic compounds (flavonols, proanthocyanins and monoterpeins) (Martz et al. 2009). An investigation on latitudinal (from 60°N to 70°N) variation of foliar flavonoids of white birch (B. pubescens) showed that the concentration of quercetin derivates correlated positively with latitude, whereas the total flavonoid levels remained constant (Stark et al. 2008). Because of a higher hydroxylation level, quercetin derivatives exhibit higher antioxidant capacity than other flavonols typically found in leaves, and they appear to be the flavonoids that are most often reported to increase with increasing light irradiation. Thus, the enhanced production of quercetin derivatives in northern populations reflects the influence of increased light irradiation in long-day conditions. However, in some plants, shift to flavonoids with a higher hydroxylation level in northern populations can also be caused by genetic adaptation. A controlled study on Oxyria digyna ecotypes from 45°N, 60°N and 78°N latitudes showed that the ratio between di- and mono-hydroxylated flavonoids increased from south to north (Nybakken et al. 2004). Lätti et al. (2008) studied the variation of anthocyanin content in 179 bilberry (V. myrtillus) clones from 20 populations growing on a south–north axis (60°21′N–68°34 N) that spanned 1000 km in Finland. Although extensive variation between populations was detected, the southern populations exhibited a significantly lower content of total anthocyanins. Furthermore, differences in the anthocyanin profiles were detected between berries growing in different latitudes. The concentrations of delphinidin and petunidin glycosides were markedly higher in the northern populations. Similarly, a higher total anthocyanin content with higher concentration of delphinidins, as well as the flavonols quercetin and myricetin was detected in bog bilberries (Vaccinium uliginosum L.) growing in northern Finland (Lätti et al. 2010). The authors conclude that the northern climate conditions appear to favour the biosynthesis of more hydroxylated anthocyanins and flavonols, which have been shown to have the greatest antioxidant capacity in vitro. Moreover, even though the final fruit colour is a combination of different factors, delphinidins are known to give bluish hues in flowers and fruits. Thus, one could speculate that the blue berries growing in the north might be more blue compared with the southern populations. These studies show evidence for the hypothesis about the higher contents of phenolic compounds in berries growing in the north. However, whether it is a result of the northern climate conditions or genetic adaptation on the growth environment is still not clear. A recent controlled experiment in controlled environment chambers that was designed to simulate six different temperature and daylength conditions, and that was performed with clonal bilberry material indicated that the bilberry clones of northern origin had a higher phenol content in all treatments (I. Martinussen et al., unpublished results). Additionally, longer day length increased the total phenol content in all samples, but the contents of some phenols were also affected by temperature.


Effect of latitude on flavonoid biosynthesis 1245

CONCLUSIONS According to the numerous studies that have been discussed here, it is obvious that the attributes characteristic to the northern climate – long days with cool night temperatures – have mainly a positive impact on the biosynthesis of flavonoids in plants although there is variation in the response between species and within individual flavonoid groups. Especially, anthocyanins seem to be sensitive to the environmental attributes connected to northern climate conditions because both quantitative and qualitative differences have been detected in the anthocyanin content of northern populations. The contents of flavonols, especially quercetin derivates, appear to increase with increased light irradiation, whereas temperature has a less prominent effect on the total flavonol content. However, there is some evidence that low temperature would increase the quercetin : kaempferol ratio. It appears that the increased total irradiation might be the most important factor because there is no clear evidence that the period of reduced FR/R ratio at the beginning and at the end of a day, which is typical to northern latitudes, would affect the accumulation of flavonoids. The depletion of the ozone layer in the polar areas has increased UV irradiation in the north, and the increase in flavonoid biosynthesis and the protective role of flavonoids against UV light have been confirmed in several experiments. However, for a better understanding of the adaptation of plants to different climates, detailed studies that focus on comparing genetically similar plants at different latitudes are still missing. Future common garden trials where plants of different origins are grown in the same place, together with the simultaneous analyses of the mother plants in the original regions, will shed light on the species-specific genetic variation. Well-designed long-term studies with various plant species are needed to complete the picture of gene–environment interaction in the biosynthesis of flavonoids.

ACKNOWLEDGMENTS We are grateful for the support by the Academy of Finland (projects 120317 and 09141). Prof Esa Hohtola is thanked for the valuable comments on the manuscript.

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