Natural Products Chemistry & Research

Flavonoids are important secondary plant metabolites with many essential functions such as attractants and antioxidant enhancing pollination and seeds dispersal and plants’ resistance to UV light, drought, cold temperature and wounding [1,2]. Flavonoids are also valuable human nutrition and health agents due to their multiple biological activities (e.g., anti-inflammatory, allergenic, bacterial, carcinogenic and viral) [3].

It has been clearly demonstrated that environmental factors, such as temperature, light, and soil moisture content, affect flavonoid biosynthesis and accumulation in many plants [4-7]. Additionally, several flavonoid biosynthesis pathway genes are expressed in various parts of flowering plants (e.g., flowers, fruits, seeds, and leaves), with some have been isolated and characterized (e.g., grape [8]; azaleas [9,10]; bilberry [11,12]; Ginkgo [13]).

Ginkgo is a traditional commercial tree species in China and it is widely planted for leaves, flowers, fruits, and timber harvesting. The medicinal value of Ginkgo leaves prompted the establishment of large scale Ginkgo plantations for the sole purpose of leaf-harvesting for flavonoids and terpene lactones extraction [14,15]. While some studies have been conducted to determine the role of environmental factors on flavonoid biosynthesis and accumulation in Ginkgo leaves, present knowledge is still incomplete due to the complex and confounding nature of field testing that precluded isolating the role of each environmental effect individually [16,17]. Furthermore, it has not yet been clarified how the genes of flavonoid biosynthesis pathway respond to various combinations of temperature and soil moisture content.

The present study is conducted to clarify and investigate the inter-relationship between temperature and soil moisture content on flavonoid accumulation as well as the expression of flavonoid biosynthesis-related genes in Ginkgo (Ginkgo bilobaL.). Phytotrons were used to provide controlled environmental conditions using 2-year-old seedlings.

Plant materials and experimental procedures

A factorial experiment with two main factors (temperature and soil moisture content), each with three levels (temperature: 15/5°C (day/ night), 25/15°C and 35/25°C designated as T1, T2 and T3, respectively, and soil moisture content: 55-60%, 40-45% and 30-35% of field capacity designated as W1, W2 and W3, respectively), thus producing 9 temperature-soil moisture content combinations expressed as W1T1, W2T1, W3T1, W1T2, W2T2, W3T2, W1T3, W2T3 and W3T3 was performed in phytotrons to allow accurate environmental control and the assessment of flavonoid accumulation and their biosynthesis-related genes expression. The present study focused on the combined effect of the main experimental factors (i.e., the 9 temperature-soil moisture content combinations) as the individual role of either temperature or soil moisture content was assessed previously [17]. Three phytotrons with identical light conditions were used and each phytotron was set to control a single temperature. Potted 2-year-old Ginkgo seedlings, two at each pot (average height = 33.0cm and diameter at ground level = 6.8mm), were transferred to the phytotrons on June, 2011, and the treatments started on July, 2011. Seedlings were grown in 12 x 15cm (diameter x height) pots in a 2:1:1 medium consisting of loam, yellow sand, and peat, respectively. The plants received basic nutrients fertilizer as needed.

The day/night temperatures were controlled automatically and soil moisture contents were controlled by weighing method. Randomly sampled leaves from each treatment combination were done at 10, 20, 30, 40 and 50days after the start of treatments. Leaf samples were divided to two parts, one to determine the flavonoid contents and the other to determine relative gene expression.

Analysis of flavonoid content

High performance liquid chromatography (HPLC) as described by the Chinese Pharmacopoeia Commission (2010) was used for flavonoid analyses [18]. A 10 μl reference solution (30 μg quercetin, 30 μg kaempferol, 20 μg isorhamnetin, and 1mL methanol) and extract solution were transferred into liquid chromatograph (Waters 2695, USA) and the contents of quercetin, kaempferol and isorhamnetin were determined. Flavonoids totals = ((quercetin content + kaempferol content + isorhamnetin content) × 2.51). Statistical significance was evaluated using the Tukey-Kramer test.

