Partial purification and characterization of a coniferyl alcohol dehydrogenase fromRhodococcus erythropolis

A nicotinamide adenine dinucleotide (NAD)-dependent coniferyl alcohol dehydrogenase was enriched 1,200-fold from crude extracts ofRhodococcus erythropolis. The purification procedure involved ion exchange chromotography, gel filtration on Biogel A 1,5 and Sephadex G-200, and hydroxyapatite treatment. The enzyme had a molecular weight of approximately 200,000 and displayed maximal activity at pH 9.0. The apparentK _m values for NAD and coniferyl alcohol were, respectively, 0.22 and 0.645 mM. Nicotinamide adenine dinucleotide phosphate (NADP) could only partially replace NAD. The enzyme was active with vanillyl alcohol and aromatic alcohols bearing the ,-unsaturated side chain of coniferyl alcohols. These aromatic alcohols included the dilignols dehydrodiconiferyl alcohol and guaiacylglycerol--coniferyl ether.     

QTLs controlling the production of transgenic and adventitious roots in Brassica oleracea following treatment with Agrobacterium rhizogenes

Brassica oleracea can be genetically engineered using Agrobacterium rhizogenes. The initial stage of this process is the production of transgenic (hairy) roots; shoots are subsequently regenerated from these roots. Previous work using gus and gfp reporter genes has shown that genotypes of B. oleracea vary in their performance for transgenic root production. Quantitative trait loci (QTLs) controlling this trait have been located in one mapping population. The current study provides evidence that performance for transgenic root production is associated with performance for adventitious (non-transgenic) root production in B. oleracea across a second mapping population. This is shown by regression analyses between performance for the two traits and the demonstration that QTLs controlling the two traits map to the same positions within the genome. Since the rate of adventitious root production does not differ significantly in the presence and absence of A. rhizogenes, there is no evidence that the expression of Agrobacterium genes induces adventitious root production. It is apparent that genotypes exhibiting high adventitious root production in the absence of A. rhizogenes will also tend to show high transgenic root production, thereby allowing the selection of lines that are more efficiently transformed.     

Transformation of <Emphasis Type="Italic">Saussurea medusa</Emphasis> for hairy roots and jaceosidin production

Axenically grown Saussurea medusa plantlets were inoculated with four Agrobacterium rhizogenes strains, and hairy root lines were established with A. rhizogenes strain R1601 in N6 medium. PCR and Southern hybridization confirmed integration of the T-DNA fragment of the Ri plasmid from A. rhizogenes into the genome of S. medusa hairy roots. In N6 medium, maximum biomass of the hairy root cultures was achieved [8 g (dry weight) per liter; growth ratio 35-fold] after 21 days of culture. The amount of jaceosidin extracted from the hairy root cultures was 46 mg/l (production ratio of 37-fold) after 27 days of culture. The maximum jaceosidin content obtained using N6 medium was higher than that obtained with Modified White, MS or B5 medium. In N6 medium, the tip segments were more efficient for hairy root growth and jaceosidin production than the middle and basal regions of the root.Abbreviations AS Acetosyringone - BA Benzyladenine - cef Cefotaxime sodium - DW Dry weight - FW Fresh weight - HPLC High-performance liquid chromatography - IAA Indole-3-acetic acid - km Kanamycin - NAA -Naphthaleneacetic acid - SDS Sodium dodecyl sulfate     

Agrobacterium rhizogenes-mediated DNA transfer inPinus halepensis Mill.

