Mutations that alter Arabidopsis flavonoid metabolism affect the circadian clock.

Flavonoids are a well-known class of specialized metabolites that play key roles in plant development, reproduction, and survival. Flavonoids are also of considerable interest from the perspective of human health, as both phytonutrients and pharmaceuticals. RNA sequencing analysis of an Arabidopsis null allele for chalcone synthase (CHS), which catalyzes the first step in flavonoid metabolism, has uncovered evidence that these compounds influence the expression of genes associated with the plant circadian clock. Analysis of promoter-luciferase constructs further showed that the transcriptional activity of CCA1 and TOC1, two key clock genes, is altered in CHS-deficient seedlings across the day/night cycle. Similar findings for a mutant line lacking flavonoid 3'-hydroxylase (F3'H) activity, and thus able to synthesize mono- but not dihydroxylated B-ring flavonoids, suggests that the latter are at least partially responsible; this was further supported by the ability of quercetin to enhance CCA1 promoter activity in wild-type and CHS-deficient seedlings. The effects of flavonoids on circadian function were also reflected in photosynthetic activity, with chlorophyll cycling abolished in CHS- and F3'H-deficient plants. Remarkably, the same phenotype was exhibited by plants with artificially high flavonoid levels, indicating that neither the antioxidant potential nor the light-screening properties of flavonoids contribute to optimal clock function, as has recently also been demonstrated in animal systems. Collectively, the current experiments point to a previously unknown connection between flavonoids and circadian cycling in plants and open the way to better understanding of the molecular basis of flavonoid action.

Mechanistic Investigation into DNA Modification by a RuII ,RhIII Bimetallic Complex.

Despite significant progress in the treatment of cancer, there remains an urgent need for more effective therapies that also have less impact on patient wellbeing. Photodynamic therapy employs targeted light activation of a photosensitizer in selected tissues, thereby reducing off-target toxicity. Our group previously reported a RuII ,RhIII bimetallic architecture that displays multifunctional covalent photomodification of DNA in the therapeutic window in an oxygen-independent manner, features that are essential for treating deep and hypoxic tumors. Herein, we explore the mechanism by which a new analogue, [(phen)2 Ru(dpp)Rh(phen)Cl2 ]3+ , or RuII -RhIII , interacts with DNA. We established that RuII -RhIII exhibits "light switch" behavior in the presence of DNA, undergoing strong electrostatic interactions that might involve groove binding. Furthermore, these noncovalent interactions play a major role in the covalent photobinding and photocleavage of DNA, which occur according to an oxygen-independent mechanism. Polymerase chain reaction (PCR) revealed that covalent modification of DNA by RuII -RhIII , especially photobinding, is critical to inhibiting amplification, thus suggesting that the complex could exert its toxic activity by interfering with DNA replication in cells. This new structural motif, with phenanthroline at all three terminal ligand positions, has a number of properties that are promising for the continued refinement of photodynamic-therapy strategies.

Diversity of unique, nonmycorrhizal endophytic fungi in cultivated Phalaenopsis orchids: A pilot study.

Orchids comprise one of the largest, most diverse, and most broadly distributed families of flowering plants and contribute significantly to habitat biodiversity. One key aspect of orchid growth and development is the formation of mycorrhizal symbioses with compatible endophytic fungi, which are maintained throughout the life of the plant. Substantial efforts to identify the fungi that form mycorrhizal symbioses across a range of orchid species have often also uncovered numerous nonmycorrhizal, endophytic fungi. These fungi could also have significant effects on orchid growth and development and are beginning to be analyzed more closely, particularly in wild species. The role of endophytic fungi in the production, distribution, and continued growth by the hobbyist of orchids is not known. As an initial step toward characterizing nonmycorrhizal endophytic fungi associated with cultivated orchids, we undertook a survey of fungi residing within roots of Phalaenopsis plants growing in home environments. Sequence analysis of ITS regions amplified from total DNA isolated from roots allowed rapid identification of endophytic fungi to the class level and may offer a useful initial screening method for beneficial species, for example, in horticultural settings. ITS-PCR sequences subsequently obtained from individual fungi cultured from surface-sterilized orchid roots corroborated the findings of the initial screen, while also providing a more complete characterization of the array of fungal taxa that were present. Although lower in diversity than has been reported for orchids growing in the wild, these endophytes have the potential to substantially enhance the growth and disease resistance of horticultural orchids.

