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http://journals.tubitak.gov.tr/biology/

Turkish Journal of Biology Turk J Biol(2016) 40: 612-622© TÜBİTAKdoi:10.3906/biy-1505-2

Flavonoid dynamic responses to different drought conditions: amount, type, and localization of flavonols in roots and shoots of Arabidopsis thaliana L.

Behrokh SHOJAIE1,2, Akbar MOSTAJERAN1,*, Mustafa GHANNADIAN2,3

1Plant Science Division, Department of Biology, Faculty of Science, University of Isfahan, Isfahan, Iran2Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran

3Department of Pharmacognosy, School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran

* Correspondence: mostajerana@yahoo.com; mostajeran@sci.ui.ac.ir

1. IntroductionPlants suffer from drought stress when they do not have access to adequate water to establish a balance between water uptake and loss (Akıncı and Lösel, 2012). In such cases, plants use various strategies to overcome water deficit in order to survive. Strategies are divided into two categories: drought avoidance and drought tolerance (Farooq et al., 2009). The strategies selected by plant species depend on drought stress (intensity and duration) and the ability of the plant to perform molecular, biochemical, and physiological modifications (Xoconostle-Cázares et al., 2010). A part of the biochemical adjustment involves changing type or amount of flavonoid compounds (Hernández et al., 2004; Yang et al., 2007; Yuan et al., 2012; Nakabayashi et al., 2014) as well as changing transport and distribution patterns in plant tissues/organs (Braidot et al., 2008; Buer et al., 2013). Flavonoids are a group of multifunctional plant secondary metabolites (Ferreyra et al., 2012) that play a key role in protecting plants against UV-radiation, pathogens, and abiotic stresses (Treutter, 2006). They determine flower, fruit, and seed colors (Ferreyra et al., 2012) and are also responsible for allelopathy, plant-

bacteria symbiosis (Treutter, 2006), and control of plant growth and development through inhibition of auxin transport (Brown et al., 2001; Buer et al., 2010; Peer et al., 2011). A part of the phenylpropanoid pathway corresponds to flavonoid biosynthesis (Winkel-Shirley, 2001). In Arabidopsis thaliana, naringenin is the first flavonoid compound, and other flavonoids are derived from it (Buer et al., 2010). In addition, kaempferol and quercetin are two main flavonoid compounds called flavonol because of the hydroxyl group in carbon 3 of ring C of the flavone backbone (Tsao, 2010). They can be glycosylated by adding rhamnose and glucose at the positions of carbon 3 and 7 and produce various glycosidic derivatives (Santelia et al., 2008; Khan et al., 2011). Moreover, the protective task of flavonoids in plants is related to their antioxidant activity (Tattini et al., 2004; Nakabayashi et al., 2014). Hydroxyl groups of flavonoids, as electron or hydrogen donors, cause the scavenging of free radicals (Han et al., 2012). Among flavonoids of Arabidopsis, kaempferol and quercetin show more radical scavenging activity than the other flavonoids (Amić et al., 2003).

Abstract: Flavonoids accumulate in plants in response to water deficit. Changes in amount, type, and localization of flavonoids under different drought conditions in Arabidopsis thaliana have not been well investigated. Therefore, in this study flavonoid patterns were investigated under water potentials of –0.2, –0.5, and –0.9 MPa at 0, 24, 48, 120, and 192 h after drought induction. Determination of amount and type of flavonoids was performed by HPLC and spectroscopy. In addition, localization of flavonoids was detected by DPBA staining and a fluorescent microscope. Only quercetin and kaempferol were detected in hydrolyzed extracts of roots and shoots. The maximum amounts of the above-mentioned flavonols were detected under severe drought stress. Under all drought conditions, there was more kaempferol than quercetin. Moreover, amounts of both flavonols and total flavonoids were greater in roots than in shoots. Different fluorescence intensities of the flavonoid-DPBA complex were observed in all seedlings from shoots to root tips. The results of this study suggest that flavonoid responses of Arabidopsis to drought stress are dynamic, and intensity and duration of drought stress could play a key role in determination of type, amount, and localization of flavonoids in response to different levels of water deficit.

