Isolation and Characterization of Two cDNAs Encoding 4-Coumarate:CoA Ligase in Lithospermum Cell Cultures

Two near full-length cDNAs (LE4CL-1, LE4CL-2), which encode4-coumarate:CoA ligase (4CL), were cloned from a library ofLithospermum erythrorhizon cell suspension cultures by the useof heterologous probe of potato 4CL. These cDNAs are 2.1 kband 2.2 kb in length, respectively. LE4CL-1 encodes 636 aminoacids, whose homologies to the 4CL protein sequences known topotato, parsley, pine and rice, were found to be 68%, 66%, 56%and 50% (identities on amino acid level), respectively, whereasthose of the predicted translation product of LE4CL-2 (594 aminoacids) to the above 4CL proteins were 49{small tilde}54%. Thesimilarity of the deduced amino acid sequences between the two4CLs from Lithospermum cell cultures was 49% in identity. Northernanalyses showed that the mRNA levels of both LE4CL-1 and LE4CL-2were much higher under illumination than in the dark, as reportedfor the 4CL genes of such plants as parsley. In comparison ofmRNA levels of LE4CL-1 and LE4CL-2, the former was demonstratedto be generally higher than the latter by means of an applicationof RT-PCR. The genomic southern blot experiments suggested thatthere are probably three copies of LE4CL-1 in the Lithospermumgenome DNA, whereas only one copy was detected for LE4CL-2. (Received May 26, 1995; Accepted August 16, 1995)     

Differential Properties of 4-Coumarate: CoA Ligase Related to Growth Suppression by Chalcone in Maize and Rice

Lignin and related metabolites have diverse and important functions for plant growth and development. 4-Coumarate: CoA ligase (4CL, EC 6.2.1.12) is one of the key enzymes in phenylpropanoid metabolism and lignin biosynthesis. In a previous study, maize (Zea maize L. cv. Yellowcorn) growth was suppressed to a greater extent by root-applied chalcone than rice (Oryza sativa L. cv. Nipponbare). The objective of this study is to clarify the relationship between the growth suppression and 4CL properties. In crude extracts, total 4CL activity and total protein content of rice were higher 1.8- and 2.7-fold than that of maize, respectively. After a gel-filtration chromatography, a single peak of 4CL activity from maize and rice was evident coincidently for both species. After anion-exchange chromatography, a single peak of 4CL activity was also apparent for both species; however, the peak of maize did not coincide with that of rice. The enzyme activity of maize and rice exhibited similar order of substrate specificities when using p-coumaric, cinnamic, caffeic, ferulic and sinapic acids substrates. Chalcone inhibited 4CL activity in maize more strongly than in rice, and 4CL kinetic data in the presence and absence of chalcone exhibited uncompetitive inhibition in both maize and rice. These results suggest that total activity and the inhibitory property of 4CL contributes to differences in growth suppression by chalcone between maize and rice, although further efforts are needed to clarify the potential of 4CL as a novel action site of the growth suppression.     

Analysis of the spatial and temporal expression pattern directed by the Populus tomentosa 4-coumarate:CoA ligase Pto4CL2 promoter in transgenic tobacco

4-Coumarate:CoA ligase (4CL) is a key enzyme in the phenylpropanoid synthesis pathway. The Pto4CL2 promoter was cloned from Populus tomentosa Carr. and fused to the reporter gene encoding β-glucuronidase (GUS); the complex expression patterns directed by the Pto4CL2 promoter were then characterized in Nicotiana tabacum Xanthi by histochemical assays. The promoter 5′-deletion and histochemical assay conducted on transformants indicated that the ?317 to ?292 nt region supports Pto4CL2 expression in the epidermis and petals and the deletion of the ?266 to ?252 nt region resulted in the loss of tissue specificity and a dramatic reduction in GUS activity. Furthermore, electrophoretic mobility shift assays testified that an adenine and cytosine-rich element (?264 to ?255 nt) and an abscisic acid-responsive element (?242 to ?235 nt) in the Pto4CL2 promoter would have functions for the complex expression profiling and efficient basal expression, respectively. These results further clarify the mode of the regulatory expression of class II 4CL promoters in higher plants.     