Expression analysis of flavonoid biosynthesis-related genes

Total RNA was isolated from the leaf samples (three samples for each treatment) following the method of [9] and cDNAs were synthesized using a cDNA synthesis kit (Clontech, USA) following the manufacturers’ manuals. We determined the expression levels of flavonoid biosynthesis-related genes, including PAL (phenylalanine ammonia-lyase), CHS (chalcone synthase), F3H (flavanone 3-hydroxylase), ANR (anthocyanidinreductase), FLS (flavonol synthase), C4H (cinnamate-4-hydroxylase), and ANS (anthocyanidin synthase), together with a fragment of GADPH (glyceraldehyde- 3-phosphate dehydrogenase) coding glyceraldehyde-3-phosphate dehydrogenase as a reference gene by quantitative real-time PCR (qRT-PCR) using an ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) and a SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) following the manufacturers’ manuals [20] (Table 1). An actin gene, having constant expression levels (data not shown), was used to normalize the raw data and to calculate the relative transcript levels. Statistical significance was evaluated using the Tukey-Kramer test.

Table 1: Primers used for the quantification of gene expression levels by qRT-PCR.

Total flavonoid content of ginkgo leaves

The temperature and soil moisture content had significant effects on total flavonoid content of Ginkgo leaves (Table 2). Additionally, all first-order interactions were significant, thus requiring interpretation based on observing the 9 temperature-soil moisture content means (Table 3). Generally, total flavonoid content varied among treatments and was substantially influenced by the treatment duration. For example, W2T1 and W3T1 produced the lowest and highest (p<0.001) flavonoid content under 10-30d and 40-50d treatments, respectively (Table 3), suggesting that a treatment combination of low temperature (T1) coupled with medium to low soil moisture content (W2 or W3) is beneficial to the biosynthesis and accumulation of flavonoid in Ginkgo leaves. Additionally, we observed gradual decrease in total flavonoid content with increasing of temperature at the same soil moisture content (Table 3). The mean flavonoid content across different soil moisture content and duration treatments under T1, T2, and T3 were 15.3, 12.4, and 9.9mg/g, respectively, indicating that lower temperature is conducive to flavonoid biosynthesis and accumulation in Ginkgo leaves. It should also be highlighted that at the same temperature condition, the total flavonoid content produced under different soil moisture contents varied and did not produce any obvious trend and the observed trend for temperature was not duplicated for soil moisture content as the mean flavonoid content across W1, W2, and W3 and duration were 12.25, 12.13, and 13.22mg/g, respectively, suggesting that lower soil moisture content is favorable but the effect is not as pronounced as that of temperature. The total flavonoid content of the same temperature-soil moisture content treatment combination also had significant variation at different treatment time, and generally the 40- and 50d durations produced higher total flavonoid content, highlighting the positive role of prolonging treatment time on flavonoid accumulation.

Table 2: Three-way analysis of variance showing the effects of temperature (T), soil moisture (W), treatments time (D), and their interactions (T×W, T×D, W×D, and T×W×D) on total flavonoid content and their expression level for seven flavonoid biosynthesis-related genes.

Table 3: Total flavonoid content in mg·g^-1 (mean ± SD) in Ginkgo leaf across five durations (10d to 50d) under nine temperature - soil moisture treatments. Means with different letters are significantly different at P< 0.05. (see Material and Methods for treatments designation).

The expression level of flavonoid biosynthesis-related upstream genes and their relationship with flavonoid accumulation

Temperature and soil moisture content treatment combinations and their duration, all affected the expression level of C4H and CHS genes significantly, but the effect of soil moisture content on the expression of PAL gene was not significant (Tables 2 and 4). All temperature-soil moisture content treatment combinations within the various treatments duration flavonoid expression levels did not produce clear trend(s) for total flavonoids and any generalization was difficult to discern (supplementary material).

Table 4: The expression level of flavonoid biosynthesis-related upstream genes (mean ± SD) in Ginkgo leaf across five durations (10d to 50d) less than nine temperature - soil moisture treatments. (See Material and Methods for treatments designation).

