Dihydroflavonol 4-Reductase Genes Encode Enzymes with Contrasting Substrate Specificity and Show Divergent Gene Expression Profiles in Fragaria Species

During fruit ripening, strawberries show distinct changes in the flavonoid classes that accumulate, switching from the formation of flavan 3-ols and flavonols in unripe fruits to the accumulation of anthocyanins in the ripe fruits. In the common garden strawberry (Fragaria×ananassa) this is accompanied by a distinct switch in the pattern of hydroxylation demonstrated by the almost exclusive accumulation of pelargonidin based pigments. In Fragaria vesca the proportion of anthocyanins showing one (pelargonidin) and two (cyanidin) hydroxyl groups within the B-ring is almost equal. We isolated two dihydroflavonol 4-reductase (DFR) cDNA clones from strawberry fruits, which show 82% sequence similarity. The encoded enzymes revealed a high variability in substrate specificity. One enzyme variant did not accept DHK (with one hydroxyl group present in the B-ring), whereas the other strongly preferred DHK as a substrate. This appears to be an uncharacterized DFR variant with novel substrate specificity. Both DFRs were expressed in the receptacle and the achenes of both Fragaria species and the DFR2 expression profile showed a pronounced dependence on fruit development, whereas DFR1 expression remained relatively stable. There were, however, significant differences in their relative rates of expression. The DFR1/DFR2 expression ratio was much higher in the Fragaria×ananassa and enzyme preparations from F.×ananassa receptacles showed higher capability to convert DHK than preparations from F. vesca. Anthocyanin concentrations in the F.×ananassa cultivar were more than twofold higher and the cyanidin:pelargonidin ratio was only 0.05 compared to 0.51 in the F. vesca cultivar. The differences in the fruit colour of the two Fragaria species can be explained by the higher expression of DFR1 in F.×ananassa as compared to F. vesca, a higher enzyme efficiency (K _cat/K _m values) of DFR1 combined with the loss of F3’H activity late in fruit development of F.×ananassa.     

新疆雪莲DFR基因的分离及同源遗传转化

二氢黄酮醇4-还原酶（dihydroflavonol 4-reductase,DFR）是植物重要的次生代谢产物花青素生物合成途径中的关键酶。运用RT-PCR和RACE技术从新疆雪莲(Saussurea involucrata Kar.et Kir.)中克隆得到DFR基因(GenBank登录号为JN092126)。DFR基因的cDNA全长序列含有1个1 029 bp的开放阅读框(ORF),编码343个氨基酸,该基因推断的蛋白与水母雪莲DFR基因推断的蛋白高度同源,相似性达到92%;以不同物种中DFR氨基酸序列进行比对分析,推断的蛋白含有与NADPH特异结合的结构域。将该基因运用农杆菌介导的叶片转化法进行同源转化,将含有转DFR基因的愈伤组织进行悬浮培养,紫外分光光度法测定愈伤组织的总黄酮含量,结果表明转基因愈伤组织的总黄酮含量明显高于非转基因愈伤组织的含量。该研究为提高新疆雪莲药用化学成分黄酮类物质及实现新疆雪莲花青素的人工生物合成的研究奠定基础,对解决天山雪莲资源匮乏提供参考。     

比利时杜鹃花RhDFR基因克隆及分析

二氢黄酮醇-4-还原酶(DFR)是植物花青素合成过程中的关键酶,能够催化二氢黄酮醇生成无色花青素。该试验以红色和白色比利时杜鹃花(Rhododendron hybridum Hort.)不同器官和不同发育时期的花瓣为实验材料,利用反转录(RT-PCR)和RACE技术克隆RhDFR基因,利用植物酶联免疫试剂盒(ELISA)测定不同发育时期的花瓣RhDFR酶活性,利用qRT-PCR技术定量分析不同器官和不同发育时期的花瓣RhDFR基因,构建pET-28a-RhDFR原核表达载体对RhDFR蛋白进行制备和纯化,为进一步探究杜鹃花DFR基因功能以及花色的分子机理奠定基础。结果表明：(1)成功获得比利时杜鹃花RhDFR基因全长1 253 bp,其开放阅读框1 035 bp,编码344个氨基酸,含有1个NADPH结合保守基序和1个底物结合区域,具有高度保守性;系统进化分析显示,比利时杜鹃花RhDFR蛋白与越橘(Vaccinium corymbosum)DFR蛋白亲缘关系最近。(2)ELISA试剂盒分析显示,比利时杜鹃花不同发育时期的花瓣DFR酶活性呈先上升后下降的趋势,并于红花初开期和白花盛开期的...     