Auxin and ethylene induce flavonol accumulation through distinct transcriptional networks.

Auxin and ethylene are key regulators of plant growth and development, and thus the transcriptional networks that mediate responses to these hormones have been the subject of intense research. This study dissected the hormonal cross talk regulating the synthesis of flavonols and examined their impact on root growth and development. We analyzed the effects of auxin and an ethylene precursor on roots of wild-type and hormone-insensitive Arabidopsis (Arabidopsis thaliana) mutants at the transcript, protein, and metabolite levels at high spatial and temporal resolution. Indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) differentially increased flavonol pathway transcripts and flavonol accumulation, altering the relative abundance of quercetin and kaempferol. The IAA, but not ACC, response is lost in the transport inhibitor response1 (tir1) auxin receptor mutant, while ACC responses, but not IAA responses, are lost in ethylene insensitive2 (ein2) and ethylene resistant1 (etr1) ethylene signaling mutants. A kinetic analysis identified increases in transcripts encoding the transcriptional regulators MYB12, Transparent Testa Glabra1, and Production of Anthocyanin Pigment after hormone treatments, which preceded increases in transcripts encoding flavonoid biosynthetic enzymes. In addition, myb12 mutants were insensitive to the effects of auxin and ethylene on flavonol metabolism. The equivalent phenotypes for transparent testa4 (tt4), which makes no flavonols, and tt7, which makes kaempferol but not quercetin, showed that quercetin derivatives are the inhibitors of basipetal root auxin transport, gravitropism, and elongation growth. Collectively, these experiments demonstrate that auxin and ethylene regulate flavonol biosynthesis through distinct signaling networks involving TIR1 and EIN2/ETR1, respectively, both of which converge on MYB12. This study also provides new evidence that quercetin is the flavonol that modulates basipetal auxin transport.

Redox, spectroscopic, and photophysical properties of Ru-Pt mixed-metal complexes incorporating 4,7-diphenyl-1,10-phenanthroline as efficient DNA binding and photocleaving agents.

The redox, spectroscopic, and photophysical properties as well as DNA interactions of the new bimetallic complexes [(Ph2phen)2Ru(BL)PtCl2](2+) (Ph2phen = 4,7-diphenyl-1,10-phenanthroline, and BL (bridging ligand) = dpp = 2,3-bis(2-pyridyl)pyrazine, or dpq = 2,3-bis(2-pyridyl)quinoxaline) were investigated. These Ru-polyazine chromophores with Ph2phen TLs (terminal ligands) and polyazine BLs are efficient light absorbers. The [(Ph2phen)2Ru(BL)PtCl2](2+) complexes display reversible Ru(II/III) oxidations at 1.57 (dpp) and 1.58 (dpq) V vs SCE (saturated calomel electrode) with an irreversible Pt(II/IV) oxidation occurring prior at 1.47 V vs SCE. Four, reversible ligand reductions occur at -0.50 dpp(0/-), -1.06 dpp(-/2-), -1.37 Ph2phen(0/-), and -1.56 V vs SCE Ph2phen(0/-). For the [(Ph2phen)2Ru(dpq)PtCl2](2+) complex, the first two reductions shift to more positive potentials at -0.23 and -0.96 V vs SCE. The electronic absorption spectroscopy is dominated in the UV region by π → π* ligand transitions and in the visible region by metal-to-ligand charge transfer (MLCT) transitions at 517 nm for [(Ph2phen)2Ru(dpp)PtCl2](2+) and 600 nm for [(Ph2phen)2Ru(dpq)PtCl2](2+). Emission spectroscopy shows that upon attaching Pt to the Ru monometallic precursor the λmax(em) shifts from 664 nm for [(Ph2phen)2Ru(dpp)](2+) to 740 nm for [(Ph2phen)2Ru(dpp)PtCl2](2+). The cis-Pt(II)Cl2 bioactive site offers the potential of targeting DNA by covalently binding the mixed-metal complex to DNA bases. The multifunctional interactions with DNA were assayed using both linear and circular plasmid pUC18 DNA gel shift assays. Both title complexes can bind to and photocleave DNA with dramatically enhanced efficiency relative to previously reported systems. The impact of the Ph2phen TL on photophysics and bioreactivity is somewhat surprising given the Ru → BL charge transfer (CT) nature of the photoreactive state in the complexes.

