Functional Promiscuity of Two Divergent Paralogs of Type III Plant Polyketide Synthases.

Plants effectively defend themselves against biotic and abiotic stresses by synthesizing diverse secondary metabolites, including health-protective flavonoids. These display incredible chemical diversity and ubiquitous occurrence and confer impeccable biological and agricultural applications. Chalcone synthase (CHS), a type III plant polyketide synthase, is critical for flavonoid biosynthesis. It catalyzes acyl-coenzyme A thioesters to synthesize naringenin chalcone through a polyketidic intermediate. The functional divergence among the evolutionarily generated members of a gene family is pivotal in driving the chemical diversity. Against this backdrop, this study was aimed to functionally characterize members of the CHS gene family from Rheum emodi, an endangered and endemic high-altitude medicinal herb of northwestern Himalayas. Two full-length cDNAs (1,179 bp each), ReCHS1 and ReCHS2, encoding unique paralogs were isolated and characterized. Heterologous expression and purification in Escherichia coli, bottom-up proteomic characterization, high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis, and enzyme kinetic studies using five different substrates confirmed their catalytic potential. Phylogenetic analysis revealed the existence of higher synonymous mutations in the intronless divergents of ReCHS. ReCHS2 displayed significant enzymatic efficiency (Vmax/Km) with different substrates. There were significant spatial and altitudinal variations in messenger RNA transcript levels of ReCHSs correlating positively with metabolite accumulation. Furthermore, the elicitations in the form of methyl jasmonate, salicylic acid, ultraviolet B light, and wounding, chosen on the basis of identified cis-regulatory promoter elements, presented considerable differences in the transcript profiles of ReCHSs. Taken together, our results demonstrate differential propensities of CHS paralogs in terms of the accumulation of flavonoids and their relative substrate selectivities.

Phytoremediation of textile dyes and effluents: Current scenario and future prospects.

Phytoremediation has emerged as a green, passive, solar energy driven and cost effective approach for environmental cleanup when compared to physico-chemical and even other biological methods. Textile dyes and effluents are condemned as one of the worst polluters of our precious water bodies and soils. They are well known mutagenic, carcinogenic, allergic and cytotoxic agents posing threats to all life forms. Plant based treatment of textile dyes is relatively new and hitherto has remained an unexplored area of research. Use of macrophytes like Phragmites australis and Rheum rhabarbarum have shown efficient removal of Acid Orange 7 and sulfonated anthraquinones, respectively. Common garden and ornamental plants namely Aster amellus, Portulaca grandiflora, Zinnia angustifolia, Petunia grandiflora, Glandularia pulchella, many ferns and aquatic plants have also been advocated for their dye degradation potential. Plant tissue cultures like suspension cells of Blumea malcolmii and Nopalea cochenillifera, hairy roots of Brassica juncea and Tagetes patula and whole plants of several other species have confirmed their role in dye degradation. Plants' oxidoreductases such as lignin peroxidase, laccase, tyrosinase, azo reductase, veratryl alcohol oxidase, riboflavin reductase and dichlorophenolindophenol reductase are known as key biodegrading enzymes which break the complex structures of dyes. Schematic metabolic pathways of degradation of different dyes and their environmental fates have also been proposed. Degradation products of dyes and their fates of metabolism have been reported to be validated by UV-vis spectrophotometry, high performance liquid chromatography, high performance thin layer chromatography, Fourier Transform Infrared Spectroscopy, gas chromatograph-mass spectroscopy and several other analytical tools. Constructed wetlands and various pilots scale reactors were developed independently using the plants of P. australis, Portulaca grandiflora, G. pulchella, Typha domingensis, Pogonatherum crinitum and Alternanthera philoxeroides. The developed phytoreactors gave noteworthy treatments, and significant reductions in biological oxygen demand, chemical oxygen demand, American Dye Manufacturers Institute color removal value, total organic carbon, total dissolved solids, total suspended solids, turbidity and conductivity of the dye effluents after phytoremediation. Metabolites of dyes and effluents have been assayed for phytotoxicity, cytotoxicity, genotoxicity and animal toxicity and were proved to be non/less toxic than untreated compounds. Effective strategies to handle fluctuating dye load and hydraulics for in situ treatment needs scientific attention. Future studies on development of transgenic plants for efficacious phytodegradation of textile dyes should be focused.