Key words: Arabidopsis thaliana, drought stress, flavonoid, kaempferol, quercetin

Received: 01.05.2015 Accepted/Published Online: 10.08.2015 Final Version: 18.05.2016

Research Article

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Drought is a common environmental stress that causes an increase in reactive oxygen species (ROS) (de Carvalho, 2008; Kavas et al., 2013). Some studies reported accumulation of flavonoids as powerful ROS scavengers in response to drought stress (Winkel-Shirley, 2002; Treutter, 2006; Nakabayashi et al., 2014). For example, under different drought conditions, enhancement of total phenolic compounds and three flavanols (epicatechin, epicatechin gallate, and epigallocatechin gallate) in leaves of Cistus clusii (Hernández et al., 2004), an increase in production of flavonoids in cell suspensions of Glycyrrhiza inflata Batal (Yang et al., 2007), a rise in levels of kaempferol in broccoli (Khan et al., 2011), and an increase in flavonoid content in different organs of motherwort plant (Wei et al., 2013) were observed.

Since there is little information about the effects of drought stress on flavonoid accumulation in roots and shoots of young Arabidopsis thaliana seedlings as a model plant, this study was performed to answer the following question: Do intensity and duration of drought stress regulate type, amount, and localization of Arabidopsis flavonoids in response to different water deficit conditions? In order to answer this question, flavonoid patterns of roots and shoots of Arabidopsis thaliana seedlings, including type of flavonoid compounds, total flavonoid content, amounts of specific flavonols, and distribution and accumulation patterns of flavonoids in seedlings grown under different water potentials (–0.2, –0.5, and –0.9 MPa) were studied at 0, 24, 48, 120, and 192 h after drought induction.

2. Materials and methods2.1. Plant material, growth conditions, and drought inductionWild-type seeds of Arabidopsis thaliana L. (Col-0) were surface sterilized. Then they were placed on Murashige and Skoog (MS) basal medium (Murashige and Skoog, 1962) solidified with 0.8% (w/v) agar (Roth, Germany), pH 5.8. After 24 h of stratification at 4 °C the seeds were grown vertically in a growth chamber at 22 ± 2 °C under a photoperiod of 16 h light and 8 h dark.

Then 4-day-old seedlings were transferred to MS media containing equal concentrations of nutrients without any osmoticum compound. On the basis of the primary experiment (data not shown), the media were supplied with 0.8%, 2%, and 4% agar in order to induce water potentials of –0.2 MPa (control condition), –0.5 MPa (mild drought stress), and –0.9 MPa (severe drought stress), respectively. After 0, 24, 48, 120, and 192 h of drought induction, 225 seedlings from each treatment were split into roots and shoots. The roots and shoots were immediately weighed separately for their fresh weight and then frozen by liquid nitrogen for further analysis.

2.2. Measurement of flavonoids by high performance liquid chromatography (HPLC)The frozen samples were ground in liquid nitrogen to obtain a very fine powder of plant tissue. Then 500 µL of cold acetone (Caledon, Canada) was added to each sample (each sample was obtained from 225 seedlings grown under a specific treatment and was equal to approximately 20–60 mg of root and 150–500 mg of shoot). Samples were incubated at 4 °C for 24 h. After being centrifuged at 13,000 rpm for 5 min, the supernatants were transferred to new tubes. The supernatants were used for analyzing aglycone flavonoids in crude extracts, and pellets were treated again with ethanol (Caledon, Canada)/deionized water (70/30, v/v) to extract glycosylated flavonoids. After evaporation of the solvent, 2 mL of HCl (2 N) (Merck, Germany) was added to each pellet, and they were incubated at 90 °C for 1 h for the hydrolyzing process. To separate the hydrolyzed flavonoids from the aqueous phase, 2 mL of ethyl acetate (Caledon, Canada) was added to each cooled acidic extract and mixed well. After formation of a two-layer mixture, the upper layer (organic phase containing hydrolyzed flavonoids) was collected and placed into a new glass vial and dried under a fume hood. Each pellet was dissolved in 200 µL of ethyl acetate and used for high performance liquid chromatography (HPLC) analysis. Samples (crude extracts and hydrolyzed extracts) were injected and analyzed by reverse-phase HPLC on a Waters system (515 HPLC pump) and Nova-Pak C18 30 × 150 mm column; (Waters, Milford, MA, USA) using phosphoric acid (Merck, Germany) (20 mM) as solvent-A and acetonitrile (Caledon, Canada)/water (70/30, v/v) as solvent-B. The HPLC program was started with A/B (80/20, v/v) for 4 min, 20%−100% B for 25 min, then A/B (0/100, v/v) for 5 min, followed by equilibrating for 10 min. The flow rate was 1 mL min–1 at 45 °C, and the injection volume for standards and samples was 20 μL. Quercetin and kaempferol were detected at 365 nm and naringenin at 290 nm. Flavonoid standards including quercetin (Sigma-Aldrich, USA), kaempferol (Sigma-Aldrich, Germany), and naringenin (Sigma-Aldrich, UK) were used for the calibration curve. All solvents were HPLC grade.2.3. Determination of total flavonoid contentTotal flavonoid determination was carried out according to AlCl3 method (Pękal and Pyrzynska, 2014) with some modifications. First, 100 µL of crude extract was added to a test tube containing 100 µL of 2% AlCl3, 20 µL of glacial acetic acid (Merck, Germany), and 200 µL of methanol (100%) (Caledon, Canada). They were then mixed well and incubated for 30 min at room temperature. Optical density of each sample was detected at 425 nm by a UV-visible spectrophotometer (UV-160, Shimadzu). Concentrations of 0.5, 1, 2, 5, 10, and 20 µg mL–1 of quercetin were used for the calibration curve.