Two Divergent Members of 4-Coumarate： Coenzyme A Ligase from Salvia miltiorrhiza Bunge： cDNA Cloning and Functional Study

4-Coumarate ： coenzyme A Ilgase （4CL） Is one of the key enzymes In phenylpropanoid metabolism leading to series of phenollcs, Including water-soluble phenolic acids, which are important compounds determining the medicinal quality of Danshen （Salvia miltiorrhiza Bunge）, a traditional Chinese medicinal herb. To Investigate the function of 4CL in the biosynthesis of water-soluble phenolic acid in Danshen, we have cloned two cDNAs （Sm4CL1 and Sm4CL2） encoding divergent 4CL members by applying nested reverse transcrlptlon-polymerase chain reaction （RT-PCR） with degenerate primers followed by 5′/3′rapid amplification of cDNA ends （RACE） （Note, these sequence data have been submitted to the GenBank database under accession numbers AY237163 and AY237164）. Either of the coding regions was inserted into a pRSET vector and a kinetic assay was performed with purified recombinant proteins. The substrate utilization profile of Sm4CL1 was distinct from that of Sm4CL2. The Km values of Sm4CL1 and Sm4CL2 to 4-coumarlc acid were （72.20±4.10） and （6.50±1.45） μmol/L, respectively. These results, In conjunction with Northern blotting and other information, imply that Sm4CL2 may play an Important role in the biosynthesis of watersoluble phenolic compounds, whereas Sm4CL1 may play a minor role in the pathway. Southern blotting analysis suggested that both Sm4CL1 and Sm4CL2 genes are present as a single copy and are located at different sites In the genome.     

Alterations in the Biosynthesis of Lignin in Transgenic Plants with Chimeric Genes for 4-Coumarate: Coenzyme A Ligase

The introduction of chimeric sense and antisense gene constructsfor 4-coumarate:coenzyme A ligase into tobacco plants causedthe reduction of the 4CL activity in the transgenic plants.In the transgenic plants, the cell walls of the xylem tissuein stems were brown and the molecular structure of lignin inthe colored cell walls was dramatically different from thatin the control plants. Analysis with different types of stainrevealed that levels of cinnamyl aldehyde residues and syringylunits in lignin were depressed in the brownish cell walls. Furthermore,the lignin content in colored tissue was lower than that inthe normal tissue. Our results indicate that 4CL has importantroles in the determination of the composition and the amountof lignin in tobacco plants. (Received December 27, 1995; Accepted July 23, 1996)     

Crystal Structures of a Piscine Betanodavirus: Mechanisms of Capsid Assembly and Viral Infection

Betanodaviruses cause massive mortality in marine fish species with viral nervous necrosis. The structure of a T = 3 Grouper nervous necrosis virus-like particle (GNNV-LP) is determined by the ab initio method with non-crystallographic symmetry averaging at 3.6 Å resolution. Each capsid protein (CP) shows three major domains: (i) the N-terminal arm, an inter-subunit extension at the inner surface; (ii) the shell domain (S-domain), a jelly-roll structure; and (iii) the protrusion domain (P-domain) formed by three-fold trimeric protrusions. In addition, we have determined structures of the T = 1 subviral particles (SVPs) of (i) the delta-P-domain mutant (residues 35−217) at 3.1 Å resolution; and (ii) the N-ARM deletion mutant (residues 35−338) at 7 Å resolution; and (iii) the structure of the individual P-domain (residues 214−338) at 1.2 Å resolution. The P-domain reveals a novel DxD motif asymmetrically coordinating two Ca²⁺ ions, and seems to play a prominent role in the calcium-mediated trimerization of the GNNV CPs during the initial capsid assembly process. The flexible N-ARM (N-terminal arginine-rich motif) appears to serve as a molecular switch for T = 1 or T = 3 assembly. Finally, we find that polyethylene glycol, which is incorporated into the P-domain during the crystallization process, enhances GNNV infection. The present structural studies together with the biological assays enhance our understanding of the role of the P-domain of GNNV in the capsid assembly and viral infection by this betanodavirus.     