Expression of SOD and APX genes positively regulates secondary cell wall biosynthesis and promotes plant growth and yield in Arabidopsis under salt stress.

Abiotic stresses cause accumulation of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) in plants. Sophisticated mechanisms are required to maintain optimum level of H2O2 that acts as signalling molecule regulating adaptive response to salt stress. CuZn-superoxide dismutase (CuZn-SOD) and ascorbate peroxidase (APX) constitute first line of defence against oxidative stress. In the present study, PaSOD and RaAPX genes from Potentilla atrosanguinea and Rheum australe, respectively were overexpressed individually as well as in combination in Arabidopsis thaliana. Interestingly, PaSOD and dual transgenic lines exhibit enhanced lignin deposition in their vascular bundles with altered S:G ratio under salt stress. RNA-seq analysis revealed that expression of PaSOD gene in single and dual transgenics positively regulates expression of lignin biosynthesis genes and transcription factors (NACs, MYBs, C3Hs and WRKY), leading to enhanced and ectopic deposition of lignin in vascular tissues with larger xylem fibres and alters S:G ratio, as well. In addition, transgenic plants exhibit growth promotion, higher biomass production and increased yield under salt stress as compared to wild type plants. Our results suggest that in dual transgenics, ROS generated during salt stress gets converted into H2O2 by SOD and its optimum level was maintained by APX. This basal level of H2O2 acts as messenger for transcriptional activation of lignin biosynthesis in vascular tissue, which provides mechanical strength to plants. These findings reveal an important role of PaSOD and RaAPX in enhancing salt tolerance of transgenic Arabidopsis via increased accumulation of compatible solutes and by regulating lignin biosynthesis.

Simultaneous over-expression of PaSOD and RaAPX in transgenic Arabidopsis thaliana confers cold stress tolerance through increase in vascular lignifications.

Antioxidant enzymes play a significant role in eliminating toxic levels of reactive oxygen species (ROS), generated during stress from living cells. In the present study, two different antioxidant enzymes namely copper-zinc superoxide dismutase derived from Potentilla astrisanguinea (PaSOD) and ascorbate peroxidase (RaAPX) from Rheum austral both of which are high altitude cold niche area plants of Himalaya were cloned and simultaneously over-expressed in Arabidopsis thaliana to alleviate cold stress. It was found that the transgenic plants over-expressing both the genes were more tolerant to cold stress than either of the single gene expressing transgenic plants during growth and development. In both single (PaSOD, RaAPX) and double (PaSOD + RaAPX) transgenic plants higher levels of total antioxidant enzyme activities, chlorophyll content, total soluble sugars, proline content and lower levels of ROS, ion leakage were recorded when compared to the WT during cold stress (4°C), besides increase in yield. In the present study, Confocal and SEM analysis in conjunction with qPCR data on the expression pattern of lignin biosynthetic pathway genes revealed that the cold stress tolerance of the transgenic plants might be because of the peroxide induced up-regulation of lignin by antioxidant genes mediated triggering.

Cytotoxic tetramic acid derivative produced by a plant type-III polyketide synthase.

The tetramic acid (2,4-pyrrolidinedione) scaffold has been recognized as an important structural feature because of its mycotoxic, antibacterial, antiviral, and antioxidant activities. This important class of natural products is reportedly produced by the type-I polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) hybrid megaenzyme systems. In contrast, the benzalacetone synthase (BAS) from Rheum palmatum is a structurally simple, plant-specific type-III PKS that catalyzes the one-step decarboxylative condensation of malonyl-CoA with 4-coumaroyl-CoA. The type-III PKS exhibits unusually broad substrate specificity and notable catalytic versatility. Here we report that R. palmatum BAS efficiently produces a series of unnatural, novel tetramic acid derivatives by the condensation of malonyl-CoA with aminoacyl-CoA thioesters chemically synthesized from L- and D-amino acids. Remarkably, the novel tetramic acid dimer D-5 formed from D-phenylalanoyl-CoA showed moderate antiproliferative activity against murine leukemia P388 cells.

A structure-based mechanism for benzalacetone synthase from Rheum palmatum.