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2.4. Fluorescent microscopyThe distribution and accumulation patterns of flavonoids in the seedlings grown under drought conditions were detected using fluorescence of the flavonoid-DPBA complex. For this step, seedlings were submerged and incubated in a staining reagent containing 2.52 mg mL–1 DPBA (diphenyl boric acid 2-amino ethyl ester) (Sigma-Aldrich, Germany) and 0.1% (v/v) Triton 100X at room temperature and under dark conditions for 2 h. Then each seedling was observed by epifluorescent microscope (Olympus BX40). WB (blue) and WG (green) filters were used for visualization of kaempferol and quercetin, respectively. All pictures were captured by an Olympus DP12 camera.2.5. Statistical analysisAll experiments were performed in a completely random design with 3 replicates. One-way ANOVA was used for treatment assay, and Duncan’s multiple range tests were used to compare the mean values.

3. Results3.1. Flavonoid quantification by HPLC analysisThe HPLC results indicated that no flavonoid compounds including quercetin, kaempferol, and naringenin were detected in crude (unhydrolyzed) extracts of roots and shoots of Arabidopsis thaliana seedlings grown under –0.2, –0.5, and –0.9 MPa water potentials up to 192 h after drought induction. In contrast, only quercetin and

kaempferol were detected in some of the hydrolyzed extracts of roots and shoots; in addition, more flavonols (almost 10–20-fold) were observed in roots than in shoots (Figures 1 and 2).3.1.1. Flavonols in rootsThe HPLC pattern of hydrolyzed extracts in roots (Figures 1a and 1b) showed that under all water potentials (except –0.9 MPa) no quercetin or kaempferol was detected before 48 h after drought induction, and then a similar upward trend in quercetin and kaempferol was observed up to 192 h. Under severe drought conditions, amounts of quercetin and kaempferol were higher than their amounts under control and mild drought conditions. The highest amounts of quercetin and kaempferol were detected under severe drought stress 192 h after drought induction. However, at 192 h there was no significant difference in the amount of quercetin for severe and mild drought stresses. In addition, both diagrams showed that under mild drought stress up to 120 h, the increase in quercetin and kaempferol is slower and more gradual than its amount under control conditions.

Furthermore, under all water potentials, the amount of kaempferol was greater than the amount of quercetin. Although the ratio of kaempferol to quercetin (about 1.5-fold) remained almost constant under severe drought stress from 48 h to 192 h, increasing and decreasing trends (about 0.2) were observed for the ratio of kaempferol to quercetin from 120 h to 192 h under control conditions and mild drought stress, respectively.