Agrobacterium rhizogenes strain LBA9402 was used to transformPinus halepensis embryos, seedlings and shoots. Mature embryos exhibited susceptibility to the agrobacterium as monitored by -glucurortidase (GUS) expression, with more than 85% showing considerable transient GUS expression in the radicle. GUS expression was also observed in cotyledons, but at a lower rate of about 24% of the embryos (1–5 spots/embryo). Stable transformation was evidenced by the regeneration of GUS-expressing roots and calli from infectedP. halepensis seedlings. Inoculum injections into intact seedling hypocotyls induced callus and root formation at the wound sites in 64% of the seedlings. Dipping seedling cuttings in a bacterial suspension resulted in adventitious root formation in 7I% of the seedling cuttings, all of which expressed GUS activity. Adventitious shoots, that were induced on 2.5-year-old seedlings by pruning and spraying with 6-benzylaminopurine, were infected by injecting of bacterial suspension into their basal side. Two months later, adventitious roots and root primordia regenerated in 74% and 40% of 2- and 5-month-old shoots, respectively. Non-transformed shoots, either without or with auxin application, failed to form roots. Polymerase chain reaction and Southern blot analyses confirmed theuidA-transgenic nature of the root and callus, as well as the presence ofrolC androlB genes in roots from infectedP. halepensis seedlings.Abbreviations BA 6-benzylaminopurine - NOS nopaline synthase - PCR polymerise chain reaction - EtOH ethanol - GUS -glucuronidase - NPTII neomycin phosphotransferase II - CaMV cauliflower mosaic virus - X-gluc 5-bromo-4-chloro-3-indolyl -D-glucuronic acid     

Unexpected behavior of coniferin in lignin biosynthesis of Ginkgo biloba L

To gain insight into the behavior of monolignol glucoside in Ginkgo biloba L., we examined glucosides potentially involved in lignin biosynthetic pathway. Coniferin (coniferyl alcohol 4O--D-glucoside) is a strong candidate for the storage form of monolignol. Coniferaldehyde glucoside may also have a role in lignin biosynthesis; this was examined with tracer experiments using labeled glucosides fed to stem segments. A series of tracer experiments showed that coniferin and coniferaldehyde glucoside were modified into coniferyl alcohol and then efficiently incorporated into lignin under the experimental conditions used. Interestingly, more than half of the administered coniferin underwent an oxidation to the aldehyde form before its aglycone; coniferyl alcohol was polymerized into lignin. This suggests that there is an alternative pathway for coniferin to enter the monolignol biosynthetic pathway, in addition to the direct pathway beginning with the deglucosylation of coniferin catalyzed by -glucosidase. Enzymatic assays revealed that coniferaldehyde glucoside was produced enzymatically from coniferin, and that coniferaldehyde glucoside can be deglucosylated to yield coniferaldehyde, which could be fated to become coniferyl alcohol . Albeit the findings cannot be taken as proof for the in-planta functioning, these results present a possibility for the existence of alternative pathway in which some of the stored coniferin is oxidized to coniferaldehyde glucoside, which is deglucosylated to generate coniferaldehyde that joins the monolignol biosynthesis pathway.     

Phenylpropanoid defence responses in transgenic Lotus corniculatus 1. Glutathione elicitation of isoflavan phytoalexins in transformed root cultures

Summary When Agrobacterium rhizogenes transformed root cultures of Lotus corniculatus were treated with glutathione, isoflavan phytoalexins accumulated in both tissue and culture medium. This accumulation of phytoalexins was preceded by a transient increase in the activity of phenylalanine ammonia lyase (PAL). Elicitation of PAL occurred throughout the growth curve of Lotus hairy roots and in different sectors of transformed root material.     

Degradation of coniferyl alcohol and other lignin-related aromatic compounds by Nocardia sp. DSM 1069

