Comparative transcriptome and metabolite profiling reveal diverse pattern of CYP-TS gene expression during corosolic acid biosynthesis in Lagerstroemia speciosa (L.) Pers.

KEY MESSAGE: Extensive leaf transcriptome profiling and differential gene expression analysis of field grown and elicited shoot cultures of L. speciosa suggest that differential synthesis of CRA is mediated primarily by CYP and TS genes, showing functional diversity. Lagerstroemia speciosa L. is a tree species with medicinal and horticultural attributes. The pentacyclic triterpene, Corosolic acid (CRA) obtained from this species is widely used for the management of diabetes mellitus in traditional medicine. The high mercantile value of the compound and limited availability of innate resources entail exploration of alternative sources for CRA production. Metabolic pathway engineering for enhanced bioproduction of plant secondary metabolites is an attractive proposition for which, candidate genes in the pathway need to be identified and characterized. Therefore, in the present investigation, we focused on the identification of cytochrome P450 (CYP450) and oxidosqualene cyclases (OSC) genes and their differential expression during biosynthesis of CRA. The pattern of differential expression of these genes in the shoot cultures of L. speciosa, elicited with different epigenetic modifiers (azacytidine (AzaC), sodium butyrate (NaBu) and anacardic acid (AA)), was studied in comparison with field grown plant. Further, in vitro cultures with varying (low to high) concentrations of CRA were systematically assessed for the expression of CYP-TS and associated genes involved in CRA biosynthesis by transcriptome sequencing. The sequenced samples were de novo assembled into 180,290 transcripts of which, 92,983 transcripts were further annotated by UniProt. The results are collectively given in co-occurrence heat maps to identify the differentially expressed genes. The combined transcript and metabolite profiles along with RT-qPCR analysis resulted in the identification of CYP-TS genes with high sequence variation. Further, instances of concordant/discordant relation between CRA biosynthesis and CYP-TS gene expression were observed, indicating functional diversity in genes.

Genome-Wide Identification, Characterization, and Expression Analysis of the HD-Zip Gene Family in Lagerstroemia for Regulating Plant Height.

The Homeodomain leucine zipper (HD-Zip) family of transcription factors is crucial in helping plants adapt to environmental changes and promoting their growth and development. Despite research on the HD-Zip family in various plants, studies in Lagerstroemia (crape myrtle) have not been reported. This study aimed to address this gap by comprehensively analyzing the HD-Zip gene family in crape myrtle. This study identified 52 HD-Zip genes in the genome of Lagerstroemia indica, designated as LinHDZ1-LinHDZ52. These genes were distributed across 22 chromosomes and grouped into 4 clusters (HD-Zip I-IV) based on their phylogenetic relationships. Most gene structures and motifs within each cluster were conserved. Analysis of protein properties, gene structure, conserved motifs, and cis-acting regulatory elements revealed diverse roles of LinHDZs in various biological contexts. Examining the expression patterns of these 52 genes in 6 tissues (shoot apical meristem, tender shoot, and mature shoot) of non-dwarf and dwarf crape myrtles revealed that 2 LinHDZs (LinHDZ24 and LinHDZ14) and 2 LinHDZs (LinHDZ9 and LinHDZ35) were respectively upregulated in tender shoot of non-dwarf crape myrtles and tender and mature shoots of dwarf crape myrtles, which suggested the important roles of these genes in regulate the shoot development of Lagerstroemia. In addition, the expression levels of 2 LinHDZs (LinHDZ23 and LinHDZ34) were significantly upregulated in the shoot apical meristem of non-dwarf crape myrtle. These genes were identified as key candidates for regulating Lagerstroemia plant height. This study enhanced the understanding of the functions of HD-Zip family members in the growth and development processes of woody plants and provided a theoretical basis for further studies on the molecular mechanisms underlying Lagerstroemia plant height.

Lagerstroemia macrocarpa extract inhibits Th2-mediated STAT6 signaling pathway in human keratinocytes.

In this study, we examined physiological functions as a key material to develop cosmeceuticals using extracts of Lagerstroemia macrocarpa Wall. Ex Kurz (L. macrocarpa). Initially, the L. macrocarpa extract was treated by different concentration and antioxidant assay (DPPH and ABTS) were performed to measure free radical scavenging ability. In the cytotoxicity experiment, the extract was treated into human epidermal keratinocytes with different concentrations to measure cytotoxicity. We found that the extract induces differentiation markers such as keratin (KRT)1, KRT2, KRT9, KRT10 in keratinocytes. Furthermore, the extract significantly induces involucrin (IVL), loricrin (LOR), claudin1 (CLDN1), and filaggrin (FLG) expression, suggesting that it may enhance skin barrier functions. Especially, the extract restored FLG expression inhibited by interleukin (IL)-4/IL-13 in in vitro atopic dermatitis-like model. Therefore, we expect L. macrocarpa extract will be an effective material to develop the therapeutic and cosmeceutical of atopic dermatitis.

