Stoichiometry of the topa quinone biogenesis reaction in copper amine oxidases

The stoichiometry of the topa quinone biogenesis reaction in phenylethylamine oxidase from Arthrobacter globiformis (AGAO) has been determined. We have shown that the 6e- oxidation of tyrosine to topa quinone (TPQ) consumes 2 mol of O2 and produces 1 mol of H2O2/mol of TPQ formed. The rate of H2O2 production is first-order (kobs = 1.0 +/- 0.2 min-1), a rate only slightly lower than the rate of TPQ formation directly determined previously (kobs = 1.5 +/- 0.2 min-1). This gives the following net reaction stoichiometry for TPQ biogenesis: E-Tyr + 2O2 --> E-TPQ + H2O2. This stoichiometry is in agreement with recently proposed mechanisms for TPQ biogenesis, and rules out several possible alternatives.     

The role of copper in bovine serum amine oxidase

Summary The role of copper in bovine serum amine oxidase was investigated by studying the effect of copper-binding inhibitors on the reactions of the pyrroloquinoline quinone carbonyl and on the reaction with oxygen. Hydrazines and hydrazides were used as carbonyl reagents and one of the hydrazines, benzylhydrazine, which was found to behave as a pseudo-substrate, was used to probe the reaction with oxygen. The presence ofN,N-diethyldithiocarbamate, a chelator that binds copper irreversibly, did not prevent the reactions at the carbonyl, but slowed down their rate and modified the conformation of the adducts. The same happened to the reaction with oxygen, which was slowed down but not abolished. Copper, which was never seen in the reduced state, thus appears to control all reactions without being directly involved in the binding of either hydrazines or oxygen. The enzyme functionality was in fact preserved upon substitution of copper with cobalt. The specific activity of the cobalt-substituted enzyme was only reduced to about 40% the native amine oxidase value. This is the first case so far in which the role of copper can be performed by a different metal ion.Abbreviations BSAO bovine serum amine oxidase - DDC N,N-diethyldithiocarbamate - PQQ pyrroloquinoline quinone     

Role of kinetin in alleviation of copper and zinc toxicity in Lupinus termis plants

The effect of exogenous kinetin application on the growth and some physiological processes of Lupinus termis plants growing in metal containing solutions with excess concentrations of Cu and Zn ion were studied. Generally, plants growing in these solutions had a lower chlorophyll (Chl.) content, leaf relative water content (RWC) and produced less biomass than the control plants. Proline content was higher in metal-treated plants than in untreated controls. Chromatography of cell-free-extracts of roots and shoots indicated three main protein peaks with molecular weights about 170, 75--70 and 5--45 kDa. These peaks were coincident with Cu or Zn maxima. Addition of kinetin reduced the decline in Chl. content in metal-treated plants, improved water status of the plants and enhanced growth of the shoots and roots. The Cu or Zn content expressed on a per mg protein basis was raised when kinetin was applied to the growing shoots. Kinetin (Kin), Cu and Zn, singly and in the presence of kinetin (Cu × Kin and Zn × Kin), significantly affected the parameters tested. Only the effects of Cu × Kin and Zn × Kin interactions on shoot fresh weight and Cu × Kin on root length were statistically insignificant. Based on the calculated coefficient of determination ( ²) the roles of Cu and Zn in affecting Chl. content and growth were dominant in comparison to kinetin. Kinetin effect was dominant for root length and proline content, but the role of the interaction was subdominant. The results of this study indicate that kinetin can alleviate the harmful effects of Cu and Zn on the growth of lupin plants through stimulation of Cu and Zn incorporation into metal-binding proteins.     

Mechanism of post-translational quinone formation in copper amine oxidases and its relationship to the catalytic turnover

Copper amine oxidases (CAOs) post-translationally construct a redox-active quinone from an amino acid side chain in their polypeptide chain. As such, these enzymes illustrate how nature is able to expand upon naturally-occurring side chains to create new, catalytically powerful functionalities. The active sites of the CAOs are highly unusual in their ability to catalyze two very different reactions: single-turnover, oxygen-dependent quinone formation, followed by catalytic oxidation (formally dehydrogenation) of amines. This review summarizes our current understanding of the pathway whereby the 2,4,5-trihydroxyphenylalanyl quinone (TPQ) cofactor is generated from the phenolic side chain of tyrosine. This reaction occurs spontaneously intermediates in the presence of O(2) and active site bound Cu(II), without the assistance of other proteins or cofactors. Ongoing work has focused on uncovering the details of the TPQ formation mechanism. A larger goal is to understand how a single active site is capable of supporting both quinone formation and subsequent catalytic turnover.     

