Euphorbia Latex Biochemistry: Complex Interactions in a Complex Environment

Plant Biosystems

ISSN: 1126-3504 (Print) 1724-5575 (Online) Journal homepage: http://www.tandfonline.com/loi/tplb20

Euphorbia latex biochemistry: Complex interactions in a complex environment

F. Pintus , R. Medda , A. C. Rinaldi , D. Spanò & G. Floris

To cite this article: F. Pintus , R. Medda , A. C. Rinaldi , D. Spanò & G. Floris (2010) Euphorbia latex biochemistry: Complex interactions in a complex environment, Plant Biosystems, 144:2, 381-391, DOI: 10.1080/11263500903396016 To link to this article: https://doi.org/10.1080/11263500903396016

Published online: 04 Aug 2010.

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Plant Biosystems, Vol. 144, No. 2, June 2010, pp. 381–391

Euphorbia latex biochemistry: Complex interactions in a complex environment

F. PINTUS1, R. MEDDA1, A. C. RINALDI2, D. SPANÒ1, & G. FLORIS1

1Dipartimento di Scienze Applicate ai Biosistemi, Università di Cagliari, Italy and 2Dipartimento di Scienze e Tecnologie Biomediche, Università di Cagliari, Italy

(TaylorReceived and Francis 25 November 2008; Accepted 7 May 2009) 10.1080/11263500903396016

Abstract Plant latex is a complex environment. Occurring in hundreds of plant species and contained in a tube system called laticifers, latex is a milky sap with a diverse composition that includes alkaloids, terpenoid compounds, other secondary metabolites and a number of enzymes. These substances are collectively believed to provide an important contribution to plant defence mechanisms by repelling and killing phytopathogens, and sealing wounded areas. This review provides insights of what is currently known about the biochemistry and molecular biology of plant latex, as studied in various model systems, above all the economically important rubber tree, Hevea brasiliensis. Selecting the Mediterranean shrub Euphorbia characias as a complementary experimental model, we have recently begun to disclose the properties of several components of the enzymatic machinery present in its latex. Although the scheme of multi-enzymatic interactions taking place in the E. characias latex depicted to date is certainly incomplete, the emerging scenario suggests that the role played by latex in plants might be significantly less passive than previously believed.

Keywords: Amine oxidase, antiquitin, calmodulin, catalase, Euphorbia, peroxidase

A large number of plant species may exudate an Plantae has not been explained to date (Pickard often milky, variously coloured sap known as latex. 2008). According to Kekwick (2001), latex occurs in some Latex is an emulsion with a diversified composition 12,500 species, belonging to 900 genera from that includes alkaloids, terpenoid compounds, poly- about 20 families – most of which are dicotyledons – meric substances, such as resins and gums, starch, oils, growing in different ecological settings. Indeed, latex and a large number of proteins and enzymatic activ- constitutes the cytoplasmic content of laticifers ities (Han et al. 2000; Kekwick 2001; Ko et al. 2003). (Hagel et al. 2008), specialised elongated cells or Currently, no universally shared view exists about the vessel-like series of cells that permeate various aerial biological role(s) of latex. A function as nutrition or tissues of the plant, including sometimes the fruits water reserve, or as an excretory product where waste and also the root system. Laticifers lack chloroplasts, plant metabolites are confined, has been repeatedly and are generally classified as articulated, that is, proposed. Most authors, however, consider it more composed of a longitudinal series of cells joined or likely that latex provides an important contribution to fused together, and as non-articulated, that is, plant defence mechanisms by repelling browsing descended from a single cell present in the embryo animals and insects, killing or controlling the growth that has grown in a branched or unbranched of microbial phytopathogens and sealing wounded manner. These structures and their features are areas (Kekwick 2001; Giordani et al. 2002). often used as elements to infer taxonomic and phylo- Most of our knowledge on the biochemistry of genetic relationships between specific plant groups latex and laticifers stems from studies on Hevea (Rudall 1994; Webster 1994; Vega et al. 2002), brasiliensis, a member of the Euphorbiaceae and an although the phyletic distribution of laticifers in the economically valuable tree as the main source of

Correspondence: G. Floris, Dipartimento di Scienze Applicate ai Biosistemi, Università di Cagliari, Cittadella Universitaria, I–09042 Monserrato (CA), Italy. Tel: +39 070 6754519. Fax: +39 070 6754523. Email: [email protected] ISSN 1126-3504 print/ISSN 1724-5575 online © 2010 Società Botanica Italiana DOI: 10.1080/11263500903396016

Published online 04 Aug 2010

382 F. Pintus et al. natural rubber. The proteome of H. brasiliensis latex has been investigated in some detail also because it contains a range of proteins that can cause allergenic reactions in sensitised persons upon regular use of products made from natural rubber, such as health- care workers wearing examination and surgical gloves (Arif et al. 2004; Wagner et al. 2007). Our knowledge of the protein functions within Hevea latex, including lectin-binding proteins, and enzymes involved in the isoprenoid pathway, continues to progress, leading to the recent sketching of a new proposed model for rubber latex coagulation (see Wititsuwannakul et al. 2008). Conversely, relatively little is known on the biochemical features of the latex of plants belonging to the large genus Euphor- bia, although several authors are working to fill this gap (e.g., Yadav et al. 2006; Mazoir et al. 2008). We have selected the Mediterranean spurge, Euphorbia characias – a shrubby, non-succulent euphorb commonly occurring in various habitats (rocky hillsides, along road verges, in open woods and in olive groves) in vast areas of the Mediterranean basin – as an alternative and complementary experimental model to study the complexity of plant latex biochemistry (Figure 1). Figure 1. EuphorbiaThe characias, the Mediterraneanplant’s spurge: (a) Plant habit; (b)latex a detail of the leaves; and has(c) latex exudate frombeen a broken branch. extensively studied with characterisation of a large number of diterpene compounds, responsible for the plant’s irritant effect (Seip & Hecker 1983; Appendino et al. 2000; Corea et al. 2004). Screening of the latex of E. characias has also revealed the presence of numerous enzymes, some of which might well be directly or indirectly involved in plant defence mechanisms. The present review reports on the isolation and characterisation of several of these molecules (and/or of their genes), namely a peroxidase regulated by the Ca2+/calmodulin system, copper amine oxidase, catalase and antiquitin (Atq), and describes how these could interact between themselves and with other latex substances to assure some form of plant protection against invading pathogens and/or environmental stresses. It is our belief that what is learned on the biochemistry of E. characias latex will ultimately contribute to a broader understanding of the metabolism and function of this plant product.