Structural Characterization of Modified Lignin in Transgenic Tobacco Plants in Which the Activity of 4-Coumarate:Coenzyme A Ligase Is Depressed

Transgenic tobacco (Nicotiana tabacum L.) plants in which the activity of 4-coumarate:coenzyme A ligase is very low contain a novel lignin in their xylem. Details of changes in hydroxycinnamic acids bound to cell walls and in the structure of the novel lignin were identified by base hydrolysis, alkaline nitrobenzene oxidation, pyrolysis-gas chromatography, and 13C-nuclear magnetic resonance analysis. In the brownish tissue of the transgenic plants, the levels of three hydroxycinnamic acids, p-coumaric, ferulic, and sinapic, which were bound to cell walls, were apparently increased as a result of down-regulation of the expression of the gene for 4-coumarate:coenzyme A ligase. Some of these hydroxycinnamic acids were linked to cell walls via ester and ether linkages. The accumulation of hydroxycinnamic acids also induced an increase in the level of condensed units in the novel lignin of the brownish tissue. Our data indicate that the behavior of some of the incorporated hydroxycinnamic acids resembles lignin monomers in the brownish tissue, and their accumulation results in dramatic changes in the biosynthesis of lignin in transgenic plants.     

4-Coumarate:CoA ligase from cell suspension cultures of Petroselinum hortense Hoffm: Partial purification,substrate specificity,and further properties

4-Coumarate:CoA ligase (EC 6.2.1.12) was isolated from 8-day-old cell suspension cultures of parsley (Petroselinum hortense Hoffm.) which had been irradiated with ultraviolet light for 15 h. The enzyme was partially purified by fractionation with MnCl₂ and (NH₄)₂SO₄ and by column chromatography on diethylaminoethyl cellulose, hydroxyapatite, and aminohexyl-Sepharose. A 90-fold increase in specific activity with an overall yield of 20% was achieved. Analytical gel electrophoresis indicated the occurrence of only one 4-coumarate:CoA ligase species in the final enzyme preparation. The enzyme was largely specific for 4-coumarate and other derivatives of cinnamic acid. 4-Coumarate had the lowest apparent K_m and the highest $\text{V}\text{K}{}_{\mathrm{m}}$ values (1.4 × 10^?5, m and 14.7 × 10⁵ pkatal × m^?1, respectively) of all substrates tested. Only the trans isomer of 4-coumarate was activated. The two cosubstrates, ATP and CoA, exhibited sigmoidal saturation kinetics, which were interpreted as indicating homotropic, allo-steric effects. A molecular weight of about 67,000 was estimated for 4-coumarate:CoA ligase. The substrate specificity of the enzyme was in agreement with its proposed function in flavonoid biosynthesis.     

光皮桦中4-香豆酸辅酶A连接酶基因Bl4CL的克隆和表达分析

以从光皮桦茎叶组织提取的mRNA为模板,根据其他已克隆到的阔叶类树种中4-香豆酸辅酶A连接酶(4CL)基因的同源序列设计兼并引物,进行RT-PCR扩增,获得部分基因片段,然后结合5’,3’RACE方法从光皮桦中扩增出1个4CL基因的全长cDNA序列,命名为Bl4CL。该基因cDNA全长为1983bp(GenBank登录号FJ410448),具有完整的开放阅读框架(69～1697bp),编码蛋白为542个氨基酸,包含一个AMP结合功能域和一个含有12个氨基酸的功能基序。与其他植物中的4CL进行同源性比对的结果显示,Bl4CL蛋白与东北白桦的同源性最高,达到了98%。该基因在光皮桦的根和茎中表达量较高,而在花和叶中的表达量低。     

Crystal Structures and Enzyme Mechanisms of a Dual Fucose Mutarotase/Ribose Pyranase

Escherichia coli FucU (Fucose Unknown) is a dual fucose mutarotase and ribose pyranase, which shares 44% sequence identity with its human counterpart. Herein, we report the structures of E. coli FucU and mouse FucU bound to l-fucose and delineate the catalytic mechanisms underlying the interconversion between stereoisomers of fucose and ribose. E. coli FucU forms a decameric toroid with each active site formed by two adjacent subunits. While one subunit provides most of the fucose-interacting residues including a catalytic tyrosine residue, the other subunit provides a catalytic His-Asp dyad. This active-site feature is critical not only for the mutarotase activity toward l-fucose but also for the pyranase activity toward d-ribose. Structural and biochemical analyses pointed that mouse FucU assembles into four different oligomeric forms, among which the smallest homodimeric form is most abundant and would be the predominant species under physiological conditions. This homodimer has two fucose-binding sites that are devoid of the His-Asp dyad and catalytically inactive, indicating that the mutarotase and the pyranase activities appear dispensable in vertebrates. The defective assembly of the mouse FucU homodimer into the decameric form is due to an insertion of two residues at the N-terminal extreme, which is a common aspect of all the known vertebrate FucU proteins. Therefore, vertebrate FucU appears to serve for as yet unknown function through the quaternary structural alteration.     