Modulation of Arabidopsis Flavonol Biosynthesis Genes by Cyst and Root-Knot Nematodes.

Although it is well established that flavonoid synthesis is induced in diverse plant species during nematode parasitism, little is known about the regulation of genes controlling flavonol biosynthesis during the plant-nematode interaction. In this study, expression of the Arabidopsis thaliana flavonol-specific transcription factor, AtMYB12, the flavonol synthase genes, AtFLS1, 2, 3, 4, and 5, and the gene encoding the central flavonoid enzyme, chalcone synthase (AtCHS), were examined in plant roots during infection by Heterodera schachtii (sugar beet cyst) and Meloidogyne incognita (root-knot) nematodes. These experiments showed that AtMYB12 was transiently upregulated at 9 dpi in syncytia associated with sugar beet cyst nematode infection and that an Atmyb12-deficient line was less susceptible to the parasite. This suggests that, rather than contributing to plant defense, this gene is essential for productive infection. However, the AtCHS and AtFLS1 genes, which are controlled by AtMYB12, did not exhibit a similar transient increase, but rather were expressly downregulated in syncytia relative to adjacent uninfected root tissue. Genetic analyses further indicated that AtFLS1 contributes to plant defense against Cyst nematode infection, while other AtFLS gene family members do not, consistent with prior reports that these other genes encode little or no enzyme activity. Together, these findings indicate a role of AtMyb12 in promoting the early stages of Cyst nematode infection, while flavonols produced through the action of AtFLS1 are essential for plant defense. On the other hand, a transient induction of AtMYB12 was not observed in galls produced during root-knot nematode infection, but this gene was instead substantially downregulated, starting at the 9 dpi sampling point, as were AtCHS and AtFLS1. In addition, both the AtMYB12- and AtFLS1-deficient lines were more susceptible to infection by this parasite. There was again little evidence for contributions from the other AtFLS gene family members, although an AtFLS5-deficient line appeared to be somewhat more susceptible to infection. Taken together, this study shows that sugar-beet cyst and root-knot nematodes modulate differently the genes involved in flavonol biosynthesis in order to successfully infect host roots and that AtFLS1 may be involved in the plant basal defense response against nematode infection.

The dynamic response of the Arabidopsis root metabolome to auxin and ethylene is not predicted by changes in the transcriptome.

While the effects of phytohormones on plant gene expression have been well characterized, comparatively little is known about how hormones influence metabolite profiles. This study examined the effects of elevated auxin and ethylene on the metabolome of Arabidopsis roots using a high-resolution 24 h time course, conducted in parallel to time-matched transcriptomic analyses. Mass spectrometry using orthogonal UPLC separation strategies (reversed phase and HILIC) in both positive and negative ionization modes was used to maximize identification of metabolites with altered levels. The findings show that the root metabolome responds rapidly to hormone stimulus and that compounds belonging to the same class of metabolites exhibit similar changes. The responses were dominated by changes in phenylpropanoid, glucosinolate, and fatty acid metabolism, although the nature and timing of the response was unique for each hormone. These alterations in the metabolome were not directly predicted by the corresponding transcriptome data, suggesting that post-transcriptional events such as changes in enzyme activity and/or transport processes drove the observed changes in the metabolome. These findings underscore the need to better understand the biochemical mechanisms underlying the temporal reconfiguration of plant metabolism, especially in relation to the hormone-metabolome interface and its subsequent physiological and morphological effects.

Multifunctional DNA interactions of Ru-Pt mixed metal supramolecular complexes with substituted terpyridine ligands.