Molecular characterization and expression analysis of dihydroflavonol 4-reductase (DFR) gene in <Emphasis Type="Italic">Saussurea medusa</Emphasis>

Dihydroflavonol 4-reductase (DFR), which catalyzes the reduction of dihydroflavonols to leucoanthocyanins, is a key enzyme in the biosynthesis of anthocyanidins, proanthocyanidins, and other flavonoids of importance in plant development and human nutrition. This study isolated a full length cDNA encoding DFR, designated as SmDFR (GenBank Accession No. EF600682), by screening a cDNA library from a red callus line of Saussurea medusa, which is an endangered, traditional Chinese medicinal plant with high pharmacological value. SmDFR was functionally expressed in yeast (Saccharomyces cerevisiae) to confirm that SmDFR can readily reduce dihydroquercetin (DHQ) and dihydrokampferol (DHK), but it could not reduce dihydromyricetin (DHM). The deduced SmDFR structure shared extensive sequence similarity with previously characterized plant DFRs and phylogenetic analysis showed that it belonged to the plant DFR super-family. SmDFR also possessed flavanone 4-reductase (FNR) activity and can catalyze the conversion of eridictyol to luteoforol. Real-time PCR analysis showed that the expression level of SmDFR was higher in flowers compared with both leaves and roots. This work greatly enhances our knowledge of flavonoid biosynthesis in S. medusa and marks a major advance that could facilitate future genetic modification of S. medusa.     

Redirection of anthocyanin synthesis in Osteospermum hybrida by a two-enzyme manipulation strategy

Modern biotechnology has developed powerful tools for genetic engineering and flower colours are an excellent object to study possibilities and limitations of engineering strategies. Osteospermum hybrida became a popular ornamental plant within the last 20 years. Many cultivars display rose to lilac flower colours mainly based on delphinidin-derived anthocyanins. The predominant synthesis of delphinidin derivatives is referred to a strong endogenous flavonoid 3',5'-hydroxylase (F3'5'H) activity. Furthermore, since dihydroflavonol 4-reductase (DFR) of Osteospermum does not convert dihydrokaempferol (DHK) to leucopelargonidin, synthesis of pelargonidin-based anthocyanins is naturally not realised. In order to redirect anthocyanin biosynthesis in Osteospermum towards pelargonidin derivatives, we introduced cDNAs coding for DFRs which efficiently convert DHK to LPg. But neither the expression of Gerbera hybrida DFR nor of Fragaria x ananassa DFR - the latter is characterised by an unusual high substrate preference for DHK - altered anthocyanin composition in flowers of transgenic plants. However, chemical inhibition of F3'5'H activity in ray florets of dfr transgenic plants resulted in the accumulation of pelargonidin derivatives. Accordingly, retransformation of a transgenic plant expressing Gerbera DFR with a construct for RNAi-mediated suppression of F3'5'H activity resulted in double transgenic plants accumulating predominantly pelargonidin derivatives in flowers.     

Response of Two Dominant Boreal Freshwater Wetland Plants to Manipulated Warming and Altered Precipitation

This study characterized the morphological and photosynthetic responses of two wetland plant species when they were subject to 2–6°C fluctuations in growth temperature and ±50% of precipitation, in order to predict the evolution of natural wetlands in Sanjiang Plain of North-eastern China. We investigated the morphological and photosynthetic responses of two dominant and competitive boreal freshwater wetland plants in Northeastern China to manipulation of warming (ambient, +2.0°C, +4.0°C, +6.0°C) and altered precipitation (−50%, ambient, +50%) simultaneously by incubating the plants from seedling to senescence within climate-controlled environmental chambers. Post-harvest, secondary growth of C. angustifolia was observed to explore intergenerational effects. The results indicated that C. angustifolia demonstrated a greater acclimated capacity than G. spiculosa to respond to climate change due to higher resistance to temperature and precipitation manipulations. The accumulated effect on aboveground biomass of post-harvest secondary growth of C. angustifolia was significant. These results explain the expansion of C. angustifolia during last 40 years and indicate the further expansion in natural boreal wetlands under a warmer and wetter future. Stability of the natural surface water table is critical for the conservation and restoration of G. spiculosa populations reacting to encroachment stress from C. angustifolia expansion.     

Typha for paludiculture—Suitable water table and nutrient conditions for potential biomass utilization explored in mesocosm gradient experiments