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Figure 1. Amounts of quercetin (a) and kaempferol (b) in hydrolyzed root extracts of Arabidopsis thaliana seedlings grown under control conditions (Ψw = –0.2 MPa) and mild (Ψw = –0.5 MPa) and severe (Ψw = –0.9 MPa) drought stresses at 0, 24, 48, 120, and 192 h after drought induction. Data are means (n = 3) and error bars are ±SD. Zero value at 0, 24, and 48 h indicated nondetected amount of quercetin and kaempferol. MPa and FW stand for megapascal and fresh weight, respectively.

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3.1.2. Flavonols in shootsAs in roots, similar patterns of quercetin and kaempferol (Figures 2a and 2b) were observed for hydrolyzed shoot extracts of Arabidopsis thaliana seedlings grown under different water potentials at 0, 24, 48, 120, and 192 h after drought induction. Under control conditions quercetin and kaempferol were detected after 120 h of drought induction, but under mild and severe drought conditions quercetin and kaempferol were detected after 48 h of drought induction. At 192 h after drought induction, the highest values for quercetin and kaempferol were measured in all drought conditions except mild drought stress.

In shoot extracts, kaempferol was also detected in higher amounts in comparison with quercetin. Under mild and severe drought stresses, the ratio of kaempferol to quercetin increased noticeably (about 1.5-fold) from 120 h to 192 h. There were significant differences in the ratio of kaempferol to quercetin under three drought conditions at 192 h, with the highest ratio (about 2.3-fold) belonging to control conditions.3.2. Total flavonoid contentThe results of total flavonoids obtained from crude (unhydrolyzed) extracts of roots and shoots of Arabidopsis thaliana seedlings (Figures 3a and 3b) showed that roots have more total flavonoid content than shoots. There was also dissimilarity between total flavonoid patterns in roots and shoots.

In roots, different amounts of total flavonoids were observed under different drought conditions at 0, 24, 48, 120, and 192 h after drought induction. Although under severe drought stress there were significant differences among total flavonoids over time, total flavonoid content of control samples showed no significant difference except at 120 h. Moreover, under severe drought stress, the amount of total flavonoids was significantly higher than its amount under control conditions or mild drought stress up to 120 h, and then it dropped sharply. However, total flavonoid content was remarkably higher under mild drought stress than severe drought stress only at 192 h.

In shoots subjected to mild drought stress, total flavonoid content increased during 192 h of drought induction. Therefore, the maximum flavonoid content was observed under mild drought stress at 192 h. In contrast, under severe drought stress and control conditions, no significant differences were seen in total flavonoids over time. 3.3. Pattern of flavonoid distribution and localization in whole seedlingsFluorescent microscope images (Figures 4 and 5) confirmed the existence of both kaempferol and quercetin in all seedlings. Although fluorescence signals of flavonol-DPBA complexes were observed in different organs in all seedlings grown under different water potentials after drought induction, there were variations in fluorescence intensities and, consequently, distribution pattern of

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Figure 2. Amounts of quercetin (a) and kaempferol (b) in hydrolyzed shoot extracts of Arabidopsis thaliana seedlings grown under control conditions (Ψw = –0.2 MPa) and mild (Ψw = –0.5 MPa) and severe (Ψw = –0.9 MPa) drought stresses at 0, 24, 48, 120, and 192 h after drought induction. Data are means (n = 3) and error bars are ±SD. Zero value at 0, 24, 48, and 120 h indicated nondetected amount of quercetin and kaempferol. MPa and FW stand for megapascal and fresh weight, respectively.

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flavonoids. High fluorescence intensities were observed in the shoot apex, hypocotyl, and root maturation zone of all seedlings. In the shoot apex, fluorescence intensity decreased slowly toward the end of the leaf blade and roots. In cotyledonary leaves and/or true leaves, the highest fluorescence intensity was observed in midrib and in other leaf veins. Moreover, a gradual reduction in fluorescence signal was evidenced in the elongation zone of all roots from the side of the maturation zone toward the root tip. In contrast, root tips, especially the root apex, showed the greatest differences in florescence intensity. At 24 h after seedlings were transferred to media with different water potentials, fluorescence of the root apex increased in comparison with 4-day-old seedlings before their transfer to new MS media with different water potentials (0 h). Then 48 h after drought induction less intensity of fluorescence was observed. In addition, fluorescence intensity did not increased up to 120 h except in seedlings grown under severe drought stress; however, at 192 h after drought induction fluorescence intensity was elevated in control and mild drought conditions. Fluorescence was also observed in lateral roots in a pattern similar to the pattern in primary roots.