Synchrotron-Based Techniques Shed Light on Mechanisms of Plant Sensitivity and Tolerance to High Manganese in the Root Environment

Plant species differ in response to high available manganese (Mn), but the mechanisms of sensitivity and tolerance are poorly understood. In solution culture, greater than or equal to 30 µm Mn decreased the growth of soybean (Glycine max), but white lupin (Lupinus albus), narrow-leafed lupin (Lupin angustifolius), and sunflower (Helianthus annuus) grew well at 100 µm Mn. Differences in species’ tolerance to high Mn could not be explained simply by differences in root, stem, or leaf Mn status, being 8.6, 17.1, 6.8, and 9.5 mmol kg^–1 leaf fresh mass at 100 µm Mn. Furthermore, x-ray absorption near edge structure analyses identified the predominance of Mn(II), bound mostly to malate or citrate, in roots and stems of all four species. Rather, differences in tolerance were due to variations in Mn distribution and speciation within leaves. In Mn-sensitive soybean, in situ analysis of fresh leaves using x-ray fluorescence microscopy combined with x-ray absorption near edge structure showed high Mn in the veins, and manganite [Mn(III)] accumulated in necrotic lesions apparently through low Mn sequestration in vacuoles or other vesicles. In the two lupin species, most Mn accumulated in vacuoles as either soluble Mn(II) malate or citrate. In sunflower, Mn was sequestered as manganite at the base of nonglandular trichomes. Hence, tolerance to high Mn was ascribed to effective sinks for Mn in leaves, as Mn(II) within vacuoles or through oxidation of Mn(II) to Mn(III) in trichomes. These two mechanisms prevented Mn accumulation in the cytoplasm and apoplast, thereby ensuring tolerance to high Mn in the root environment.Manganese (Mn) is an essential element for plant growth, but its availability differs greatly in space and time, depending largely on the nature and amount of Mn minerals present and on the soil’s pH and redox potential. With an elaborate chemistry, Mn forms complexes with many organic and inorganic ligands. In soils, Mn has three common oxidation states, Mn(II), Mn(III), and Mn(IV), which form hydrated oxides of mixed valency; Mn is present also as numerous carbonates, silicates, sulfates, and phosphates (Lindsay, 1979). Cationic Mn²⁺ is the most common form readily absorbed by plant roots (Clarkson, 1988). The toxicity of Mn occurs in acid or waterlogged soils high in Mn minerals.Many plants have mechanisms to accommodate the large differences in Mn²⁺ in soils. At low available Mn, uptake is increased in some Poaceae by excretion of phytosiderophores of the mugineic acid family (Takahashi et al., 2003), with root phytase exudation also potentially important for acquisition of Mn when Mn availability is limited (George et al., 2014). Mechanisms in other plants include the ability of roots to decrease rhizosphere pH or excrete organic ligands (Neumann and Romheld, 2012; Lambers et al., 2015). However, the relative importance of the many complexes on Mn uptake remains unclear. Toxicity results from high Mn in leaf cell walls (Wissemeier et al., 1992; Wissemeier and Horst, 1992) and through adverse effects on symplastic proteins (Führs et al., 2008). Many plants have mechanisms that limit the adverse effects of high Mn²⁺ in soils, with numerous ligands involved in its translocation and that of other essential cations (Haydon and Cobbett, 2007). Edwards and Asher (1982) classified a range of crop and pasture species based on their ability to deal with high Mn as those that (1) limit Mn from entering the roots, (2) retain Mn in the roots, or (3) tolerate high Mn in the shoots. At the extreme are plants that hyperaccumulate more than 10,000 mg Mn kg^–1 on a dry mass (DM) basis in foliar tissues without metabolic damage (Fernando et al., 2013; van der Ent et al., 2013). Based on 15% DM of leaves, this equates to 12.1 mmol kg^–1 on a fresh mass (FM) basis. Celosia argentia, a species adapted to growth on Mn-contaminated mine tailings, accumulated more than 20,000 mg kg^–1 Mn in leaves (Liu et al., 2014). Tolerance of high Mn in shoots of some