Interaction between Orobanche crenata and its host legumes: unsuccessful haustorial penetration and necrosis of the developing parasite

BACKGROUND AND AIMS: Orobanche species represent major constraints to crop production in many parts of the world as they reduce yield and alter root/shoot allometry. Although much is known about the histology and effect of Orobanche spp. on susceptible hosts, less is known about the basis of host resistance to these parasites. In this work, histological aspects related to the resistance of some legumes to Orobanche crenata have been investigated in order to determine which types of resistance responses are involved in the unsuccessful penetration of O. crenata. METHODS: Samples of resistance reactions against O. crenata on different genotypes of resistant legumes were collected. The samples were fixed, sectioned and stained using different procedures. Sections were observed using a transmission light microscope and by epi-fluorescence. KEY RESULTS: Lignification of endodermal and pericycle host cells seems to prevent parasite intrusion into the root vascular cylinder at early infection stages. But in other cases, established tubercles became necrotic and died. Contrary to some previous studies, it was found that darkening at the infection site in these latter cases does not correspond to death of host tissues, but to the secretion of substances that fill the apoplast in the host-parasite interface and in much of the infected host tissues. The secretions block neighbouring host vessels. This may interfere with the nutrient flux between host and parasite, and may lead to necrosis and death of the developing parasite. CONCLUSIONS: The unsuccessful penetration of O. crenata seedlings into legume roots cannot be attributed to cell death in the host. It seems to be associated with lignification of host endodermis and pericycle cells at the penetration site. The accumulation of secretions at the infection site, may lead to the activation of xylem occlusion, another defence mechanism, which may cause further necrosis of established tubercles.     

Structure of root nodules and nitrogen fixation in Egyptian wild herb legumes

Five wild herb legumes (Trifolium resupinatum, Melilotus indica, Medicago intertexta, Trigonella hamosa, and Alhagi murarum) were collected from cultivated lands of the Nile Valley, and compared with clover (Trifolium alexandrinum), a cultivated forage legume. The wild herb legumes exhibited great variation in nodulation percentage, nodule number, nodule mass and acetylene reduction activity with regard to locality. Nodulation of T. resupinatum and M. indica ranged between 50 - 100% and 33 - 100%, respectively, compared to 50 - 100% for T. alexandrinum. The number of nodules formed on T. resupinatum was 9 - 128 and that of M. indica 6 - 39, compared to 13 - 122 nodule per plant for T. alexandrinum. Nodule mass was correlated with nodule number. In M. indica, a small number of nodules was compensated with high specific nitrogenase activity. The herb legumes formed nodules of small size, varying shape (globose, cylindrical, branched, etc.), and of different types (crotalaroid and astragaloid). Microscopic examination of root-nodules from T. resupinatum, M. indica and M. intertexta, showed that these legumes formed indeterminate and effective nodules, containing apical meristems, central symbiotic tissue with characteristic zonation and peripheral vascular bundles. The nodules harboured bacteroids with pleiomorphic morphology. This revised version was published online in July 2006 with corrections to the Cover Date.     

New perspectives on the role of amine oxidases in physiopathology

Summary.  In the paper here presented we summarize some results obtained in our laboratory in the last few years on new structural and functional aspects of some amine oxidases (AOs), which have to be taken into consideration in defining new strategies of controlling the cellular physiopathology. In particular, the ability of Cu-AO purified from vegetal sources or from bovine serum to bind different cellular targets inducing in them conformational as well as chemical modifications are described and the consequences of this interaction on cellular functions are discussed. This is the case of the protective effect of Cu-AO against the damage induced by free radicals, cell enrichment with Cu-AO, induction of cataract and the leukocyte-endothelia interaction. The role of Cu and FAD-amine oxidases related as to the protection or damage of cells is also discussed. In this context the involvement of MAOs in the modulation of the mitochondrial functions and in the induction of apoptosis is described and some aspects of the molecular mechanism of AO inhibition by H₂O₂ and metronidazole analyzed. Received January 9, 2002 Accepted June 27, 2002 Published online November 14, 2002 Acknowledgements This paper was supported by C.N.R. grant n° G002FD1 and MURST grant (Giovani Ricercatori, 2000). Authors' address: Prof. Bruno Mondovì, Department of Biochemical Science, University of Rome “La Sapienza”, P.le Aldo Moro 5, I-00185 Rome, Italy     