The coupling of a light absorbing unit to a bioactive site allows for the development of supramolecules with multifunctional interactions with DNA. A series of mixed metal supramolecular complexes that couple a DNA-binding cis-Pt(II)Cl(2) center to a ruthenium chromophore via a polyazine bridging ligand have been prepared, and their DNA interactions have been studied, [(TL)RuCl(dpp)PtCl(2)](PF(6)) (TL = tpy (2,2':6',2''-terpyridine), MePhtpy (4'-(4-methylphenyl)-2,2':6',2''-terpyridine), or (t)Bu(3)tpy (4,4',4''-tri-tert-butyl-2,2':6',2''-terpyridine and dpp = 2,3-bis(2-pyridyl)pyrazine). This series provides for unique tridentate coordinated Ru(II) systems to photocleave DNA with preassociation with the DNA target via coordination of the Pt(II) center. Electronic absorption spectroscopy of the complexes displays intense ligand-based pi-->pi* transitions in the UV region and metal to ligand charge transfer (MLCT) transitions in the visible region. The Ru(dpi)-->dpp(pi*) MLCT transitions occur at 545 nm, red-shifted relative to the 520 nm maxima for the monometallic synthons, [(TL)RuCl(dpp)](PF(6)). The title RuPt complexes display reversible Ru(II/III) oxidative couples at 1.10, 1.10, and 1.01 V vs Ag/AgCl for TL = tpy, MePhtpy, and (t)Bu(3)tpy, respectively. The TL(0/-) reduction occurred at -1.43, -1.44, and -1.59 V vs Ag/AgCl for TL = tpy, MePhtpy, and (t)Bu(3)tpy, respectively. These complexes display a dpp(0/-) couple (-0.50 -0.55, and -0.59 V) significantly shifted to positive potential relative to their monometallic synthons (-1.15, -1.16, and -1.22 V), consistent with the bridging coordination of the dpp ligand. Coupling of (TL)Ru(II)Cl(BL) subunit to a cis-Pt(II)Cl(2) site provides for the application of photochemically inactive Ru(II)(tpy)-based chromophores in DNA photocleavage applications. The [(TL)RuCl(dpp)PtCl(2)](+) complexes display covalent binding to DNA and photocleavage upon irradiation with visible light modulated by TL identity. The redox, spectroscopic, DNA-binding, and photocleavage properties of a series of supramolecular complexes are presented.

Metabolic channeling in plants.

The organization of cooperating enzymes into macromolecular complexes is a central feature of cellular metabolism. A major advantage of such spatial organization is the transfer of biosynthetic intermediates between catalytic sites without diffusion into the bulk phase of the cell. This so-called "metabolic channeling" offers unique opportunities for enhancing and regulating cellular biochemistry. Studies in a number of plant primary and secondary metabolic systems continue to contribute to our understanding of the nature and importance of this phenomenon. This article reviews advances in four systems: the cysteine synthase complex, the Calvin cycle, cyanogenic glucoside biosynthesis, and the phenylpropanoid pathway. Each of these systems is providing new evidence for the importance of enzyme organization in cellular biochemistry as well as exclusive insights into the molecular basis of enzyme complex assembly. This review also explores current prospects for understanding metabolon structure, assembly, and biological function.

Photochemical methods to assay DNA photocleavage using supercoiled pUC18 DNA and LED or xenon arc lamp excitation.

Methods for the study of DNA photocleavage are illustrated using a mixed-metal supramolecular complex [{(bpy)(2)Ru(dpp)}(2)RhCl(2)]Cl(5). The methods use supercoiled pUC18 plasmid as a DNA probe and either filtered light from a xenon arc lamp source or monochromatic light from a newly designed, high-intensity light-emitting diode (LED) array. Detailed methods for performing the photochemical experiments and analysis of the DNA photoproduct are delineated. Detailed methods are also given for building an LED array to be used for DNA photolysis experiments. The Xe arc source has a broad spectral range and high light flux. The LEDs have a high-intensity, nearly monochromatic output. Arrays of LEDs have the advantage of allowing tunable, accurate output to multiple samples for high-throughput photochemistry experiments at relatively low cost.

Biochemical and genetic characterization of Arabidopsis flavanone 3beta-hydroxylase.