The oxidative damage of the Lagerstroemia indica chlorosis mutant gl1 involves in ferroptosis.

Photooxidation is the major physiological performance of the Lagerstroemia indica chlorosis mutant gl1 under field conditions. The mechanisms of the progressive symptoms of oxidative damage from the lower older leaves to the upper mature leaves are complicated and still unclear. The aim of this work was to investigate the physiological mechanisms of oxidative stress from the perspective of the photosynthetic metabolites. The phytosynthetic metabolites of gl1 mutant changed significantly compared to wild type (WT) L. indica, such as by increasing phenolics, decreasing soluble sugar, protein and ascorbate, and redistributing antioxidant enzyme activities. The co-accumulation of phenolics and guaiacol-POD in gl1 mutant promote the removal of H2O2, as well the increase of phenoxyl radicals levels. Furthermore, the ion balance was significantly disturbed and Fe accumulated the most among these fluctuating nutrients in the leaves of gl1 mutant. The accumulated Fe was found neither in the chloroplasts nor in the cell wall of the leaves and became unshielded Fe, which favors the Fenton/Haber-Weiss reaction and stabilizes the phenoxyl radicals in metal complexation. The results suggested that the increase of phenolics and Fe accumulation were obviously involved in oxidative damage of gl1 mutant.

Lagerstroemia indica extract regulates human hair dermal papilla cell growth and degeneration via modulation of ß-catenin, Stat6, and TGF-ß signaling pathway.

BACKGROUND: Lagerstroemia indica (L. indica) is reported to have diverse biological activities including anti-inflammatory, anti-cancer, neuro-regulatory, antidiabetic, and antioxidant activity. AIMS: The purpose of this study is to examine the potential of hair growth promotion and/or hair loss prevention by L. indica extract. PATIENTS/METHODS: The effects of L. indica on hair growth have been studied in human hair follicle dermal papillary (hHFDP) cells and follicular organ culture ex vivo by cell proliferation assay, PCR, western blot analysis, and reporter gene activity assay. Moreover, a clinical trial was conducted in healthy volunteers. RESULTS: Lagerstroemia indica significantly promoted the proliferation of hHFDP cells, which was associated with increased expression of TCF/LEF, VEGF, and Gli1 mRNA, and inhibition of STAT6 and Smad2 mRNA. Treatment with L. indica also increased the TCF/LEF reporter gene activity but downregulated the SBE- and STAT6-luciferase activities. The expression of total ß-catenin, CDK4, and CDK2 were elevated, while that of STAT6 and SMAD2/3 was suppressed upon treatment with L. indica. In human hair follicles organ culture, L. indica significantly inhibited hair follicular degeneration. The clinical trial showed a statistically significant rise in total hair count in test group (n = 24) after 24 weeks of applying the hair tonic enriched with L. indica (141.46 ± 21.27 number/cm2 , p < 0.05). CONCLUSION: We suggest that L. indica extract prevents hair loss as well as stimulate hair growth by regulating the Wnt-ß-catenin, JAK3-STAT6, and TGF-ß1-Smad signaling pathways, and may be further developed as a novel functional cosmetic for preventing hair loss.

Effect of Banaba (Lagerstroemia speciosa) on Metabolic Syndrome, Insulin Sensitivity, and Insulin Secretion.