At the same soil moisture content, the expression level of PAL, C4H, and CHS genes gradually declined with increasing of temperature (Table 4). At the same temperature, the PAL and C4H expression levels were highest at W3 while W1 produced the highest gene expression for CHS. Generally, under the three temperatures used, the mean PAL, C4H, and CHS gene expression of different soil moisture treatments declined (PAL: 1.41, 1.16, 0.96 (T1, T2, T3); C4H: 1.47, 1.08, 0.93; CHS: 1.85, 1.61 and 0.88), indicating that lower temperature is conducive for their expression in Ginkgo leaves. On the other hand, under the three studied soil moisture contents, the mean PAL, C4H, and CHS gene expression of different temperature treatments produced different trends (PAL: 1.19, 1.15, 1.20 (W1, W2, W3); C4H: 1.11, 1.02, 1.35; CHS: 1.83, 1.23, 1.28), suggesting that lower soil moisture is favorable for C4H, higher soil moisture is beneficial for CHS, and soil moisture has no obvious effect on PAL gene expression (Table 2 and 4).

The expression level of flavonoid biosynthesis-related downstream genes and their relationship with the flavonoid accumulation

Temperature and soil moisture content as well as treatments duration, all significantly affected the expression level of the flavonoid biosynthesisrelated downstream genes (F3H, FLS, ANS, and ANR); however, the expression level varied across the same temperature-soil moisture combinations (Table 2 and 5). Additionally, prolonging treatment time was not associated with any clear increasing or decreasing trends across the four studied genes, indicating that a particular treatment combination (i.e., interaction) is required for each specific gene.

Table 5: The expression level of flavonoid biosynthesis-related downstream genes (mean ± SD) in Ginkgo leaf across five durations (10d to 50d) under nine temperature - soil moisture treatments. (see Material and Methods for treatments designation).

The flavonoid family is divided into six main classes; namely, flavonols, flavones, lavan-3-ols, isoflavones, flavanones, and anthocyanidins [21]. The metabolism of flavonoids follows a complex pathway, and some environmental factors such as temperature, water status, light condition, and nitrogen all have been demonstrated to affect flavonoids accumulation [22-25]. The present study demonstrated that lower temperature and to a lesser extent lower soil moisture content is favourable to flavonoid accumulation in Ginkgo leaves, and more specifically W2T1 and W3T1 treatments produced the highest total flavonoid content (Table 3). Studies related to temperature effect on flavonoid content in other plants generally concluded that lower temperature is beneficial to the biosynthesis and accumulation of flavonoid supporting our observations. For example, elevated bunch temperature was associated with total anthocyanin, proanthocyanidin, and quercetin concentrations reduction in grape berry skins (Vitis vinifera cv. Merlot and Cabernet-Sauvignon [26-27]). Similar observation was made in maize (Zea mays) where seedlings growing under short-term, low-temperature treatment showed substantial changes in anthocyanin content [4]. Modeling flavonoid biosynthesis and degradation indicated that they are sensitive to temperature [24]. Conversely, the role of soil moisture on flavonoid content in plants did not produce uniform results. For example, water stress resulted in increasing the expression of anthocyanin biosynthetic pathway genes in grape berries; however, with limited effect on the biosynthesis of proanthocyanidin and flavonol [6]. Opposite trend was observed for epigallocatechin gallate, ascorbic acid, and α-tocopherol concentrations of Cistus clusii leaves which increased by 2.8, 2.6, and 3.3 fold; respectively, in response to drought stress [22]. Finally, no clear trend was detected for leaf polyphenol concentrations in Ligustrum vulgare leaves under reduced water treatment [28].

