Jerzy Zoń

Ferulate cross-linking in cell walls isolated from maize cell suspensions

Abstract Cross-linking of arabinoxylans by ferulate dehydrodimers and incorporation of feruloylated arabinoxylans into lignin were modelled with maize walls (Zea mays cv Black Mexican) containing 5.3–18.0 mg g−1 of total ferulates. The proportion of dehydrodimers to total ferulates increased from ca 20 to 45% when dilute hydrogen peroxide was added to walls containing bound peroxidase. About 45% of the dehydrodimers were coupled by 8-5 linkages, with 8-8, 8-O-4 and 5-5 coupled dehydrodimers each comprising 10–25% of the total. The quantity of ferulates released by saponification were reduced by 83–95% when exogenously supplied hydroxycinnamyl alcohols were polymerized into walls by wall-bound peroxidase and in situ generated hydrogen peroxide. Only 40% of the ferulates incorporated into lignin were recovered following hydrolysis of ether linkages. These results indicate that primary walls in grasses become extensively cross-linked by ferulic and dehydrodiferulic acids during lignification, and that only a portion of ferulates in lignified tissues are measurable by current solvolytic methods.

Phenylalanine Ammonia‐Lyase: The Use of Its Broad Substrate Specificity for Mechanistic Investigations and Biocatalysis—Synthesis of L‐Arylalanines

Several fluoro- and chlorophenylalanines were found to be good substrates of phenylalanine ammonia-lyase (PAL/EC 4.3.1.5) from parsley. The enantiomerically pure L-amino acids were obtained in good yields by reaction of the corresponding cinnamic acids with 5M ammonia solution (buffered to pH 10) in the presence of PAL. The kinetic constants for nine different fluoro- and chlorophenylalanines do not provide a rigorous proof for but are consistent with the previously proposed mechanism comprising an electrophilic attack of the methylidene-imidazolone cofactor of PAL at the aromatic nucleus as a first chemical step. In the resulting Friedel-Crafts-type sigma complex the beta-protons are activated for abstraction and consequently the pro-S is abstracted by an enzymic base. Results from semi-empirical calculations combined with a proposed partial active site model showed a correlation between the experimental kinetic constants and the change in polarization of the pro-S Cbeta-H bond and heat of formation of the sigma complexes, thus making the electrophilic attack at the neutral aromatic ring plausible. Furthermore, while 5-pyrimidinylalanine was found to be a moderately good substrate of PAL, 2-pyrimidinylalanine was an inhibitor.

Significance of phenols in cadmium and nickel uptake

The effects of 2-aminoindane-2-phosphonic acid (AIP), a potent phenylalanine ammonia-lyase (PAL) inhibitor, on the accumulation of cadmium and nickel in chamomile (Matricaria chamomilla) were examined in this study. In vitro assay of AIP effect showed a 90% reduction in PAL activity. In plants cultured for 7 days in Cd or Ni solutions with AIP, PAL activity was higher in both shoots and roots (in comparison with metals without AIP), and was correlated with changes in free phenylalanine content. Individual amino acids were both positively and negatively affected by AIP, with the accumulation of tyrosine and proline showing increases in some variants. Contents of soluble phenols and flavonoids were not considerably affected, while amounts of coumarin-related compounds, cell wall-bound phenols and phenolic acids were substantially reduced in AIP-treated variants. Lignin accumulation decreased in controls and increased in Cd variants in response to AIP. Shoot Cd content was depleted, but shoot Ni was elevated by AIP. Total root content of Cd and Ni decreased in +AIP variants. AIP also caused more expressive changes in hydrogen peroxide and superoxide content in Cd than in Ni variants. Our results indicate that phenols have important roles in the uptake of Cd and Ni. The present findings are discussed in the context of available data regarding AIPs effect on phenols.

Naphthalene-based linkers for metal phosphonates: synthesis, structure, and interesting conformational flexibility influence on final lanthanum hybrids

Two diphosphonic acids based on naphthalene scaffold, namely naphthalene-1,4-diphosphonic acid, C10H6(PO3H2)2 [1, H4NDP(1,4)] and 1-(phosphono)naphthalene-4-(methylphosphonic) acid, C10H6(PO3H2)(CH2PO3H2) [2, H4NDP(1,4C)] were obtained. Their supramolecular self-assembly were studied as well as solvent effect for the second one (2a, 2b). These potential building blocks in coordination polymer chemistry were engaged with a La3+ ion in a room temperature reaction to produce new single crystalline metal phosphonates: (I) [La{C10H6(PO3H)2}{C10H6(PO3H)(PO3H2)}(H2O)2]n·nH2O (3), a 2D coordination polymer with the I1O1 structure and {42.6}2{44.64.82} topology of the layer, and (II) [La{C10H6(PO3H1.5)(CH2PO3H)}2(H2O)7]·2H2O (4), a discrete complex. These drastic differences in hybrid material structures can be explained by ligand structures analysis including their conformational flexibility.