Determination of 4-chloroindoleacetic acid methyl ester in Vicieae species by gas chromatography-mass spectrometry

The natural chlorinated auxin, 4-chloroindoleacetic acid methyl ester, was identified in immature seeds of Lathyrus sativus L., Lathyrus maritimus (L.) Bigel and Lathyrus odoratus L. by thin layer chromatography and gas chromatography-mass spectrometry. In immature seeds of Vicia sativa L. and Lens culinaris Medik. the hormone was identified by selected ion monitoring. The hormone was determined quantitatively using pentadeuterated 4-chloroindoleacetic acid methyl ester as internal standard. Contents varied from 1 mg/kg fresh weight in Lathyrus sativus to 0.02 mg/kg in Lens culinaris. Lathyrus maritimus also contained indoleacetic acid methyl ester (0.3 mg/kg) besides the chlorinated analogue.     

Expression and occurrence of uracil-DNA glycosylase in higher plants

Uracil-DNA glycosylase (UDG) is the first enzyme in the base excision repair pathway for removal of uracil in DNA. DNA repair capacity is likely to be a critical factor in mutagenesis and thereby in the capacity to prevent genetic damage and unwanted variation. We have studied expression of UDG in 9 higher plant species. The highest expression of UDG was measured in Solanum tuberosum. A comparison of 6 Solanum tuberosum cultivars showed that the specific activity ranged from 30 pmol mg¹ protein min^?1 in the cultivar Laila to 80 pmol mg^?1 protein min^?1 in the cultivar Ostara. Measurement of UDG in Begonia X cheimantha gave no indications of enzyme activity. The possible effects of no or low UDG activity is discussed. In vitro cultures of Solanum tuberosum and Thymus vulgaris were used to examine the effect of auxin and cytokinin on the UDG activity. Axillary shoots of Solanum tuberosum were cultured on medium including 20 variations in hormone concentration. Auxin (1-naphtaleneacetic acid) increased the expression of UDG. Plants cultured on medium supplemented with 3 mg 1^?1 1-naphtaleneacetic acid showed a specific UDG activity which was approximately 3-fold higher than the activity in controls. The cytokinin benzyladenine reduced the specific UDG activity at concentrations in the range 0.25–10 mg 1^?1. In vitro cultured Saintpaulia ionantha was used to examine UDG activity during initiation, conditioning and multiplication cycles. In general, highest expression of UDG was measured in the conditioning cycle on hormone free medium. Measurement of UDG expression during single subculture periods, clearly showed that UDG expression may vary over one culture period. Expression of UDG was in general highest three weeks after transfer to fresh medium. Of different seedling organs from 0- to 15-day-old Brassica napus L., roots and hypocotyls showed the highest UDG activities. In cotyledons a very low and nearly constant specific activity was observed. In 12-day-old seedlings the activity in roots was approximately 20 times higher than the activity in cotyledons.     

Influence of genes determining supernodulation on root colonization by the mycorrhizal fungus Glomus mosseae in Pisum sativum and Medicago truncatula mutants

Pisum sativum (pea) mutants of the wild type cv. Frisson and six supernodulating Medicago truncatula mutants of the wild-type cv. Jemalong line J5 for their ability to form endomycorrhizas. The six mutants of M. truncatula were shown to be allelic mutants of the same gene Mtsym12, whereas distinct genes (sym28 and sym29) are known to determine the supernodulation character of the P64 and P88 pea mutants, respectively. Mutant P88 of pea and the majority of the M. truncatula mutants were significantly more colonized by the mycorrhizal fungus Glomus mosseae than their corresponding wild types, 4 weeks and 30 days after inoculation, respectively. These differences were expressed essentially in transversal intensity rather than in length intensity of root colonization and appeared to correspond to an increase in arbuscule formation. Results are discussed in relation to the mutated genes and, in particular, whether the observed effects are due indirectly to plant physiological modifications or are a direct result of possible common factors of regulation of nodulation and mycorrhizal development. Accepted: 9 February 2000     