Flavanone 3beta-hydroxylase (F3H; EC 1.14.11.9) is a 2-oxoglutarate dependent dioxygenase that catalyzes the synthesis of dihydrokaempferol, the common precursor for three major classes of 3-hydroxy flavonoids, the flavonols, anthocyanins, and proanthocyanidins. This enzyme also competes for flux into the 3-deoxy flavonoid branch pathway in some species. F3H genes are increasingly being used, often together with genes encoding other enzymes, to engineer flavonoid synthesis in microbes and plants. Although putative F3H genes have been cloned in a large number of plant species, only a handful have been functionally characterized. Here we describe the biochemical properties of the Arabidopsis thaliana F3H (AtF3H) enzyme and confirm the activities of gene products from four other plant species previously identified as having high homology to F3H. We have also investigated the surprising "leaky" phenotype of AtF3H mutant alleles, uncovering evidence that two related flavonoid enzymes, flavonol synthase (EC 1.14.11.23) and anthocyanidin synthase (EC 1.14.11.19), can partially compensate for F3H in vivo. These experiments further indicate that the absence of F3H in these lines enables the synthesis of uncommon 3-deoxy flavonoids in the Arabidopsis seed coat.

Identification of MOS9 as an interaction partner for chalcone synthase in the nucleus.

Plant flavonoid metabolism has served as a platform for understanding a range of fundamental biological phenomena, including providing some of the early insights into the subcellular organization of metabolism. Evidence assembled over the past three decades points to the organization of the component enzymes as a membrane-associated complex centered on the entry-point enzyme, chalcone synthase (CHS), with flux into branch pathways controlled by competitive protein interactions. Flavonoid enzymes have also been found in the nucleus in a variety of plant species, raising the possibility of alternative, or moonlighting functions for these proteins in this compartment. Here, we present evidence that CHS interacts with MOS9, a nuclear-localized protein that has been linked to epigenetic control of R genes that mediate effector-triggered immunity. Overexpression of MOS9 results in a reduction of CHS transcript levels and a metabolite profile that substantially intersects with the effects of a null mutation in CHS. These results suggest that the MOS9-CHS interaction may point to a previously-unknown mechanism for controlling the expression of the highly dynamic flavonoid pathway.

In vivo inhibition of E. coli growth by a Ru(II)/Pt(II) supramolecule [(tpy)RuCl(dpp)PtCl2](PF6).

Supramolecular complexes consisting of ruthenium chromophores and a cisplatin unit represent an emerging class of bioactive molecules of interest as anti-cancer agents. Although the ability of Ru(II)/Pt(II) heteronuclear complexes to bind to DNA has been demonstrated, the in vivo activity of these complexes has not yet been reported. In the present work, we report the anti-bacterial activity of the complex [(tpy)RuCl(dpp)PtCl(2)](PF(6)) (where dpp=2,3-bis(2-pyridyl)pyrazine, tpy=2,2':6',2''-terpyridine). The impact on bacterial cell growth of exposure to different concentrations of [(tpy)RuCl(dpp)PtCl(2)](PF(6)) and cisplatin was studied. The bioactivity of this complex was found to be due to the presence of the cis-PtCl(2) moiety, as the monometallic synthon [(tpy)RuCl(dpp)](PF(6)) did not inhibit bacterial cell growth.

Exogenous Auxin Elicits Changes in the Arabidopsis thaliana Root Proteome in a Time-Dependent Manner.

Auxin is involved in many aspects of root development and physiology, including the formation of lateral roots. Improving our understanding of how the auxin response is mediated at the protein level over time can aid in developing a more complete molecular framework of the process. This study evaluates the effects of exogenous auxin treatment on the Arabidopsis root proteome after exposure of young seedlings to auxin for 8, 12, and 24 h, a timeframe permitting the initiation and full maturation of individual lateral roots. Root protein extracts were processed to peptides, fractionated using off-line strong-cation exchange, and analyzed using ultra-performance liquid chromatography and data independent acquisition-based mass spectrometry. Protein abundances were then tabulated using label-free techniques and evaluated for significant changes. Approximately 2000 proteins were identified during the time course experiment, with the number of differences between the treated and control roots increasing over the 24 h time period, with more proteins found at higher abundance with exposure to auxin than at reduced abundance. Although the proteins identified and changing in levels at each time point represented similar biological processes, each time point represented a distinct snapshot of the response. Auxin coordinately regulates many physiological events in roots and does so by influencing the accumulation and loss of distinct proteins in a time-dependent manner. Data are available via ProteomeXchange with the identifier PXD001400.