Nocardia sp. DSM 1069 was grown on mineral salt media with coniferyl alcohol, 4-methoxybenzoic acid, 3-methoxybenzoic acid or veratric acid as sole sources of carbon and energy. During incubation on coniferyl alcohol, the formation of coniferyl aldehyde, ferulic acid and quantitative accumulation of vanillic acid and proteocatechuic acid could be achieved with mutants. Washed cell suspensions of N. sp. grown on 4-methoxybenzoic acid, oxidized 4-hydroxybenzoic acid and protocatechuic acid. Cells grown on veratric acid, oxidized vanillic acid, isovanillic acid, and protocatechuic acid. Cell extracts were shown to cleave protocatechuic acid by ortho-fission.A mutant without protocatechuate 3,4-dioxygenase activity was not influenced in its growth on 3 methoxybenzoic acid. Cell free extracts of cells grown on 3-methoxybenzoic acid were shown to catalyze the oxidation of gentisic acid (2,5-dihydroxybenzoic acid). The resulting ring cleavage product was further metabolized by a glutathione dependent reaction.The specificity of the demethylation reactions has been investigated with a mutant unable to grow on vanillic acid. This mutant was not impaired in the degradation of isovanillic acid, 4-methoxy-, or 3-methoxybenzoic acid, whereas growth of this mutant on veratric acid (3,4-dimethoxybenzoic acid) was only half as much as that of the wild type. Concomitantly with growth on veratric acid this mutant accumulated vanillic acid with a yield of about 50%.A pathway for the catabolism of coniferyl alcohol, involving oxidation and shortening of the side chain, and of 4-methoxybenzoic acid and veratric acid with protocatechuic acid as intermediate is being proposed. A second one is proposed for the degradation of 3-methoxybenzoic acid with gentisic acid as intermediate.     

Enhanced production of antimicrobial sesquiterpenes and lipoxygenase metabolites in elicitor-treated hairy root cultures of Solanum tuberosum

Potato (Solanum tuberosum) hairy root cultures, established by infecting potato tuber discs with Agrobacterium rhizogenes, were used as a model system for the production of antimicrobial sesquiterpenes and lipoxygenase (LOX) metabolites. Of the four sesquiterpene phytoalexins (rishitin, lubimin, phytuberin and phytuberol) detected in elicitor-treated hairy root cultures, rishitin (213 g g^–1 dry wt) was the most predominant followed by lubimin (171 g g^–1 dry wt). The elicitors also induced LOX activity (25-fold increase) and LOX metabolites, mainly 9-hydroxyoctadecadienoic acid and 9-hydroxyoctadecatrienoic acid, in potato hairy root cultures. The combination of fungal elicitor plus cyclodextrin was the most effective elicitor treatment, followed by methyl jasmonate plus cyclodextrin in inducing sesquiterpenes and LOX metabolites.     

Increased podophyllotoxin production in Podophyllum hexandrum cell suspension cultures after feeding coniferyl alcohol as a β-cyclodextrin complex

Summary Cell suspension cultures, derived from roots of Podophyllum hexandrum Royle (Berberidaceae), accumulate podophyllotoxin. In this study the use of -cyclodextrin in feeding the poorly water-soluble precursor coniferyl alcohol to these cultures is described. By complexation with -cyclodextrin, a solution of 3 mM coniferyl alcohol could be fed, resulting in enhanced podophyllotoxin accumulation. The same concentration of non-complexed suspended coniferyl alcohol had only little effect on the podophyllotoxin accumulation. -Cyclodextrin itself was proven to be non-toxic for the cells. It did not influence the podophyllotoxin content and it was not metabolized or used as a carbon source by the cells. For comparison, coniferin, the water-soluble -D-glucoside of coniferyl alcohol, was also fed in the same concentration. The effect of coniferin on the podophyllotoxin accumulation was stronger than that of coniferyl alcohol complexed with -cyclodextrin, but coniferin is not commercially available.Abbreviations -CD -cyclodextrin - NAA naphthaleneacetic acid     

Indigo production in hairy root cultures of Polygonum tinctorium Lour.

The transformed root culture of Polygonum tinctorium Lour. was established by infecting leaf explants with Agrobacterium rhizogenes A4. These cultures were examined for their growth and indigo content under various culture conditions. Among the four different culture media tested, SH medium showed the highest yield for root growth (28 mg dry wt/30 ml) and indigo production (152 g/dry wt). In SH medium, 30 g sucrose l^–1, 2500 mg KNO₃ l^–1, 300 mg NH₄H₂PO₄ l^–1 were the best conditions for indigo production at pH 5.7. The production of indigo in hairy roots slightly increased with the addition of 200 mg chitosan l^–1 (186 g/dry wt) and 20 U pectinase l^–1 (181 g/dry wt).     