Drainage has turned 650,000 km² of peatlands worldwide into greenhouse gas sources. To counteract climate change, large‐scale rewetting is necessary while agricultural use of rewetted areas, termed paludiculture, is still possible. However, more information is required on the performance of suitable species, such as cattail, in the range of environmental conditions after rewetting. We investigated productivity and biomass quality (morphological traits and tissue chemical composition) of Typha angustifolia and Typha latifolia along gradients of water table depth (−45 to +40 cm) and nutrient addition (3.6–400 kg N ha⁻¹ a⁻¹) in a six‐month mesocosm experiment with an emphasis on their high‐value utilization, e.g., as building material, paper, or biodegradable packaging. Over a wide range of investigated conditions, T. latifolia was more productive than T. angustifolia. Productivity was remarkably tolerant of low nutrient addition, suggesting that long‐term productive paludiculture is possible. Low water tables were beneficial for T. latifolia productivity and high water tables for T. angustifolia biomass quality. Rewetting will likely create a mosaic of different water table depths. Our findings that the yield of T. angustifolia and tissue chemical composition of T. latifolia were largely unaffected by water table depth are therefore promising. Depending on intended utilization, optimal cultivation conditions and preferable species differ. Considering yield or diameter, e.g., for building materials, T. latifolia is generally preferable over T. angustifolia. A low N, P, K content, high Si content and high C/N‐ratio can be beneficial for processing into disposable tableware, charcoal, or building material. For these utilizations, T. angustifolia is preferable at high water tables, and both species should be cultivated at a low nutrient supply. When cellulose and lignin contents are relevant, e.g., for paper and biodegradable packaging, T. angustifolia is preferable at high water tables and both species should be cultivated at nutrient additions of about 20 kg N ha⁻¹ a⁻¹.     

Angiotensin II Stimulates Thick Ascending Limb Superoxide Production via Protein Kinase Cα-dependent NADPH Oxidase Activation

Angiotensin II (Ang II) stimulates thick ascending limb (TAL) O production, but the receptor(s) and signaling mechanism(s) involved are unknown. The effect of Ang II on O is generally attributed to the AT₁ receptor. In some cells, Ang II stimulates protein kinase C (PKC), whose α isoform (PKCα) can activate NADPH oxidase. We hypothesized that in TALs, Ang II stimulates O via AT₁ and PKCα-dependent NADPH oxidase activation. In rat TALs, 1 nm Ang II stimulated O from 0.76 ± 0.17 to 1.97 ± 0.21 nmol/min/mg (p < 0.001). An AT₁ antagonist blocked the stimulatory effect of Ang II on O (0.87 ± 0.25 nmol/min/mg; p < 0.006), whereas an AT₂ antagonist had no effect (2.16 ± 0.133 nmol/min/mg; p < 0.05 versus vehicle). Apocynin, an NADPH oxidase inhibitor, blocked Ang II-stimulated O by 90% (p < 0.01). Ang II failed to stimulate O in TALs from p47^phox−/− mice (p < 0.02). Monitored by fluorescence resonance energy transfer, Ang II increased PKC activity from 0.02 ± 0.03 to 0.13 ± 0.02 arbitrary units (p < 0.03). A general PKC inhibitor, GF109203X, blocked the effect of Ang II on O (1.47 ± 0.21 versus 2.72 ± 0.47 nmol/min/mg with Ang II alone; p < 0.03). A PKCα- and β-selective inhibitor, Gö6976, also blocked the stimulatory effect of Ang II on O (0.59 ± 0.15 versus 2.05 ± 0.28 nmol/min/mg with Ang II alone; p < 0.001). To distinguish between PKCα and PKCβ, we used tubules expressing dominant-negative PKCα or -β. In control TALs, Ang II stimulated O by 2.17 ± 0.44 nmol/min/mg (p < 0.011). In tubules expressing dominant-negative PKCα, Ang II failed to stimulate O (change: −0.30 ± 0.27 nmol/min/mg). In tubules expressing dominant-negative PKCβ1, Ang II stimulated O by 2.08 ± 0.69 nmol/min/mg (p < 0.002). We conclude that Ang II stimulates TAL O production via activation of AT₁ receptors and PKCα-dependent NADPH oxidase.     

Cymbidium hybrida dihydroflavonol 4-reductase does not efficiently reduce dihydrokaempferol to produce orange pelargonidin-type anthocyanins

Some angiosperms are limited to a range of possible flower colors. This limitation can be due to the lack of an anthocyanin biosynthetic gene or to the substrate specificity of a key anthocyanin biosynthetic enzyme, dihydroflavonol 4-reductase (DFR). Cymbidium hybrida orchid flowers primarily produce cyanidin-type (pink to red) anthocyanins and lack the pelargonidin-type (orange to brick-red) anthocyanins. To investigate the underlying molecular mechanism of this flower color range, we cloned a Cymbidium DFR gene and transformed it into a DFR- petunia line. We found that the Cymbidium DFR did not efficiently reduce dihydrokaempferol (DHK), which is an essential step for pelargonidin production. Phylogenetic analysis of a number of DFR sequences indicate that the inability to catalyze DHK reduction has occurred at least twice during angiosperm evolution. Our results indicate that developing a pelargonidin-type orange flower color in Cymbidium may require the transformation of a DFR gene that can efficiently catalyze DHK reduction.     