Variation of DNA photocleavage efficiency for [(TL)2Ru(dpp)]Cl2 complexes where TL=2,2'-bipyridine, 1,10-phenanthroline, or 4,7-diphenyl-1,10-phenanthroline.

The complexes [(bpy)(2)Ru(dpp)]Cl(2), [(phen)(2)Ru(dpp)]Cl(2), and [(Ph(2)phen)(2)Ru(dpp)]Cl(2) (where dpp=2,3-bis(2-pyridyl)pyrazine, bpy=2,2'-bipyridine, phen=1,10-phenanthroline, Ph(2)phen=4,7-diphenyl-1,10-phenanthroline) have been investigated and found to photocleave DNA via an oxygen-mediated pathway. These light absorbing complexes possess intense metal-to-ligand charge transfer (MLCT) transitions in the visible region of the spectrum. The [(TL)(2)Ru(dpp)](2+) systems populate (3)MLCT states after visible light excitation, giving rise to emissions in aqueous solution centered at 692, 690, and 698nm for TL=bpy, phen, and Ph(2)phen respectively. The (3)MLCT states and emissions are quenched by O(2), producing a reactive oxygen species. These complexes photocleave DNA with varying efficiencies, [(Ph(2)phen)(2)Ru(dpp)](2+)>[(phen)(2)Ru(dpp)](2+)>[(bpy)(2)Ru(dpp)](2+). The presence of the polyazine bridging ligand will allow these chromophores to be incorporated into larger supramolecular assemblies.

A new class of Ru(II) polyazine agents with potential for photodynamic therapy.

Appending anthracene units to [(bpy)2Ru(dpp)](2+) results in Ru(II) agents that exhibit dynamic photoreactivity towards DNA and protein. [(Anthbpy)(bpy)Ru(dpp)](2+) and [(Anthbpy)2Ru(dpp)](2+) are the first metal-organic Ru(II) agent with dpp ligands shown to photomodify DNA in the presence or absence of oxygen, while also binding protein in an oxygen-dependent manner.

Evolutionary correlations in flavonoid production across flowers and leaves in the Iochrominae (Solanaceae).

Plant reproductive and vegetative tissues often use the same biochemical pathways to produce specialized metabolites. In such cases, selection acting on the synthesis of specific products in a particular tissue could result in correlated changes in other products of the pathway, both in the same tissue and in other tissues. This study examined how changes in floral anthocyanin pigmentation affect the production of other compounds of the flavonoid pathway in flowers and in leaves. Focusing on the Iochrominae, a clade of Solanaceae with a wide range of flower colors, liquid chromatography coupled with mass spectrometry and UV detection was used to profile and quantify the variation in two classes of flavonoids, anthocyanins and flavonols. Purple, red, orange and white-flowered Iochrominae produced all of the six common anthocyanidin types, as well as several classes of flavonols. Differences in anthocyanin and flavonol production were significantly correlated in flowers, particularly with respect to B ring hydroxylation pattern. However, these differences in floral flavonoids were not strongly related to differences in leaf chemistry. Specifically, most species made only flavonols (not anthocyanins) in leaves, and these comprised the two most common flavonols, quercetin and kaempferol, regardless of the color of the flower. These results suggest that shifts in flower color may occur without significant pleiotropic consequences for flavonoid production in vegetative tissues. Similar studies in other systems will be important for testing the generality of this pattern in other groups of flowering plants.

Exploring the activity of a polyazine bridged Ru(ii)-Pt(ii) supramolecule in F98 rat malignant glioma cells.

The mixed-metal supramolecular complex, [(Ph2phen)2Ru(dpp)PtCl2]2+, displays significant DNA modification, cell growth inhibition, and toxicity towards F98 malignant glioma cells following visible light irradiation. The design of this complex affords superior cellular uptake and antiproliferative activity compared to the classic chemotherapeutic agent, cisplatin.