Mn hyperaccumulators has been found to occur through binding to ligands (such as malate or citrate) or sequestration in the vacuole (Fernando et al., 2010).Characteristic symptoms of Mn toxicity include chlorotic and distorted leaves with small necrotic lesions. These lesions have been shown in cowpea (Vigna unguiculata) to contain oxidized Mn and callose (Wissemeier et al., 1992), which forms as a reaction to high intracellular Ca (Kartusch, 2003). The necrotic lesions result mainly from oxidized phenolics (Wissemeier and Horst, 1992) and increased peroxidase activity in the apoplast (Horst et al., 1999). With a critical solution concentration for toxicity (10% growth reduction) of no more than 9 µm Mn, Edwards and Asher (1982) found that cotton (Gossypium hirsutum), bean (Phaseolus vulgaris), cowpea, and soybean (Glycine max) were the most sensitive species of 13 crop and pasture plants grown for 18 to 31 d at constant Mn in solution culture. By contrast, the critical concentration for sunflower (Helianthus annuus) was 7 times higher at 65 µm Mn. Sunflower was the first species found to tolerate high Mn through its sequestration in the trichomes on stems, petioles, and leaves (Blamey et al., 1986). The suspected accumulation of Mn was confirmed using wavelength dispersive x-ray spectroscopy with darkening inferred as due to insoluble higher oxides of Mn. Similarly, high Mn results in darkened trichomes of cucumber (Cucumis sativus) leaves due to oxidized Mn, as shown by the colorimetric benzidine test (Horiguchi, 1987). Watermelon (Citrullus lanatus; Elamin and Wilcox, 1986b), but not muskmelon (Citrullus melo; Elamin and Wilcox, 1986a), grown at high Mn also develops small dark spots around the leaf trichomes. Other species that sequester Mn in the trichomes include common nettle (Urtica dioica; Hughes and Williams, 1988) and Alyssum murale, a Ni hyperaccumulator (Broadhurst et al., 2009; McNear and Küpper, 2014). Thus, some plants in four families, Asteraceae, Cucurbitaceae, Urticaceae, and Brassicaceae, tolerate high Mn in shoots through Mn sequestration in or around the trichomes. The mechanisms may differ, however, because the high Mn present during development of common nettle stinging hairs decreases as plants mature (Hughes and Williams, 1988).Recently developed techniques, including those based on synchrotron radiation, allow investigations of the distribution and speciation of Mn in planta, with most research to date focused on Mn hyperaccumulators (Fernando et al., 2013). For example, Fernando et al. (2010) used x-ray absorption near-edge spectroscopy (XANES) to confirm the widely accepted view that Mn(II) predominates in seven Mn hyperaccumulators. Synchrotron-based x-ray fluorescence microspectroscopy (µ-XRF) was used by McNear and Küpper (2014) to show that the basal region of trichomes of A. murale plants grown at no more than 10 µm Mn contained Mn(II) complexed with phosphate. At 50 µm Mn in solution, however, the increased amount of Mn that had accumulated around the trichomes was present as Mn(III). Few studies, however, have used synchrotron-based techniques to investigate the mechanisms of Mn toxicity and tolerance in agronomic species despite their importance for food production in regions where soils are acidic or intermittently waterlogged. One study on cowpea, with a critical toxicity concentration of only 2 µm Mn (Edwards and Asher, 1982), has shown an accumulation of Mn-citrate in the root cap and associated mucigel within 5 min of exposure to 150 µm Mn (Kopittke et al., 2013).This study aimed to determine the distribution and speciation of Mn in fresh roots, stems, and leaves of four crop species, soybean, white lupin (Lupinus albus), narrow-leafed lupin (Lupinus angustifolius), and sunflower, which differ in tolerance to high Mn. It was hypothesized that Mn distribution and speciation would differ between Mn-sensitive soybean and the three other species. Furthermore, we considered it likely that the Mn tolerance mechanism of sunflower would differ from those of the two lupin species, which do not have darkened trichomes when grown at high Mn.