Isolation of the promoter of a root cap expressed pectinmethylesterase gene from Pisum sativum L. (rcpme1) and its use in the study of gene activity

A genomic clone of a pea pectinmethylesterase encoding gene, rcpme1, was isolated; the promoter region was found to include regions of homology to phenylalanine ammonia lyase (PAL) and nodulin gene promoters. Agrobacterium rhizogenes mediated hairy roots were used for rcpme1 expression and functional analysis in pea. Patterns of rcpme1 expression in cultured hairy roots, measured using uidA encoding -glucuronidase (GUS) as a reporter gene, were distinct from patterns which occur in normal pea roots. No reporter gene expression occurred in transgenic Arabidopsis thaliana, whose roots do not produce border cells. Border cell number from transgenic hairy roots expressing rcpme1 anti-sense mRNA under the control of its 2.75 kb 5 flanking sequence was reduced by > 50%. Nodulation genes of Rhizobium leguminosarum were used as a marker to document that roots with reduced production of border cells and other root cap exudates have a corresponding reduction in levels of biologically active signal molecules. Direct measurements were used to confirm that most of the exudate harvested from young, unwounded roots of normal pea plants is derived from the root tip region where rcpme1 is expressed. The potential application of the rcpme1 gene as a molecular marker for root exudate production is discussed.     

Assessment of the efficiency of cotransformation of the T-DNA of disarmed binary vectors derived from Agrobacterium tumefaciens and the T-DNA of A. rhizogenes

Co-transfer of Agrobacterium rhizogenes T-DNA and T-DNA from the A. tumefaciens binary vector pBin19 (Bevan, 1984) was studied in detail using Nicotiana rustica. High frequencies of co-transfer of T-DNA's were observed, even when no selection pressure was exerted. Increased levels of pBin19 T-DNA were found in hairy root cultures with selection at higher levels of kanamycin sulphate (50–200 g ml^–1). Several other species were also transformed by A. rhizogenes carrying pBin19 and A. rhizogenes harbouring a different binary factor, pAGS125 (Van den Elzen et al., 1985), was used to transform N. rustica hairy roots to confer hygromycin B resistance.     

Hairy root cultures of Anethum graveolens (dill): establishment, growth, time-course study of their essential oil and its comparison with parent plant oils

Transformed root cultures of Anethum graveolens were induced by inoculation of aseptically grown seedlings with Agrobacterium rhizogenes carrying plasmid pRi 1855. The main component of the essential oils from the fruits and from the roots of the parent plant was carvone, whereas -phellandrene and apiole were dominant in the oil from, respectively, the aerial parts and the hairy roots. The essential oils from the fruits, aerial parts and roots of the parent plant were at 2%, 0.3% and 0.06% (v/w), respectively, but only 0.02% (v/w) in the hairy root cultures. Growth of the hairy root cultures reached 600 mg dry wt/50 ml medium after 50 days. The essential oil composition did not change significantly during their growth.     

Microscopic studies of cell divisions induced in alfalfa roots by Rhizobium meliloti

We have used spot-inoculation and new cytological procedures to observe the earliest events stimulated in alfalfa (Medicago sativa L.) roots by Rhizobium meliloti. Roots were inoculated with 1–10 nl of concentrated bacteria, fixed in paraformaldehyde, and after embedding and sectioning stained with a combination of acridine orange and DAPI (4-6-diamidino-2-phenylindole hydrochloride). Normal R. meliloti provoke cell dedifferentiation and mitosis in the inner cortex of the root within 21–24 h after inoculation. This activation of root cells spreads progressively, leading to nodule formation. In contrast, the R. meliloti nodA and nodC mutants do not stimulate any activation or mitosis. Thus the primary and earliest effect of Rhizobium nod gene action is plant cellular activation. A rapid, whole-mount visualization by lactic acid shows that the pattern of nodule form varies widely. Some R. meliloti strains were found to be capable of stimulating on alfalfa roots both normal nodules and a hybrid structure intermediate between a nodule and a lateral root.     