Benzalacetone synthase (BAS), a plant-specific type III polyketide synthase (PKS), catalyzes a one-step decarboxylative condensation of malonyl-CoA and 4-coumaroyl-CoA to produce the diketide benzalacetone. We solved the crystal structures of both the wild-type and chalcone-producing I207L/L208F mutant of Rheum palmatum BAS at 1.8 A resolution. In addition, we solved the crystal structure of the wild-type enzyme, in which a monoketide coumarate intermediate is covalently bound to the catalytic cysteine residue, at 1.6 A resolution. This is the first direct evidence that type III PKS utilizes the cysteine as the nucleophile and as the attachment site for the polyketide intermediate. The crystal structures revealed that BAS utilizes an alternative, novel active-site pocket for locking the aromatic moiety of the coumarate, instead of the chalcone synthase's coumaroyl-binding pocket, which is lost in the active-site of the wild-type enzyme and restored in the I207L/L208F mutant. Furthermore, the crystal structures indicated the presence of a putative nucleophilic water molecule which forms hydrogen bond networks with the Cys-His-Asn catalytic triad. This suggested that BAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. These findings provided a structural basis for the functional diversity of the type III PKS enzymes.

Early low-temperature responsive mitogen activated protein kinases RaMPK1 and RaMPK2 from Rheum australe D. Don respond differentially to diverse stresses.

Rheum grows luxuriantly in a niche of low temperature (LT) at high altitudes in Himalayan belt. The plant is expected to harbor novel genes particularly for tolerance to LT. Using differential display, two cDNAs RaMPK1 and RaMPK2, showing homology to mitogen-activated protein kinases (MAPKs) were isolated. As compared to RaMPK1, RaMPK2 exhibited strong up-regulation in response to LT. RaMPK1 was novel in terms of possessing a small glutamine and proline rich region at the N-terminal end. Secondly, though RaMPK1 showed homology with salicylic acid (SA) responsive MAPKs, the gene was down-regulated by SA but activated by jasmonate (JA). Abscisic acid (ABA) and polyethylene glycol (PEG) also down-regulated RaMPK1. RaMPK2 showed down-regulation within 5 min of exposure to JA and SA treatments, followed by gradual increase in expression. Expression of RaMPK2 was wavy in response to ABA and PEG treatment. Results are discussed in light of the novelty of these MAPKs.

Metabolism of sulphonated anthraquinones in rhubarb, maize and celery: the role of cytochromes P450 and peroxidases.

Sulphonated anthraquinones are precursors of many synthetic dyes and pigments, recalcitrant to biodegradation, and thus contaminating many industrial effluents and rivers. In the development of a phytotreatment to remove sulphonated aromatic compounds, rhubarb (Rheum rhaponticum), a plant producing natural anthraquinones, as well as maize (Zea mays) and celery (Apium graveolens), plants not producing anthraquinones, were tested for their ability to metabolise these xenobiotics. Plants were cultivated under hydroponic conditions, with or without sulphonated anthraquinones, and were harvested at different times. Either microsomal or cytosolic fractions were prepared. The monooxygenase activity of cytochromes P450 towards several sulphonated anthraquinones was tested using a new method based on the fluorimetric detection of oxygen consumed during cytochromes P450-catalysed reactions. The activity of cytosolic peroxidases was measured by spectrophotometry, using guaiacol as a substrate. Results indicated that the activity of cytochromes P450 and peroxidases significantly increased in rhubarb plants cultivated in the presence of sulphonated anthraquinones. A higher activity of cytochromes P450 was also detected in maize and celery exposed to the pollutants. In these two plants, a peroxidase activity was also detected, but without a clear difference between the control plants and the plants exposed to the organic contaminants. This research demonstrated the existence in rhubarb, maize and celery of biochemical mechanisms involved in the metabolism and detoxification of sulphonated anthraquinones. Taken together, results confirmed that rhubarb might be the most appropriate plant for the phytotreatment of these organic pollutants.

The role of cytochromes P450 and peroxidases in the detoxification of sulphonated anthraquinones by rhubarb and common sorrel plants cultivated under hydroponic conditions.