To evaluate the effect of Banaba (Lagerstroemia speciosa) on metabolic syndrome (MetS), insulin sensitivity, and insulin secretion. A randomized, double-blind, placebo-controlled clinical trial was carried out in 24 patients with diagnosis of MetS according to the International Diabetes Federation criteria. Body weight, waist circumference, and blood pressure were evaluated. Fasting plasma glucose (FPG) and insulin concentrations were measured every 30 min during 2 h after a 75-g dextrose load. Lipid profile was determined before and after the pharmacological intervention. Twelve patients received Banaba (500 mg) twice a day, before breakfast and dinner for 12 weeks. The remaining 12 patients received placebo at the same dosage. Body mass index, area under the curve (AUC) of glucose and insulin, insulin sensitivity, total insulin secretion, and the first phase of insulin secretion were calculated. After Banaba administration, there were significant decreases in systolic blood pressure (SBP) (121.5 ± 12.9 vs. 116.3 ± 9.8 mmHg, P = .050), FPG (5.9 ± 0.4 vs. 5.7 ± 0.4 mmol/L, P = .034), triglycerides (TG) (2.3 ± 0.4 vs. 1.7 ± 0.5 mmol/L, P = .021), very low-density lipoprotein (VLDL) (0.5 ± 0.1 vs. 0.3 ± 0.1 mmol/L, P = .021), AUC of insulin (50,675 ± 14,309 vs. 37,983 ± 19,298 mmol/L, P = .017), and insulinogenic index (0.4 ± 0.2 vs. 0.3 ± 0.2, P = .047). Eight patients (67%) of the Banaba group showed remission of MetS. In the placebo group, there was a downward trend toward statistical significance in the Stumvoll index (910.3 ± 514.1 vs. 651.0 ± 405.2, P = .062). Banaba administration leads to remission of MetS and a significant decrease in SBP, FPG, TG, VLDL, AUC of insulin, and total insulin secretion. Clinical Trial Registration number: NCT02767869.

Transcriptome-based analysis reveals that the biosynthesis of anthocyanins is more active than that of flavonols and proanthocyanins in the colorful flowers of Lagerstroemia indica.

Mechanisms associated with the control of flower color in crape myrtle varieties have yet to be sufficiently elucidated, which has tended to hamper the use of modern molecular and genetic strategies in the breeding programs for this plant. The whole transcriptome of four L. indica varieties characterized by different flower colors (white, light purple, deep purplish pink, and strong red) was sequenced, and we performed bioinformatic, quantitative PCR, and co-expression analyses of R2R3 MYB transcription factor and anthocyanin/flavonol pathway genes. We obtained a total of 49,980 transcripts with full-length coding sequences. Both transcriptome and qPCR analyses revealed that anthocyanin/flavonol pathway genes were differentially expressed among the four different flowers types, with the expression of LiPAL, LiCHS, LiCHI, LiDFR, LiANS/LDOX, and LiUFGT being induced in colorful flowers, whereas that of LiF3´5´H, LiFLS, and LiLAR was found to be inhibited. Base on phylogenetic analysis, seven R2R3 MYB transcriptional factors were identified as putative regulators of flower color. The molecular characteristics and co-expression patterns indicated that these MYBs differentially modulate their target genes, with two probably acting as activators, three as repressors, and one contributing to the regulation of vacuolar pH. The findings of this study indicate that the anthocyanin biosynthesis is more active than the flavonol and proanthocyanin in the colorful flowers. These observations provide new genomic information on L. indica and contribute gene resources for the flower color-targeted breeding of crape myrtle.

Candidate reference genes for quantitative gene expression analysis in Lagerstroemia indica.

Quantitative gene expression analysis by qPCR requires reference genes for normalization. Lagerstroemia indica (crape myrtle) is a popular ornamental plant in the world, but suitable endogenous reference genes are lacking. To find suitable reference genes, we evaluated the stabilities of nine candidate genes in six experimental data sets: six different tissues, three leaf colors, nine flower colors, and under three abiotic stresses (salt, drought, cold) using four statistical algorithms. A target gene LiMYB56 (homolog of Arabidopsis MYB56) was used to verify the authenticity and accuracy of the candidate reference genes. The results showed that the combination of two stably expressed reference genes, rather than a single reference gene, improved the accuracy of the qPCR. LiEF1α-2 + LiEF1α-3 was best for the tissue, salt treatment, and drought treatment sets; LiEF1α-2 + LiEF1α-1 was optimal for leaf color; LiEF1α-2 + LiACT7 was optimal for cold treatment; and LiUBC + LiEF1α-1 was best for the flower color set. Notably, LiEF1α-2 had high expression stability in all six experimental sets, implying it may be a good reference gene for expression studies in L. indica. Our results will facilitate future gene expression studies in L. indica.

Label-free comparative proteomic and physiological analysis provides insight into leaf color variation of the golden-yellow leaf mutant of Lagerstroemia indica.