Limited distribution of natural cyanamide in higher plants: occurrence in Vicia villosa subsp. varia, V. cracca, and Robinia pseudo-acacia

Cyanamide (NH2CN) has recently been proven to be a natural product, although it has been synthesized for over 100 years for agricultural and industrial purposes. The distribution of natural cyanamide appears to be limited, as indicated by our previous investigation of 101 weed species. In the present study, to investigate the distribution of natural cyanamide in Vicia species, we monitored the cyanamide contents in V. villosa subsp. varia, V. cracca, and V. amoena during their pre-flowering and flowering seasons. It was confirmed that V. cracca was superior to V. villosa subsp. varia in accumulating natural cyanamide, and that V. amoena was unable to biosynthesize this compound under laboratory condition examined. The localization of cyanamide in the leaves of V. villosa subsp. varia seedlings was also clarified. In a screening study to find cyanamide-biosynthesizing plants, only Robinia pseudo-acacia was found to contain cyanamide among 452 species of higher plants. We have investigated 553 species to date, but have so far found the ability to biosynthesize cyanamide in only three species, V. villosa subsp. varia, V. cracca and R. pseudo-acacia.     

Effects of artificial defoliation on reproductive allocation in the common vetch,Vicia sativa (Fabaceae: Papilionoideae)

Previous field experiments have shown that, for the annual herb Vicia sativa, leaf area lost to herbivory results in reduced number of fruit (pods) produced per plant as well as fewer seeds per pod. We conducted a controlled garden experiment to determine the precise relationship between level of defoliation and various measures of maternal fitness through fruit and seed. We employed manual clipping of individual leaflets of newly produced leaves to 25% increments of damage (from 0% to 100%) over the entire period of development and flowering of these annual herbs, harvesting pods when filled but not dehisced. We found significant reduction in number of pods, number of seeds, total seed mass, and individual seed mass with leaf area lost. Even with the highest levels of defoliation over the life of the individual, plants still produced a substantial number of pods and seeds. Seeds produced by plants in all treatment groups showed similar percentages of germination and time to germination.     

Subcellular aspects of the protective effect of spermine against atrazine in pea plants

The action of the exogenously applied tetraamine spermine in reversing the effect of atrazine stress on intact pea plants (Pisum sativum L., cv. Koray) was investigated at the ultrastructural level. The results indicate that atrazine increases cell senescence by lipid peroxidation and loss of unsaturated fatty acids from thylakoid membranes of pea plant chloroplasts. Spermine acts as a polyfunctional effector. It stabilises the molecular composition of the membranes by preventing lipid peroxidation and as a consequence protects the conformation of the thylakoid system and structural integrity of the chloroplasts. Spermine treatment also contributes to the process of neutralisation of the free radicals by peroxisomes.     

Regulation of assimilation and senescence by the fruit in monocarpic plants

Intercellular acidic isoperoxidases (EC 1.11.1.7) isolated from exponentially growing lupin ( Lupinus albus . L. cv. multolupa) hypocotyls are under the control of exogenously applied auxins. Application of auxins leads to a short-term reduction in the level of free intercellular peroxidases, and this effect is associated with a binding of these free peroxidases to the cell walls, probably mediated by an acidification of the cell wall. The ratio of free intercellular peroxidases to the total intercellular peroxidase activity, varies along the axis of exponentially growing hypocotyls. It has a V-shaped distribution with the minimum value in the elongation III-zone, where high levels of auxins have previously been implied in differentiation. This minimum value coincides spatially with the first signs of cell wall thickening in the hypocotyl cells and, paradoxically, it is out of phase with respect to the maximal cell elongation. On the other hand, the ratio of free intercellular peroxidases reaches its maximal values in both the most undiffercntiated phloem cells and the differentiated xylem cells. High levels of free intercellular peroxidase activity in phloem cells are hard to explain, since phloem cell walls remain unlignified during almost all stages of differentiation. However, association of free intercellular peroxidase activity with xylem cells is clearly associated with the lignification of the xylem cell walls. The physiological significance of the binding vs release of intercellular peroxidase is discussed in relation to the catalytic properties and stability at acidic pH of both the bound and free forms of this enzyme.     