BACKGROUND, AIM AND SCOPE: Sulphonated anthraquinones are precursors of many synthetic dyes and pigments, recalcitrant to biodegradation and thus not eliminated by classical wastewater treatments. In the development of a phytotreatment to remove sulphonated aromatic compounds from dye and textile industrial effluents, it has been shown that rhubarb (Rheum rabarbarum) and common sorrel (Rumex acetosa) are the most efficient plants. Both species, producing natural anthraquinones, not only accumulate, but also transform these xenobiotic chemicals. Even if the precise biochemical mechanisms involved in the detoxification of sulphonated anthraquinones are not yet understood, they probably have cross talks with secondary metabolism, redox processes and plant energy metabolism. The aim of the present study was to investigate the possible roles of cytochrome P450 monooxygenases and peroxidases in the detoxification of several sulphonated anthraquinones. MATERIALS AND METHODS: Both plant species were cultivated in a greenhouse under hydroponic conditions, with or without sulphonated anthraquinones. Plants were harvested at different times and either microsomal or cytosolic fractions were prepared. The monooxygenase activity of cytochromes P450 toward several sulphonated anthraquinones was tested using a new method based on the fluorimetric detection of oxygen consumed during cytochromes P450-catalysed reactions. The activity of cytosolic peroxidases was measured by spectrophotometry, using guaiacol as a substrate. RESULTS: A significant activity of cytochromes P450 was detected in rhubarb leaves, while no (rhizome) or low (petioles and roots) activity was found in other parts of the plants. An induction of this enzyme was observed at the beginning of the exposition to sulphonated anthraquinones. The results also indicated that cytochromes P450 were able to accept as substrate the five sulphonated anthraquinones, with a higher activity toward AQ-2,6-SS (0.706 nkat/mg protein) and AQ-2-S (0.720 nkat/mg protein). An activity of the cytochromes P450 was also found in the leaves of common sorrel (1.212 nkat/mg protein (AQ-2,6-SS)), but no induction of the activity occurred after the exposition to the pollutant. The activity of peroxidases increased when rhubarb was cultivated in the presence of the five sulphonated anthraquinones (0.857 nkat/mg protein). Peroxidase activity was also detected in the leaves of the common sorrel (0.055 nkat/mg protein), but in this plant, no significant difference was found between plants cultivated with and without sulphonated anthraquinones. DISCUSSION: Results indicated that the activity of cytochromes P450 and peroxidases increased in rhubarb in the presence of sulphonated anthraquinones and were involved in their detoxification mechanisms. CONCLUSIONS: These results suggest the existence in rhubarb and common sorrel of specific mechanisms involved in the metabolism of sulphonated anthraquinones. Further investigation should be performed to find the next steps of this detoxification pathway. RECOMMENDATIONS AND PERSPECTIVES: Besides these promising results for the phytotreatment of sulphonated anthraquinones, it will be of high interest to develop and test, at small scale, an experimental wastewater treatment system to determine its efficiency. On the other hand, these results reinforce the idea that natural biodiversity should be better studied to use the most appropriate species for the phytotreatment of a specific pollutant.

Structure function analysis of benzalacetone synthase from Rheum palmatum.

Benzalacetone synthase (BAS) is a plant-specific chalcone synthase (CHS) superfamily type III polyketide synthase (PKS) that catalyzes a one-step decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA. The diketide forming activity of Rheum palmatum BAS is attributed to the characteristic substitution of the conserved active-site Phe215 with Leu (numbering in Medicago sativa CHS). To further understand the structure and function of R. palmatum BAS, four site-directed mutants (C197T, C197G, G256L, and S338V) were newly constructed. All the mutants did not change the product pattern, however, the activity was 2-fold increased in S338V, while reduced to half in G256L mutant. On the other hand, the C197 mutants were functionally almost identical to wild-type BAS, excluding the possibility that the second active-site Cys is involved in the enzyme reaction. Instead, homology modeling suggested a possibility that, unlike the case of CHS, BAS utilizes an alternative pocket to lock the coumaroyl moiety for the diketide formation reaction.

Enzymatic formation of quinolone alkaloids by a plant type III polyketide synthase.

[Structure: see text] Benzalacetone synthase from Rheum palmatum efficiently catalyzed condensation of N-methylanthraniloyl-CoA (or anthraniloyl-CoA) with malonyl-CoA (or methylmalonyl-CoA) to produce 4-hydroxy-2(1H)-quinolones, a novel alkaloidal scaffold produced by a type III polyketide synthase (PKS). Manipulation of the functionally divergent type III PKSs by a nonphysiological substrate thus provides an efficient method for production of pharmaceutically important quinolone alkaloids.