GL1 is a golden-yellow leaf mutant that cultivated from natural bud-mutation of Lagerstroemia indica and has a very low level of photosynthetic pigment under sunlight. GL1 can gradually increase its pigment content and turn into pale-green leaf when shading under sunshade net (referred as Re-GL1). The mechanisms that cause leaf color variation are complicated and are not still unclear. Here, we have used a label-free comparative proteomics to investigate differences in proteins abundance and analyze the specific biological process associated with mechanisms of leaf color variation in GL1. A total of 245 and 160 proteins with different abundance were identified in GL1 vs WT and GL1 vs Re-GL1, respectively. Functional classification analysis revealed that the proteins with different abundance mainly related to photosynthesis, heat shock proteins, ribosome proteins, and oxidation-reduction. The proteins that the most significantly contributed to leaf color variation were photosynthetic proteins of PSII and PSI, which directly related to photooxidation and determined the photosynthetic performance of photosystem. Further analysis demonstrated that low jasmonic acid content was needed to golden-yellow leaf GL1. These findings lay a solid foundation for future studies into the molecular mechanisms that underlie leaf color formation of GL1. BIOLOGICAL SIGNIFICANCE: The natural bud mutant GL1 of L. indica is an example through changing leaf color to cope with complex environment. However, the molecular mechanism of leaf color variation are largely elusive. The proteins with different abundance identified from a label-free comparative proteomics revealed a range of biological processes associated with leaf color variation, including photosynthesis, oxidation-reduction and jasmonic acid signaling. The photooxidation and low level of jasmonic acid played a primary role in GL1 adaptation in golden-yellow leaf. These findings provide possible pathway or signal for the molecular mechanism associated with leaf color formation and as a valuable resource for signal transaction of chloroplast.

Exploring the Molecular Mechanism underlying the Stable Purple-Red Leaf Phenotype in Lagerstroemia indica cv. Ebony Embers.

Lagerstroemia indica is an important ornamental tree worldwide. The development of cultivars with colorful leaves and increased ornamental value represents one of the current main research topics. We investigated the anthocyanin profiles in two contrasting cultivars for leaf color phenotypes and explored the underlying molecular basis. Both cultivars display purple-red young leaves (Stage 1), and when the leaves mature (Stage 2), they turn green in HD (Lagerstroemia Dynamite) but remain unchanged in ZD (Lagerstroemia Ebony Embers). Seven different anthocyanins were detected, and globally, the leaves of ZD contained higher levels of anthocyanins than those of HD at the two stages with the most pronounced difference observed at Stage 2. Transcriptome sequencing revealed that in contrast to HD, ZD tends to keep a higher activity level of key genes involved in the flavonoid-anthocyanin biosynthesis pathways throughout the leaf developmental stages in order to maintain the synthesis, accumulation, and modification of anthocyanins. By applying gene co-expression analysis, we detected 19 key MYB regulators were co-expressed with the flavonoid-anthocyanin biosynthetic genes and were found strongly down-regulated in HD. This study lays the foundation for the artificial manipulation of the anthocyanin biosynthesis in order to create new L. indica cultivars with colorful leaves and increased ornamental value.

Oxidosqualene cyclase and CYP716 enzymes contribute to triterpene structural diversity in the medicinal tree banaba.

Pentacyclic triterpenes (PCTs) represent a major class of bioactive metabolites in banaba (Lagerstroemia speciosa) leaves; however, biosynthetic enzymes and their involvement in the temporal accumulation of PCTs remain to be studied. We use an integrated approach involving transcriptomics, metabolomics and gene function analysis to identify oxidosqualene cyclases (OSCs) and cytochrome P450 monooxygenases (P450s) that catalyzed sequential cyclization and oxidative reactions towards PCT scaffold diversification. Four monofunctional OSCs (LsOSC1,3-5) converted the triterpene precursor 2,3-oxidosqualene to either lupeol, ß-amyrin or cycloartenol, and a multifunctional LsOSC2 formed α-amyrin as a major product along with ß-amyrin. Two CYP716 family P450s (CYP716A265, CYP716A266) catalyzed C-28 oxidation of α-amyrin, ß-amyrin and lupeol to form ursolic acid, oleanolic acid and betulinic acid, respectively. However, CYP716C55 catalyzed C-2α hydroxylation of ursolic acid and oleanolic acid to produce corosolic acid and maslinic acid, respectively. Besides, combined transcript and metabolite analysis suggested major roles for the LsOSC2, CYP716A265 and CYP716C55 in determining leaf ursane and oleanane profiles. Combinatorial expression of OSCs and CYP716s in Saccharomyces cerevisiae and Nicotiana benthamiana led to PCT pathway reconstruction, signifying the utility of banaba enzymes for bioactive PCT production in alternate plant/microbial hosts that are more easily tractable than the tree species.