4. Discussion4.1. Flavonoid quantification by HPLC analysisThe high antioxidant activity and structural properties of flavonoids (Amić et al., 2003; Han et al., 2012) give plants a remarkable ability to deal with various conditions

(Solecka, 1997). Studies show that flavonoids could have a determinant role in response to different stresses such as drought (Petrussa et al., 2013). In this regard, overexpression of some genes involved in flavonoid metabolism and overaccumulation of flavonoids caused an increase in drought tolerance in Arabidopsis thaliana (Nakabayashi et al., 2014).

In the present study, flavonoid compounds including naringenin, quercetin, and kaempferol were not detected in crude (unhydrolyzed) extracts of roots or shoots of Arabidopsis thaliana seedlings, whereas quercetin and kaempferol in hydrolyzed extracts were measured by HPLC, especially in seedlings grown under drought stress at 120 and 192 h after drought induction. Buer et al. (2007) also reported that quercetin and kaempferol were not detected in the unhydrolyzed extracts of roots or aerial tissue (excluding kaempferol) of Arabidopsis seedlings. The lack of detected flavonoids in crude (unhydrolyzed) extracts could be related to the very low amounts of naringenin and two other aglycone flavonoids (quercetin and kaempferol) that were below the detection threshold of our HPLC apparatus. The very low amounts of flavonoids in unhydrolyzed extracts may be due to quick conversion of naringenin as the first and key compound of the flavonoid biosynthesis pathway into other compounds, particularly quercetin and kaempferol. Furthermore, these aglycone flavonoids could be rapidly glycosylated (Buer et al., 2010) to reduce the toxicity of aglycone flavonoids and increase their water solubility and chemical stability

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Figure 3. Total flavonoids in unhydrolyzed extracts of root (a) and shoot (b) of Arabidopsis thaliana seedlings grown under control conditions (Ψw = –0.2 MPa) and mild (Ψw = –0.5 MPa) and severe (Ψw = –0.9 MPa) drought stresses at 0, 24, 48, 120, and 192 h after drought induction. Data are means (n = 3) and error bars are ±SD. MPa and FW stand for megapascal and fresh weight, respectively.

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Figure 4. Distribution pattern of kaempferol based on fluorescence of kaempferol-DPBA complex in Arabidopsis thaliana seedlings grown under control conditions (Ψw = –0.2 MPa) and mild (Ψw = –0.5 MPa) and severe (Ψw = –0.9 MPa) drought stresses at 0, 24, 48, 120, and 192 h after drought induction. WB blue filter was used for visualization of kaempferol-DPBA complex. Magnification of all images except pictures with higher magnification (200×) was 100×. Scale bars = 20 µm. LR and Ψw stand for lateral root and water potential, respectively. Arrows show lateral roots.

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Figure 5. Distribution pattern of quercetin based on fluorescence of quercetin-DPBA complex in Arabidopsis thaliana seedlings grown under control conditions (Ψw = –0.2 MPa) and mild (Ψw = –0.5 MPa) and severe (Ψw = –0.9 MPa) drought stresses at 0, 24, 48, 120, and 192 h after drought induction. WG green filter was used for visualization of quercetin-DPBA complex. Magnification of all images except pictures with higher magnification (200×) was 100×. Scale bars = 20 µm. LR and Ψw stand for lateral root and water potential, respectively. Arrows show lateral roots.