Effect of inhibition of biosynthesis of phenylpropanoids on sessile oak somatic embryogenesis

Abstract The inhibition of phenylpropanoid biosynthesis by 2-aminoindan-2-phosphonic acid (AIP) in embryogenic culture of sessile oak (Quercus petraea (Matt.) Liebl.) was associated with a strong reduction of the amount of cinnamic acid derivatives, decline in lignin content and changes in the proportion of different types of somatic embryos. The decrease in the level of phenylpropanoid lignin precursors after AIP application decreased the amount of embryos with deep green cotyledons that are in control cultures characterized by high content of lignin. Their proportion in the total yield represented 30% and 10% in the control and AIP media, respectively. On the contrary, the reduced endogenous pool of cinnamic acids stimulated the cell division activity and the formation of early stages of somatic embryos. Globular, heart- and torpedo-shaped stages of somatic embryos represented in AIP-treated culture about 25% from the total yield of embryos, while only 10% in the control culture. Higher amounts of free putrescine (Put), spermidine and spermine and significantly lower levels of their conjugated forms were characteristic for embryos cultured for 7 weeks on AIP medium (related to controls). Significant increase in Put contents in cotyledonary embryos after 7-week culture with AIP might reflect the stress caused by lowered levels of phenylpropanoids in treated cells.

The cleavage of 1-amino-2′-nitrobenzylphosphonates in a basic medium. Formation of the 3-amino-2,1-benzisoxazole derivatives

Abstract Treatment of 1-amino-2′-nitrobenzylphosphonic acids with aqueous sodium hydroxide caused a C-P bond cleavage, with formation of 3-amino-2,1-benzisoxazole derivatives ( 3 ). The leaving phosphorus moiety was identified here as phosphoric acid. In the case of basic hydrolysis of corresponding esters, new cyclic phosphorus compounds (derivatives of benzoxazaphosphorin-3,1,2 P v -one-2) were obtained. The cyclic products were formed as a result of the subsequent reaction of anthranil derivatives with leaving phosphorus fragment, presumably metaphosphate. These benzoxazaphosphorins (compounds 4 ) were converted by means of aqueous hydrochloric acid to 3-amino-2,1-benzisoxazole derivatives.

Experimental and ab initio calculated structures of 2-aminoindane-2-phosphonic acid, a potent inhibitor of phenylalanine ammonia-lyase, and theoretical studies of its binding to the model enzyme structure

The structure of 2-aminoindane-2-phosphonic acid (AIP) was studied using X-ray crystallography, NMR spectroscopy, molecular modelling, IR spectroscopy, and potentiometric titration. In the solid state, a thermodynamically stable conformer of AIP is one in which the phosphonic group occupies the equatorial position and the amino group the axial position. The NMR data suggest that fast equilibrium in solution between the two conformers of AIP is significantly shifted toward the equatorial conformer (EC). Both solid state studies, that is X-ray analysis and IR spectroscopy of AIP, revealed the presence of hydrogen-bonded water. Ab initio calculations in the gas phase indicate only a small barrier between the two possible conformations of AIP. Binding studies of both conformers, in various protonation states, to the model of the phenylalanine ammonia-lyase structure suggest that only the axial phosphonic group conformer is docked specifically. Indications from modelling are that phenylalanine ammonia-lyase binds AIPs conformer with higher specificity and that the molecular reorganisation required can be responsible for the experimentally observed time-dependent inhibition.