Genetic transformation of <Emphasis Type="Italic">Gentiana macrophylla</Emphasis> with <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>: growth and production of secoiridoid glucoside gentiopicroside in transformed hairy root cultures

Hairy root cultures of Gentiana macrophylla were established by infecting the different explants four Agrobacterium rhizogenes strains namely A₄GUS, R1000, LBA 9402 and ATCC11325, and hairy root lines were established with A. rhizogenes strain R1000 in 1/2 MS + B₅ medium. Initially, 42 independent hairy root clones were maintained and seven clones belongs to different category were evaluated for growth, morphology, integration and expression of Ri T-DNA genes, and alkaloid contents in dry root samples. On the basis of total root elongation, lateral root density and biomass accumulation on solid media, hairy root clones were separated into three categories. PCR and Southern hybridization analysis revealed both left and right T-DNA integration in the root clones and RT-PCR analysis confirmed the expression of hairy root inducible gene. GUS assay was also performed to confirm the integration of left T-DNA. The accumulation of considerable amounts of the root-specific secoiridoid glucosides gentiopicroside was observed in GM1 ( and ) and the GM2 ( and DNA) type clones in considerably higher amount whether as two but callus-type clones (GM3) accumulated much less or only very negligible amounts of gentiopicroside. Out of four media composition the 1/2 MS + B₅ vitamin media was found most suitable. We found that initial establishment of root cultures largely depends on root:media ratio. Maximum growth rate was recorded in 1:50 root:media ratio. The maximum biomass in terms of fresh weight (33-fold) was achieved in 1/2 MS + B₅ media composition after 35 days in comparison to sixfold increase in control. The biomass increase was most abundant maximum from 15 to 30 days. Influence of A. rhizogenes strains and Ri plasmid of hairy root induction, the possible role of the T_L-DNA and T_R-DNA genes on growth pattern of hairy root, initial root inoculum:media ratio and effect of media composition is discussed.     

Efficient transformation of potato (Solanum tuberosum L.) using a binary vector in Agrobacterium rhizogenes

Summary We transformed three potato (Solanum tuberosum L.) genotypes by using A. rhizogenes or a mixture of A. rhizogenes and A. tumefaciens. Inoculations of potato stem segments were performed with Agrobacterium rhizogenes AM8703 containing two independent plasmids: the wild-type Ri-plasmid, pRI1855, and the binary vector plasmid, pBI121. In mixed inoculation experiments, Agrobacterium rhizogenes LBA1334 (pRI1855) and Agrobacterium tumefaciens AM8706 containing the disarmed Ti-plasmid (pAL4404) and the binary vector plasmid (pBI121) were mixed in a 11 ratio. The T-DNA of the binary vector plasmid pBI121 contained two marker genes encoding neomycin phosphotransferase, which confers resistance to kanamycin, and -glucuronidase. Both transformation procedures gave rise to hairy roots on potato stem segments within 2 weeks. With both procedures it was possible to obtain transformed hairy roots, able to grow on kanamycin and possessing -glucuronidase activity, without selection pressure. The efficiency of the A. rhizogenes AM8703 transformation, however, was much higher than that of the mixed transformation. Up to 60% of the hairy roots resulting from the former transformation method were kanamycin resistant and possessed -glucuronidase activity. There was no correlation between the height of the kanamycin resistance and that of the -glucuronidase activity in a root clone. Hairy roots obtained from a diploid potato genotype turned out to be diploid in 80% of the cases. Transformed potato plants were recovered from Agrobacterium rhizogenes AM8703-induced hairy roots.     