Transcriptome analysis of the genes regulating phytohormone and cellular patterning in Lagerstroemia plant architecture.

Plant architecture is a popular research topic because plants with different growth habits that may generate economic or ornamental value are in great demand by orchards and nurseries. However, the molecular basis of the architecture of woody perennial plants is poorly understood due to the complexity of the phenotypic and regulatory relationships. Here, transcriptional profiling of dwarf and non-dwarf crapemyrtles was performed, and potential target genes were identified based on the phenotype, histology and phytohormone metabolite levels. An integrated analysis demonstrated that the internode length was explained mainly by cell number and secondarily by cell length and revealed important hormones in regulatory pathway of Lagerstroemia architecture. Differentially expressed genes (DEGs) involved in phytohormone pathways and cellular patterning regulation were analysed, and the regulatory relationships between these parameters were evaluated at the transcriptional level. Exogenous indole-3-acetic acid (IAA) and gibberellin A4 (GA4) treatments further indicated the pivotal role of auxin in cell division within the shoot apical meristem (SAM) and suggested an interaction between auxin and GA4 in regulating the internode length of Lagerstroemia. These results provide insights for further functional genomic studies on the regulatory mechanisms underlying Lagerstroemia plant architecture and may improve the efficiency of woody plant molecular breeding.

Solar catalysed activity against methyl orange dye, cytotoxicity activity of MCF-7 cell lines and identification of marker compound by HPTLC of Lagerstroemia speciosa.

The investigation was aimed to quantify the Gallic acid present in Lagerstroemia speciosa leaves (Lythraceae). The High-Performance Thin Layer Chromatography (HPTLC) quantification was performed for acetone (AE), methanolic (ME) and chloroform (CE) extract of leaves of L. speciosa. The pre-coated silica gel 60 F254 was used for complete separation of compounds using the mobile phase pet. Ether: ethyl acetate: formic acid (5:5:1v/v).The validation of the extracts was carried out using ICH guidelines for precision, repeatability and accuracy showing the Rf 0.49 against standard Gallic acid. Linearity range for Gallic acid was done from 200 to 1000ng/spot (AE) and200 ng to 600ng/spot (ME), with Correlation, coefficient r=0.99 (AE) and 0.54 (ME) in the said concentrations. The composition in crude leaf extract was determined to be of 49.712mg (AE) and 20.125mg (ME), while it was not found in chloroform extract against standard Gallic acid. Hence the proposed method was very simple, precise, accurate and easy for the screening of the bioactive compounds present in the acetone and methanolic extracts of the leaves of L. speciosa. It was observed that the acetone extract subjected to cytotoxicity showed promising activity at higher concentrations (100 and 200µg/ml) showed 92.9% and 87.13% inhibition against MCF-7 cell lines respectively. The photocatalytic activity of the acetone and methanolic extracts of methyl orange was found to be 90.25% (190min) and 89.03% (180min) respectively. Therefore this can be used as an indicator of purity of herbal drugs and formulation containing L. speciosa.

Isolation of quercetin from the methanolic extract of Lagerstroemia speciosa by HPLC technique, its cytotoxicity against MCF-7 cells and photocatalytic activity.

The flavonoids present in the leaves of Lagerstroemia speciosa were extracted, characterized by spectral methods and studied for its cytotoxicity activity against MCF-cell lines and photocatalytic activity against azo dye. Direct and sequential soxhlet extraction was performed and its concentrated crude extract was subjected to high performance liquid chromatography. The yield obtained by the isolated compound (MEI-quercetin) from leaves of L. speciosa was found to be 1.8g from the methanolic extract. The phytochemical analysis and the Rf value of the isolated flavonoid was found to be 3.59. The isolated compound was characterized by Infrared Spectroscopy, NMR and Mass. Based on the characterization, the structure was elucidated as quercetin - a flavonoid. The isolated compound showed the significant in vitro cytotoxicity activity against MCF-7 cell lines at 500µg/ml when compared to the crude extract. Among the various concentrations (25, 50, 100, 250, and 500µg/ml), at higher concentration the cell viability was pronounced and also compared with that of the control. It was first time to report that the isolated flavonoid showed photocatalytic against azo dye-methyl orange. The dye degradation was monitored by UV-Vis spectrophotometry. The isolated compound showed dye degradation of 91.66% with the crude extract 82.47% at 160min. Hence in the present findings, the photocatalytic degradation of MO dye under UV irradiation was investigated over isolated compound of L. speciosa. Hence we expect that this can be used to treat the waste water in near future based on the photocatalytic technique.