Flavonoid biosynthesis in Ginkgo leaves requires coordinated expression of genes encoding enzymes in the core phenylpropanoid pathway, such as PAL and 4-coumarate CoA ligase, and enzymes in branch pathways, such as CHS and CHI (chalconeisomerase). These genes are divided into structural genes that encode the key enzymes of the flavonoid pathway and regulatory genes that are required for flavonoid biosynthesis. The structural genes are separated into upstream genes, such as PAL, C4H, CHS, and CHI, which catalyze the process from phenylalanine to hydroxycinnamic acid and 4-Coumaroyl-CoA, and downstream genes, such as F3H, FLS, ANS, and ANR, which catalyze the process from 4-Coumaroyl-CoA to flavonoids. PAL is a link between primary and secondary metabolism and high PAL expression is often found in parallel with high levels of flavonoids [29]. In our study, we found PAL expression levels at 15/5°C (day/night) was 1.46 and 1.20 times than that at 35/25 and 25/15°C, respectively, while PAL expression levels at different soil moisture content treatments did not differ as they were not significantly different from each other (Table 3). We also found that the changing trend of PAL expression in different treatments at different durations were identical with that of flavonoid content, and higher PAL expression coincided with higher flavonoid content. These results imply that lower temperature improves the expression of PAL gene, while soil moisture content has a negligible effect on its expression, as evident by the clear relation between PAL expression and the biosynthesis of flavonoid. Similar observations of PAL increased expression were reported after exposure to low temperature in Arabidopsis thaliana leaves [30] and tomato (Solanum lycopersicum) plants [31]. Additionally, the unclear role of moisture effect on PAL gene expression was also supported by the conflicting observations in Camellia sinensis [32] and Scutellaria baicalensis [33] which showed decreased and increased PAL expression under water deficit conditions, respectively.

C4H and CHS, the other two upstream genes of flavonoid biosynthesis are well investigated as C4H is the key enzyme in the second step of phenylpropanoid pathway as it controls the synthesis of ρ-coumaric acid from trans-cinnamic acid [34], while CHS is known to encode the first committed enzyme in flavonoid biosynthesis. CHS has been used extensively as a model for identifying and studying elements controlling the expression of flavonoid biosynthetic genes [5]. In the present study, we found lower temperature to be conducive for the expression of C4H and CHS genes in Ginkgo leaves as total flavonoid content was highest at lower temperature. Treatment duration; however, tended to change C4H and CHS gene expression as their expression levels were consistent with total flavonoid content, further supporting the close relation between flavonoids biosynthesis and C4H and CHS expression. Similar results were reported in grape skin (V. labruscana) [7] and tomato plants (Solanum lycopersicum cv. Suzanne) [31] showing increased CHS expression in response to lower temperatures. Our results also demonstrated the presence of consistent relationship between soil moisture and C4H expression and flavonoid content (i.e., higher expression under lower soil moisture) as well as the lack of relationship for CHS as expression was higher under high soil moisture, results are at variance with what has been reported in Camellia sinensis [32] and Scutellaria baicalensis [33] in which increased expression was reported under water deficit.

F3H, FLS, ANS, and ANR are downstream genes of flavonoid biosynthesis, which catalyze hydroxyl cinnamoyl CoA to generate different kinds of flavonoids. F3H is a key enzyme at a diverging point of flavonoid pathway leading to the production of different pigments, such as phlobaphene, proanthocyanidin, and anthocyanin. FLS is an essential enzyme of flavonols biosynthesis as it converts dihydrokaempferol or dihydroquercetin to the corresponding flavonols, kaempferol and quercetin, respectively, and it has a close relation with the accumulation of flavonols. ANS and ANR play important role in anthocyanidin and proanthocyanidin biosynthesis, respectively, and both affect flavonoid biosynthesis and their content in plants. Our results showed that the best temperature and soil moisture conditions to stimulate the expression of these downstream genes are different and F3H, FLS, ANS, and ANR preferred different combinations (i.e., interaction). F3H and FLS favoured lower, ANR responded well to higher, while ANS reacted similarly to both low and high temperature. The same scenario was observed to soil moisture with FLS and F3H favouring low and ANS and ANR responded well to high soil moisture content. Similar results were reported for F3H and FLS genes with increased expression in grape berries under water deficit [6].

The present study demonstrated that temperature and soil moisture content, to a lesser extent, has significant effects on the accumulation of flavonoid and gene expression of flavonoid biosynthesis. Total flavonoid content was higher in lower temperature and lower soil moisture content in Ginkgo leaves, and that flavonoid biosynthesisrelated genes were up-regulated by lower or higher temperature as well as lower or higher soil moisture, suggesting that temperature and soil water content have a synergistic effect on the expression of genes within the flavonoid biosynthesis pathway. These results should contribute to a complete understanding of the relationships between environmental factors and flavonoid accumulation in Ginkgo leaves.

We thank the National Natural Science Foundation of China, The Science and Technology Department of China, and The State Forestry Bureau.