Effects of self-pollination and maternal resources on reproduction and offspring performance in the wild lupine, Lupinus perennis (Fabaceae)

We examined the effects of self-pollination and resource addition to maternal plants of Wild Lupine on seed production in the field, and on offspring performance in the greenhouse. Although 24% of flowers set fruits when open-pollinated, only 11% of flowers set fruits when self-pollinated. Self-pollination significantly reduced fruit and seed production per inflorescence and increased aborted seeds per fruit. Resource addition to maternal plants significantly increased fruit and seed number in the field. Moreover, selfed plants exhibited greater variability in seed production in the absence of resource addition to the maternal plant. We planted a total of 1,306 of the seeds from this experiment in the greenhouse. While self-pollination did not affect the proportion of seeds emerging, it slowed seedling emergence by 5–10%, and reduced offspring biomass by 25–35%. Interestingly, resource addition to the maternal plants significantly decreased proportion of seedlings surviving after 5 months. Moreover, offspring from maternal plants with resource addition expressed more inbreeding depression in the seedling stage compared to offspring from maternal plants without resource addition, for which more inbreeding depression occurred during seed maturation and emergence. These results indicate that conservation efforts using benign environments to increase number of seeds or offspring may face compensating reductions in survivorship at other life stages.     

Previous infestation of pea aphids Acyrthosiphon pisum on broad bean plants resulted in the increased performance of conspecific nymphs on the plants

Abstract

We studied the effects of previous infestation of broad bean plants by pea aphids Acyrthosiphon pisum on the performance of conspecific nymphs on the plants and the involvement of jasmonic acid (JA)-related defenses. The time needed for newly emerged nymphs to become reproductive adults on broad bean plants previously infested by conspecifics (pre-infested plants) was significantly shorter than on uninfested (control) broad bean plants. The total numbers of nymphs produced by aphids on preinfested and control plants were not significantly different. Preinfested plants produced significantly less endogenous JA than that control plants did. To test the effect of JA decreases, we conducted experiments on the developmental duration of nymphs on broad bean plants treated with JA (JA-treated plants) before infestation. The time needed for nymphs to become reproductive adults on JA treated preinfested broad bean plants was not significantly different from that on JA-treated control plants. The results suggested a possible parental care by pea aphids: the adult aphids manipulated JA-related defenses in broad bean plants that had positive effects for their offspring.     

The use of a principal axis model to examine individual plant harvest index in four grain legumes

BACKGROUND AND AIMS: A principal axis model (PAM) has been proposed to enable the selection of crop ideotypes. The PAM enables plant-to-plant variability within crops to be quantified and compared. The aim of this paper is to validate the PAM for four grain legumes. METHODS: Four grain legumes (Cicer arietinum, Lens culinaris, Lupinus angustifolius, Pisum sativum) were used to quantify the influence of plant-to-plant variability on crop yields. To create variability, populations of 10, 100 and 400 plants m(-2) were established 'on-the-square' with sowing depths of 2, 5 and 10 cm. Further, a central plant was treated with nitrogen and the impact of this on its four neighbouring plants was examined. Seeds were sown and plants harvested individually by hand. KEY RESULTS: Mean individual plant seed weight (SWT) and plant weight (PWT) decreased as plant population increased but there was a consistent and strong (R2 > 0.90) linear relationship between SWT and PWT, with a negative SWT-axis intercept in all species. These components form the basis of the principal axis model (PAM). The PAM was used to summarize the performance of individual plants within a crop and quantify the variability caused by N treatment and the lowest and highest yielding individual plants. A negative SWT-axis intercept indicated that a minimum plant weight (MPW) was required for seed production and therefore the relationship between plant harvest index (PHI) and PWT was asymptotic. The heaviest MPW was calculated for plants grown at the lowest plant population and it was species-dependent, being higher in the larger seeded species. CONCLUSIONS: Agronomic or physiological characteristics that lead to variability in PWT within a population will decrease PHI, and crop yield. The PAM may be useful in breeding programmes to identify plant phenotypes that minimize this plant-to-plant variability.     