Appearance and immunohistochemical localization of UDP-glucose: coniferyl alcohol glucosyltransferase in spruce (Picea abies (L.) Karst.) seedlings

UDP-glucose: coniferyl alcohol glucosyltransferase was detected in hypocotyls of spruce (Picea abies (L.) Karst.) seedlings. Enzyme activity rose after germination to maximal activity on about the 10th day and then rapidly declined. An antiserum against the glucosyltransferase isolated from cambial sap of spruce (Schmid and Grisebach, 1982, Eur. J. Biochem. 123, 363–370) was employed for localization of the enzyme in cross sections of hypocotyls from 10-d-old spruce seedlings, using the immunofluorescent technique. The results show that the transferase is located predominantly in the epidermal and subepidermal layers and in the vascular bundles. Intracellularly, the enzyme is located in the parietal cytoplasmic layer. The results corroborate the assumption that coniferin (coniferyl alcohol -D-glucoside) participates in lignification of spruce.     

Hairy root cultures of Taxus × media var. Hicksii Rehd. as a new source of paclitaxel and 10-deacetylbaccatin III

Hairy root cultures of Taxus × media var. Hicksii were established by infection of the plantlets with the Agrobacterium rhizogenes strain LBA 9402. The paclitaxel accumulation in hairy root cultures increased after 100 M methyl jasmonate treatment from 69 to 210 g g^–1 dry wt while the 10-deacetybaccatin III content was not affected by the elicitor.     

Characterization of human S-adenosyl-homocysteine hydrolase in vitro and identification of its potential inhibitors

Human S-adenosyl-homocysteine hydrolase (SAHH, E.C.3.3.1.1) has been considered to be an attractive target for the design of medicines to treat human disease, because of its important role in regulating biological methylation reactions to catalyse the reversible hydrolysis of S-adenosylhomocysteine (SAH) to adenosine (Ado) and l-homocysteine (Hcy). In this study, SAHH protein was successfully cloned and purified with optimized, Pichia pastoris (P. pastoris) expression system. The biological activity results revealed that, among the tested compounds screened by ChemMapper and SciFinder Scholar, 4-(3-hydroxyprop-1-en-1-yl)-2-methoxyphenol (coniferyl alcohol, CAS: 458-35-5, ZINC: 12359045) exhibited the highest inhibition against rSAHH (IC₅₀=?34?nM). Molecular docking studies showed that coniferyl alcohol was well docked into the active cavity of SAHH. And several H-bonds formed between them, which stabilized coniferyl alcohol in the active site of rSAHH with a proper conformation.     

Influence of phosphate and nitrate supply on root hair formation of rape,spinach and tomato plants

Summary Experiments with tomato, rape and spinach in nutrient solutions have shown that the formation of root hairs is strongly influenced by phosphate and nitrate supply. Decreasing the phosphate concentration of the nutrient solution from 100 to 2 M P resulted in an increase of root hair length from 0.1–0.2 to 0.7 mm of the three plant species. Root hair density also increased by a factor of 2–4 when the P concentration was lowered from 1000 to 2 M. The variation of these two root properties raised the root surface area by a factor of 2 or 3 compared to plants well supplied with P. Root hair length was closely related to the phosphate content of the root and shoot material. On the other hand, spinach plants grown in a split-root experiment produced root hairs in solutions of high P concentration (1000M P) if the major part of the total root system was exposed to low P concentration (2 M P). It is therefore concluded that the formation of root hairs does not depend on directly the P concentration at the root surface but on the P content of the plant.Similar experiments with nitrate also resulted in an increase in length and density of root hairs with the decrease of concentration below 1000 M. In this case marked differences between plant species occurred. At 2 M compared to 1000 M NO₃ root hair length of tomato increased by a factor of 2, of rape by a factor of 5 and of spinach by a factor of 9. Root hair length was correlated, but not very closely, to the total nitrogen content of the plants. It is concluded, that the influence of nutrient supply on the formation of root hairs is a mechanism for regulating the nutrient uptake of plants.Dedicated to Prof. Dr. E. Welte on the occasion of his 70th anniversary.