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(Jones and Vogt, 2001; Treutter, 2006). The increase in the amount of glycosylated flavonoids could be an appropriate way to cope with free radicals produced during drought stress, organogenesis, and growth without creating a negative effect from its accumulation.4.1.1. Flavonols in rootsDetection of quercetin and kaempferol (in hydrolyzed extract) at 24 h after drought induction in roots grown under –0.9 MPa water potential led to the idea that severe drought stress could induce synthesis and accumulation of glycosylated flavonoids earlier than mild drought stress and control conditions. Moreover, the increase in flavonols under both drought stresses may confirm that drought promotes accumulation of flavonoids in roots. Accumulation of quercetin and kaempferol under control conditions could be attributed to the role of flavonoids as multifunctional compounds in plant growth and development (Misyura et al., 2012). Flavonoids can serve a plant via regulation of ROS, which are byproducts of metabolic pathways in some cellular organelles. Under nonstress conditions, the progression of plant growth and development causes a rise in ROS levels; as a result, the level of antioxidant compounds, such as flavonoids, can increase for scavenging ROS (Boguszewska and Zagdańska, 2012). Other possible reasons for the enhancement of flavonoids during plant growth and development under control conditions (nonstress conditions) are the significance of flavonoids in the regulation of processes such as auxin transport (Buer et al., 2010; Peer et al., 2011), protein activity, and transcription (Brunetti et al., 2013). Therefore, the amount of flavonoids may increase along with growth and development due to the need for these processes.

A ratio of about 1.5-fold of kaempferol to quercetin (in hydrolyzed extract) under severe drought stress indicated that kaempferol may increase more than quercetin under severe drought stress and that biosynthesis of flavonoids shifted toward kaempferol. This may be related to the greater antioxidant power of kaempferol in comparison with quercetin (Amić et al., 2003). However, the reduction of 0.2 in this ratio under mild drought stress could be related to the same tendency for biosynthesis or accumulation of both of the above-mentioned flavonoids in seedlings grown under mild drought stress. Yuan et al. (2012) also reported variation in accumulation of flavonoid compounds under drought stress. Under control conditions, the ratio of kaempferol to quercetin at 120 h increased up to approximately 1.5-fold at 192 h. Therefore, as previously mentioned, under control conditions by increasing ROS during the growth and development stages and the need for higher antioxidant activity, the seedlings probably preferred the accumulation kaempferol over quercetin due to the greater antioxidant power of kaempferol (Amić et al., 2003).

4.1.2. Flavonols in shootsThe lower amounts of flavonols found in shoots compared with roots may be due to the transport of flavonoids to the roots. In previous studies, transport of flavonoids from shoots to roots has been reported (Buer et al., 2007). In addition, conversion of flavonols to anthocyanins could be another reason for lower accumulation of flavonols in shoots (Misyura et al., 2012). We observed accumulation of anthocyanins in shoots grown under different water potentials and growth stages (data not shown). However, the patterns of quercetin and kaempferol and their ratios under drought conditions could be a confirmation of the importance of kaempferol in shoots. Moreover, the ratio of kaempferol to quercetin could be a determinant factor in response to different stress conditions such as drought. In support of this idea, it was reported that high ratio of quercetin to kaempferol caused photoprotection in Petunia leaves (Ryan et al., 2002). Consistent with the results of our study, diversity in the amount of accumulated flavonoids in response to drought stress has been reported in leaves of Ligustrum vulgare (Tattini et al., 2004).4.2. Total flavonoids In this study, total flavonoids means all flavonoid compounds in unhydrolyzed extracts of roots and shoots; flavonols such as quercetin and kaempferol are considered a part of total flavonoid compounds. In addition, kaempferol and quercetin, as measured by HPLC, were obtained from hydrolyzed extracts; total flavonoids were measured using unhydrolyzed extracts. Therefore, the amount of total flavonoids may not correlate with the sum of kaempferol and quercetin as measured by HPLC.

Total flavonoid patterns in roots and shoots could indicate that synthesis or accumulation of total flavonoids depends on drought intensity and may be regulated by duration of drought stress. The same result was reported for total flavonoids of some plants grown under drought stress (Yamaguchi et al., 2010; Yuan et al., 2012; Wei et al., 2013). Since roots are the first site to sense the drought signal (Comstock, 2002), higher accumulation of total flavonoids in the roots may be attributed to the role of roots in signal perception.4.3. Distribution and localization pattern of flavonoidsFluorescence patterns obtained from the flavonoid-DPBA complex (Figures 4 and 5) indicate that flavonoid localization may be regulated by development and tissue/organ type as well as drought intensity. The results of localization patterns of flavonoids in this study are similar to those of other studies (Peer et al., 2001). Moreover, the existence of fluorescence gradients from shoot apex toward roots and cotyledonary leaves or true leaves could be evidence for biosynthesis of flavonoids in shoots and their transport to other organs via vascular tissue. In previous studies, biosynthesis of flavonoids in shoots was confirmed (Buer et al., 2007).