狮子山铜尾矿植物对铜的吸收及土壤特的影响

我国铜矿储藏丰富,铜矿开采带来巨大经济利益的同时,也对生态环境造成极大的破坏,这种恶劣的环境严重阻碍了植物的定居,但是自然界物种繁多,总有一些植物能适应这种环境而生存下来.本文通过对狮子山优势植物吸收和积累铜的分析,发现这些植物均能富集较多的铜,在土壤铜含量很高的情况下,依然生长旺盛,没有出现受害症状,成为尾矿上的优势种,并形成了单优群落或多优小群落.这些植物的存在改变了土壤的理化特性,降低了土壤中的重金属的含量,提高了土壤的全N、全P、全K和有机质含量,一定程度上改善了土壤的不良环境,在尾矿的植被恢复和土壤修复中起着非常重要的作用.     

Effect of water stress on root meristems in woody and herbaceous plants during the first stage of development

We investigated the effect of water stress on the root system architecture of pine saplings and pea seedlings during the first stage of development. Attention was focused on meristematic tissue situated at the root tip because of the leading role played by the tissue in the planning of root system architecture. The data showed that both species are extremely sensitive and that plants arrest their growth immediately during water stress treatment. When stress treatment was not intense, both species recovered growth but presented modifications in the root system architecture. In pine saplings, the modification in root system architecture was the consequence of fine root meristems not recovering from water stress. The saplings survived by producing new lateral meristems from the cortical tannin zone above the fine root tip. In the case of pea seedlings, the meristematic tissues in the primary root arrested proliferation during water stress although they recovered when the event occurred during the first hours of germination. The response was different when water stress was enforced on older seedlings. In this case, root meristems never completely recovered their proliferation despite the increase in proline content observed in the cells. The modification of root system architecture in pea seedlings depended on the arrest of primary root elongation and the formation of new root laterals. As regards the primary roots, water stress treatment induced along the axis the formation of irregular ‘swellings’ in the cortical zone above the meristematic zone. Anatomical investigations suggested that such swellings may have derived from the changes in elongation direction of derivatives. The formation of new laterals was observed in hydroponic cultures when water stress treatment was enforced slowly and prolonged for a long time. The production of new lateral meristems may have been a similar response of woody and herbaceous plants to water stress conditions. It is not known whether these new meristems present characteristics of resistance to water stress. This revised version was published online in June 2006 with corrections to the Cover Date.     

Replacement of nitrate by ammonium as the nitrogen source increases the salt sensitivity of pea plants. II. Inter- and intracellular solute compartmentation in leaflets

Pea plants (Pisum sativum L. cv. ‘Kleine Rheinländerin’) grown on ammonium or nitrate as the sole nitrogen source were treated with 50 mol m⁻³ NaCl. Four days after salt addition, ammonium-grown plants developed the first visible damage symptoms (wilting of leaflets, starting from the margins). Salt-treated, nitrate-grown plants were not affected during the experimental period. In order to obtain a better understanding of this differential salt sensitivity, we investigated the inter- and intracellular ion compartmentation of leaflets under both nutritional conditions by analysing ion concentrations in the apoplastic space, in chloroplasts and in protoplasts. When the leaves of nitrate- and ammonium-grown plants had attained similar sodium and chloride contents (after different times of exposure to salinity), the latter had a considerably lower chloroplastic chloride (and also sulphate) concentration. The results suggest that the intracellular compartmentation capacity of ammonium-grown plants is considerably lower than that of nitrate-grown plants. Ion toxicity appeared to initiate breakdown of metabolism in parts of the mesophyll tissue of ammonium-grown plants, causing an abrupt release of solutes into the apoplast, which coincided with the appearance of visible damage. Although the ammonium concentrations in leaves increased dramatically in the later phases of damage development, they were too low to cause the collapse of electrochemical gradients at the time at which damage became visible. Thus, the reason for a lower compartmentation capacity under ammonium nutrition remains as yet unclear.