Active site residues governing substrate selectivity and polyketide chain length in aloesone synthase.

Aloesone synthase (ALS) and chalcone synthase (CHS) are plant-specific type III poyketide synthases sharing 62% amino acid sequence identity. ALS selects acetyl-CoA as a starter and carries out six successive condensations with malonyl-CoA to produce a heptaketide aloesone, whereas CHS catalyses condensations of 4-coumaroyl-CoA with three malonyl-CoAs to generate chalcone. In ALS, CHS's Thr197, Gly256, and Ser338, the active site residues lining the initiation/elongation cavity, are uniquely replaced with Ala, Leu, and Thr, respectively. A homology model predicted that the active site architecture of ALS combines a 'horizontally restricting' G256L substitution with a 'downward expanding' T197A replacement relative to CHS. Moreover, ALS has an additional buried pocket that extends into the 'floor' of the active site cavity. The steric modulation thus facilitates ALS to utilize the smaller acetyl-CoA starter while providing adequate volume for the additional polyketide chain extensions. In fact, it was demonstrated that CHS-like point mutations at these positions (A197T, L256G, and T338S) completely abolished the heptaketide producing activity. Instead, A197T mutant yielded a pentaketide, 2,7-dihydroxy-5-methylchromone, while L256G and T338S just afforded a triketide, triacetic acid lactone. In contrast, L256G accepted 4-coumaroyl-CoA as starter to efficiently produce a tetraketide, 4-coumaroyltriacetic acid lactone. These results suggested that Gly256 determines starter substrate selectivity, while Thr197 located at the entrance of the buried pocket controls polyketide chain length. Finally, Ser338 in proximity of the catalytic Cys164 guides the linear polyketide intermediate to extend into the pocket, thus leading to formation of the hepataketide in Rheum palmatum ALS.

The first plant type III polyketide synthase that catalyzes formation of aromatic heptaketide.

A cDNA encoding a novel plant type III polyketide synthase (PKS) was cloned from rhubarb (Rheum palmatum). A recombinant enzyme expressed in Escherichia coli accepted acetyl-CoA as a starter, carried out six successive condensations with malonyl-CoA and subsequent cyclization to yield an aromatic heptaketide, aloesone. The enzyme shares 60% amino acid sequence identity with chalcone synthases (CHSs), and maintains almost identical CoA binding site and catalytic residues conserved in the CHS superfamily enzymes. Further, homology modeling predicted that the 43-kDa protein has the same overall fold as CHS. This provides new insights into the catalytic functions of type III PKSs, and suggests further involvement in the biosynthesis of plant polyketides.

Aromatic and pyrone polyketides synthesized by a stilbene synthase from Rheum tataricum.

A cDNA encoding a stilbene synthase, RtSTS, was isolated from the rhizomes of Tatar rhubarb, Rheum tataricum L. (Polygonaceae), a medicinal plant containing stilbenes and other polyketides. Recombinant RtSTS was expressed in E. coli and assayed with acetyl-coenzyme A (CoA), n-butyryl-CoA, isovaleryl-CoA, n-hexanoyl-CoA, cinnamoyl-CoA and p-coumaroyl-CoA as primers of polyketide synthesis. RtSTS synthesized resveratrol and a trace amount of naringenin chalcone from p-coumaroyl-CoA, supporting the enzyme's identification as a resveratrol-type stilbene synthase (EC 2.3.1.95). Bis-noryangonin and p-coumaroyl triacetic acid lactone (CTAL)-type pyrones were observed in minor amounts in the reaction with p-coumaroyl-CoA and as major products with cinnamoyl CoA. As well, such pyrones, and not aromatic polyketides, were identified as the only products in assays with aliphatic and benzoyl CoA esters. Acetonyl-4-hydroxy-2-pyrone, a pyrone synthesized from acetyl-CoA, was identified as a new product of a stilbene synthase. Using Northern blot analysis, RtSTS transcript was found to be highly expressed in R. tataricum rhizomes, with low transcript levels also present in young leaves. This expression pattern correlated with the occurrence of resveratrol, which was detected in higher amounts in R. tataricum rhizomes compared with leaves and petioles using HPLC. Few stilbene synthases have been found in plants, and the identification of RtSTS provides additional sequence and catalytic information with which to study the evolution of plant polyketide synthases.