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The intensity of fluorescence in root elongation zone of seedlings grown under severe drought stress was greater than in other seedlings. Since these seedlings had the shortest primary roots, as shown in our previous study (Shojaie et al., 2015), it may be that drought stress via modification of flavonoid accumulation in the root elongation zone regulates root growth. The role of flavonoids in regulation of auxin transport as a main factor in root growth and development can support this idea (Murphy et al., 2000; Brown et al., 2001; Peer et al., 2011). Furthermore, there are some reports that indicate that higher flavonoid concentrations could inhibit plant growth (Brown et al., 2001; Mahajan et al., 2011). As a result, it seems that flavonoids aid the survival of Arabidopsis seedlings under severe drought conditions in two ways. First, they increase the antioxidant ability of seedlings to scavenge free radicals. Second, the excessive accumulation of flavonoids, especially in the root elongation zone, could inhibit primary root elongation to aid in survival.

In the root tips a different intensity of fluorescence and, consequently, various patterns of flavonoid accumulation under different drought conditions showed that root tips could play a key role in sensing and responding to different water potentials. The importance of root tips in response to drought was reported by Shimazaki et al. (2005). Thus, flavonoids could be signal molecules that play an important role in perception or transduction of signals because they can regulate the level of ROS as the main factor in stress signaling (Karuppanapandian et al., 2011).

Although biosynthesis of flavonoids occurs in shoots, some studies showed that flavonoids can be synthetized in roots of Arabidopsis seedlings (Brown et al., 2001; Saslowsky and Winkel-Shirley, 2001). An increase in fluorescence intensity in root tips at 24 h after transference to new medium with different water potentials may indicate that mechanical stimulus also has an effect on flavonoid accumulation. The higher fluorescence intensity of the DPBA-flavonoid complex at the site of lateral root formation demonstrates that flavonoids may stimulate lateral root initiation and can promote lateral root formation. Maximum lateral root number in seedlings grown under severe drought stress, as shown in our previous study (Shojaie et al., 2015), may confirm this idea. Peer et al. (2001) also demonstrated the accumulation of quercetin in mature secondary roots of wild-type Arabidopsis seedlings. Flavonoid compounds may have a positive effect on lateral root formation.

The results of this study showed different patterns of flavonoids in type, amount, and localization in roots and shoots of Arabidopsis thaliana seedlings grown under control (Ψw = –0.2 MPa), mild (Ψw = –0.5 MPa), and severe (Ψw = –0.9 MPa) drought conditions at 0, 24, 48, 120, and 192 h after drought induction. According to the results, no flavonoid compounds (naringenin, quercetin, or kaempferol) were detected in unhydrolyzed extracts obtained from roots and shoots of Arabidopsis thaliana seedlings. Only quercetin and kaempferol were measured in hydrolyzed extracts of roots and shoots of the seedlings grown under different levels of drought, especially 120 and 192 h after drought induction. These results indicated low amounts of naringenin and aglycone flavonoids, as well as accumulation of glycosidic flavonoids in seedlings. The maximum level of flavonols (quercetin and kaempferol) belonged to seedlings grown under severe drought stress, which may be related to the high antioxidant power of these flavonols. In addition, the amount of kaempferol was greater than the amount of quercetin under all drought conditions which represented a high tendency in both roots and shoots for accumulation of kaempferol. Roots also accumulated more flavonoid compounds than shoots did. Different fluorescence intensities of the flavonoids-DPBA complex from shoot to root tip in all seedlings indicated that localization of flavonoids (kaempferol and quercetin) might be regulated by developmental mechanisms, tissue/organ type, and drought conditions. Changes in flavonoid metabolism in response to different drought conditions are dynamic and depend on the intensity and duration of drought stress. Therefore, intensity and duration of drought stress could modulate type, amount, and localization of flavonoids in roots and shoots of Arabidopsis thaliana seedlings.

AcknowledgmentsThanks are expressed to the University of Isfahan for financial support and the Department of Biology for space and cooperation. Special thanks to Professor MR Rahiminejad, for cooperation in use of the epifluorescent microscope. We are grateful to the Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy, Isfahan University of Medical Sciences, for collaboration on part of the work.

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