Facile biosynthesis, characterization, and solar assisted photocatalytic effect of ZnO nanoparticles mediated by leaves of L. speciosa.

Synthesis of metal oxide nanoparticles using novel methodologies always attracts great importance in research. The use of plant extract to synthesize nano-particle has been considered as one of the eco-friendly methods. This paper describes the biosynthetic route of preparation of zinc oxide nanoparticles (ZnO NPs) from the Lagerstroemia speciosa leaf extract. This approach appears to be low-cost preparation and alternative method to conventional methods. Highly stable and hexagonal phase ZnO NPs with average particle size of 40nm were synthesized and characterized by UV-Vis absorption spectroscopy (surface Plasmon resonance), Fourier transform infrared spectroscopy (surface functionalities), X-ray Diffraction analysis (crystallinity), TEM and SEM (size and morphology), Energy Dispersive X-ray spectroscopy (elemental composition), Thermogravimetric analysis (weight loss) and Zeta potential (stability). The preliminary phytochemical experiments identify the possible chemical groups present in leaves extract. The photocatalytic properties of ZnO NPs were studied using UV-Vis spectroscopy by exposing methyl orange to sunlight and it is found to be degraded up to 93.5% within 2h. The COD values were significantly reduced from 5600mg/L to 374mg/L after 100min of solar radiation. The hemolytic activity of synthesized zinc oxide nanoparticles was performed on human erythrocyte cells. Thus the present study provides a simple and eco-friendly method for the preparation of multifunctional property of ZnO NPs utilizing the biosynthetic route.

Plant-based corosolic acid: future anti-diabetic drug?

Diabetes is one of the nation's most prevalent, debilitating and costly diseases. For diabetes, frequent insulin treatment is very expensive and may increase anti-insulin antibody production, which may cause unwanted side effects. Corosolic acid may also have some efficacy in the treatment of diabetes, but without induction of anti-insulin antibodies. Recently, corosolic acid from Lagerstroemia speciosa L. leaf extracts has been reported to act via an indirect mechanism (unlike insulin) in animal experiments. The insulin-complementary anti-diabetic therapeutic value observed in these Japanese preliminary clinical trials has led to renewed interest in the biosynthesis of this compound. So far, there has been no clear evidence for a corosolic acid biosynthetic pathway in plants. This article provides possible roles of corosolic acid and hypothetical information on the biosynthetic pathway in plants.

Responses of epidermal phenolic compounds to light acclimation: in vivo qualitative and quantitative assessment using chlorophyll fluorescence excitation spectra in leaves of three woody species.

Chlorophyll fluorescence (ChlF) excitation spectra were measured to assess the UV-sunscreen compounds accumulated in fully expanded leaves of three woody species belonging to different chemotaxons, (i.e. Morus nigra L., Prunus mahaleb L. and Lagerstroemia indica L.), grown in different light microclimates. The logarithm of the ratio of ChlF excitation spectra (logFER) between two leaves acclimated to different light microclimates was used to assess the difference in epidermal absorbance (EAbs). EAbs increased with increasing solar irradiance intercepted for the three species. This epidermal localisation of UV-absorbers was confirmed by the removal of the epidermis. It was possible to simulate EAbs as a linear combination of major phenolic compounds (Phen) identified in leaf methanol extracts by HPLC-DAD. Under UV-free radiation conditions, shaded leaves of M. nigra accumulated chlorogenic acid. Hydroxybenzoic acid (HBA) derivatives and hydroxycinnamic acid (HCA) derivatives greatly increased with increasing PAR irradiance under the low UV-B conditions found in the greenhouse. These traits were also observed for the HCA of the two other species. Flavonoid (FLAV) accumulation started under low UV-A irradiance, and became maximal in the adaxial epidermis of sun-exposed leaves outdoors. A decrease in the amount of HCA was observed concomitantly to the intense accumulation of FLAV for both leaf sides of the three species. Judging from the logFER, under low UV-B conditions, larger amounts of HCA are present in the epidermis in comparison to FLAV for the three species. Upon transition from the greenhouse to full sunlight outdoors, there was a decrease in leaf-soluble HCA that paralleled FLAV accumulation in reaction to increasing solar UV-B radiation in the three species. In M. nigra, that contains large amounts of HCA, the logFER analysis showed that this decrease occurred in the adaxial epidermis, whereas the abaxial epidermis, which is protected from direct UV-B radiation, continued to accumulate large amounts of HCA.