Investigation of substrate specificity of wildtype and mutant BphKLB400 (a glutathione S-transferase) from Burkholderia LB400

The bphK gene located in the bph operon of Burkholderia LB400 encodes a protein, BphK^LB400, with significant sequence similarity to glutathione-S-transferases (GST), a group of enzymes involved in the detoxification of many endobiotic and xenobiotic substances. Comparison of the amino acid sequence of BphK^LB400 with GST from other polychlorinated biphenyl (PCB)-degrading bacteria identified a number of highly conserved amino acids in the C-terminal region of the protein that may be associated with substrate specificity. In this study, two of these conserved amino acids in BphK^LB400 (amino acids 152 and 180) were selected for mutation, using site-directed mutagenesis, and substrate specificity assays. BphK^LB400 (wildtype and mutant) was over-expressed in Escherichia coli where the bphK gene (wildtype and mutant) is under the expression of a lac promoter and is induced by isopropyl thiogalactoside, and bacterial cell extracts were prepared for GST activity assays. Mutations at amino acids 152 and 180 were shown to affect GST activity of BphK^LB400 using 1-chloro-2,4-dinitrobenzene, the model substrate for GST activity assays; 4-chlorobenzoate and 3-chlorobenzoate, intermediates in the polychlorinated biphenyl (PCB) degradation pathway, and 2,4-dichlorophenoxyacetate and atrazine, commonly used herbicides; as substrates. A BphK^LB400 mutant (Ala180Pro) is identified in this study as having increased activity towards all substrates tested. This mutant may have potential in bioremediation.     

Polydatin prevents angiotensin II-induced cardiac hypertrophy and myocardial superoxide generation

Our studies and others recently demonstrate that polydatin, a resveratrol glucoside, has antioxidative and cardioprotective effects. This study aims to investigate the direct effects of polydatin on Ang II-induced cardiac hypertrophy to explore the potential role of polydatin in cardioprotection. Our results showed that in primary cultured cardiomyocytes, polydatin blocked Ang II-induced cardiac hypertrophy in a dose-dependent manner, which were associated with reduction in the cell surface area and [³H]leucine incorporation, as well as attenuation of the mRNA expressions of atrial natriuretic factor and β-myosin heavy chain. Furthermore, polydatin prevented rat cardiac hypertrophy induced by Ang II infusion, as assessed by heart weight-to-body weight ratio, cross-sectional area of cardiomyocyte, and gene expression of hypertrophic markers. Further investigation demonstrated that polydatin attenuated the Ang II-induced increase in the reactive oxygen species levels and NADPH oxidase activity in vivo and in vitro. Polydatin also blocked the Ang II-stimulated increases of Nox4 and Nox2 expression in cultured cardiomyocytes and the hearts of Ang II-infused rats. Our results indicate that polydatin has the potential to protect against Ang II-mediated cardiac hypertrophy through suppression of NADPH oxidase activity and superoxide production. These observations may shed new light on the understanding of the cardioprotective effect of polydatin.     

Engineering the meso-Diaminopimelate Dehydrogenase from Symbiobacterium thermophilum by Site Saturation Mutagenesis for d-Phenylalanine Synthesis

In order to enlarge the substrate binding pocket of the meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum to accommodate larger 2-keto acids, four amino acid residues (Phe146, Thr171, Arg181, and His227) were targeted for site saturation mutagenesis. Among all mutants, the single mutant H227V had a specific activity of 2.39 ± 0.06 U · mg⁻¹, which was 35.1-fold enhancement over the wild-type enzyme.     

Production of red-flowered plants by genetic engineering of multiple flavonoid biosynthetic genes

Orange- to red-colored flowers are difficult to produce by conventional breeding techniques in some floricultural plants. This is due to the deficiency in the formation of pelargonidin, which confers orange to red colors, in their flowers. Previous researchers have reported that brick-red colored flowers can be produced by introducing a foreign dihydroflavonol 4-reductase (DFR) with different substrate specificity in Petunia hybrida, which does not accumulate pelargonidin pigments naturally. However, because these experiments used dihydrokaempferol (DHK)-accumulated mutants as transformation hosts, this strategy cannot be applied directly to other floricultural plants. Thus in this study, we attempted to produce red-flowered plants by suppressing two endogenous genes and expressing one foreign gene using tobacco as a model plant. We used a chimeric RNAi construct for suppression of two genes (flavonol synthase [FLS] and flavonoid 3′-hydroxylase [F3′H]) and expression of the gerbera DFR gene in order to accumulate pelargonidin pigments in tobacco flowers. We successfully produced red-flowered tobacco plants containing high amounts of additional pelargonidin as confirmed by HPLC analysis. The flavonol content was reduced in the transgenic plants as expected, although complete inhibition was not achieved. Expression analysis also showed that reduction of the two-targeted genes and expression of the foreign gene occurred simultaneously. These results demonstrate that flower color modification can be achieved by multiple gene regulation without use of mutants if the vector constructs are designed resourcefully. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.