Inhibition of phenylpropanoid biosynthesis increases cell wall digestibility, protoplast isolation, and facilitates sustained cell division in American elm (Ulmus americana)

BackgroundProtoplast technologies offer unique opportunities for fundamental research and to develop novel germplasm through somatic hybridization, organelle transfer, protoclonal variation, and direct insertion of DNA. Applying protoplast technologies to develop Dutch elm disease resistant American elms (Ulmus americana L.) was proposed over 30 years ago, but has not been achieved. A primary factor restricting protoplast technology to American elm is the resistance of the cell walls to enzymatic degradation and a long lag phase prior to cell wall re-synthesis and cell division.ResultsThis study suggests that resistance to enzymatic degradation in American elm was due to water soluble phenylpropanoids. Incubating tobacco (Nicotiana tabacum L.) leaf tissue, an easily digestible species, in aqueous elm extract inhibits cell wall digestion in a dose dependent manner. This can be mimicked by p-coumaric or ferulic acid, phenylpropanoids known to re-enforce cell walls. Culturing American elm tissue in the presence of 2-aminoindane-2-phosphonic acid (AIP; 10-150 μM), an inhibitor of phenylalanine ammonia lyase (PAL), reduced flavonoid content, decreased tissue browning, and increased isolation rates significantly from 11.8% (±3.27) in controls to 65.3% (±4.60). Protoplasts isolated from callus grown in 100 μM AIP developed cell walls by day 2, had a division rate of 28.5% (±3.59) by day 6, and proliferated into callus by day 14. Heterokaryons were successfully produced using electrofusion and fused protoplasts remained viable when embedded in agarose.ConclusionsThis study describes a novel approach of modifying phenylpropanoid biosynthesis to facilitate efficient protoplast isolation which has historically been problematic for American elm. This isolation system has facilitated recovery of viable protoplasts capable of rapid cell wall re-synthesis and sustained cell division to form callus. Further, isolated protoplasts survived electrofusion and viable heterokaryons were produced. Together, these results provide the first evidence of sustained cell division, callus regeneration, and potential application of somatic cell fusion in American elm, suggesting that this source of protoplasts may be ideal for genetic manipulation of this species. The technological advance made with American elm in this study has potential implications in other woody species for fundamental and applied research which require availability of viable protoplasts.

Copper Uptake Is Differentially Modulated by Phenylalanine Ammonia-lyase Inhibition in Diploid and Tetraploid Chamomile

The effect of phenylalanine ammonia-lyase (PAL) inhibition by 2-aminoindane-2-phosphonic acid (AIP) in copper-exposed diploid and tetraploid chamomile (Matricaria chamomilla ) roots has been studied in a short-term experiment (24 h). Cu evoked stronger induction of PAL activity and accumulation of soluble phenols, flavonols (quercetin and kaempferol), and lignin in diploid plants, whereas AlCl3-reactive flavonoids and phenolic acids did not differ with respect to ploidy. Amounts of hydrogen peroxide and superoxide also preferentially increased in diploid. Surprisingly, PAL activity was restored in both +AIP cultivars, being inversely correlated with the accumulation of free phenylalanine. Notwithstanding this, total soluble phenols and flavonols were more depleted in Cu+AIP diploid roots. Soluble proteins decreased in response to Cu, and AIP had no effect. Among free amino acids, proline increased more visibly in Cu+AIP diploid, suggesting that this could be a protective mechanism in conditions with depleted content of phenols. Decrease in potassium content was ploidy-independent, calcium increased in all Cu variants, and Fe increased in Cu-exposed tetraploid. Shoot Cu content did not differ in Cu-exposed cultivars, but diploid roots contained more Cu. AIP decreased root Cu but increased shoot Cu amounts in diploid, whereas tetraploid plants did not exhibit similar responses. These data indicate that inhibition of root phenolic metabolism by AIP was effective enough, allowing Cu to accumulate in diploid shoots. The present findings are discussed in the context of available data about AIP effects and with respect to the role of phenols in metal uptake.

Beyond Single-Wavelength SHG Measurements: Spectrally-Resolved SHG Studies of Tetraphosphonate Ester Coordination Polymers

Powder second-harmonic generation (SHG) efficiencies are usually measured at single wavelengths. In the present work, we provide a proof of concept of spectrally resolved powder SHG measured for a newly obtained series of three non-centrosymmetric coordination polymers (CPs). CPs are constructed from tetrahedral linker-tetraphenylmethane-based tetraphosphonate octaethyl ester and cobalt(II) ions of mixed, octahedral (Oh), and tetrahedral (Td), geometries and different sets of donors (CoO6 vs CoX3O). Isostructurality of the obtained materials allowed for the determination of anion-dependent tunability of SHG optical spectra and their relationship with solid-state absorption spectra.