Peroxidase and calmodulin The superfamily of haem-containing peroxidases (EC 1.11.1.7, donor: hydrogen peroxide oxidoreductase) is a widely distributed group of enzymes found in bacteria, fungi, plants and animals that utilise hydrogen peroxide or other peroxides to catalyse a number of oxidative reactions (Welinder 1992). Non-animal Figure 1. Euphorbia characias, the Mediterranean spurge: (a) peroxidases can be divided into three classes on the Plant habit; (b) a detail of the leaves; and (c) latex exudate from a broken branch. basis of their amino acid sequence: Class I contains

Euphorbia latex biochemistry 383 bacterial, fungal and plant intracellular enzymes from mitochondria and chloroplasts, such as ascorbate peroxidase and cytochrome c peroxidase; Class II consists of secreted fungal peroxidases, good examples of these being manganese peroxidase and lignin- degrading peroxidase; Class III is made up of secreted plant peroxidases, with horseradish peroxidase (HRP) as its best-known member. Plant peroxidases – for which crystal structures and a number of site-directed mutants have become available in several cases along the years – are found in the cytosol, vacuole, apoplast or cell wall, and participate in crucial physiological events, such as development and growth induction, polymerisation of cell wall lignin and suberin precursors, auxin catabolism, wound healing and defence against pathogen infection (Passardi et al. 2005). Typically, Class III peroxidases may exist under an extremely high number of isoforms within the same species, potentially implicated in different functions (Veitch 2004). The HRP isozyme C, for example, one of the more than 30 isoforms of HRP, classified as acidic, neutral and basic forms, has been the archetypal example of Class III higher plant peroxidases. HRP- C is a single glycosylated polypeptide chain containing high-spin Fe3+ in a protoporphyrin IX pentacoordinated to a “proximal” histidine ligand that functions to stabilise the higher oxidation states of the iron atom (Poulos et al. 1993). Another histidine, known as the “distal”, functions as an acid– base catalyst to accept one proton from the peroxide. Two mol of Ca2+/mol of enzyme are also present in native HRP-C, and the Ca2+-binding sites are known as the proximal and distal site, respectively, accord- Figure 2. Structure of the calcium-binding sites in ELP. The 2+ ing to their location relative to the porphyrin plane. proximal and distal Ca -binding sites in Euphorbia latex peroxidase have been inferred from an analysis of the protein sequence, Calcium ions have been proposed to play a role in and comparison with the structural features of HRP. Only the maintaining the integrity of haem pocket structure, direct coordination environment is shown, but several other ami- which is the key to high catalytic activity. no acids close to the Ca2+ ions are also highly conserved, and are A Class III peroxidase (ELP) was isolated and char- probably important for maintaining the correct geometry of the 2+ acterised from the latex of E. characias (Medda et al. binding sites and of the haem pocket. Both Ca ions are seven- coordinated. In ELP, the distal Ca2+ is loosely bound but is nec- 2003). ELP is a single glycosylated polypeptide chain essary for full enzyme activity. of 347 residues with a relative molecular mass of 47 kDa. It contains a ferric iron–protoporphyrin IX in a quantum mechanically mixed-spin state, pentacoor- Figureisnecessary shown, 2. butfor StructureThe severalfull enzyme ofother the activity. calcium-bindingamino acids Euphorbia close sites to the in ELP.Ca 2+ Theions proximal are also highlyand distal conserved, Ca 2+-binding and are sitesenzyme probably in Euphorbia important latex for peroxidase maintaining have the beencorrecthas, inferred geometry from of an the analysis binding atof sitesthe protein and of variancesequence, the haem andpocket. comparison Both Ca with2+ ions the arestructural seven-coordinated. featureswith of H RP.In ELP, Only the the distal direct otherCa coordination2+ is loosely environment bound but is dinated to a “proximal” histidine ligand. The ELP known plant peroxidases, low specific activity for sequence (GenBank accession number AY586601) classical peroxidase substrates, whereas its activity permits to identify two highly conserved histidine resi- seems to be closely regulated by exogenous Ca2+ ions. 2+ dues coordinated to the haem (His50 and His179, distal After addition of Ca , the kcat increased 100 times and proximal, respectively). Like other secreted plant with a decrease of KM for H2O2. Analysis of the steady peroxidases, purified ELP has one mol of Ca2+/mol state by stopped-flow measurements suggested that of enzyme and two calcium binding sites (Figure 2). the main effect of Ca2+ ions is to favour the oxidation 2+ The proximal Ca ion is strongly bound to Thr180, of the ferric enzyme by H2O2 to form Compound I, Asp256, Thr259, Ile262, Asp265, and plays a critical role a reaction intermediate. Calcium binding to the distal in conferring structural stability to the haem environ- low-affinity site probably induces a reorientation of ment and in retaining the enzyme active site geometry the distal His, thereby changing the almost inactive (Mura et al. 2005). The Ca2+ ligands at the distal site form of Euphorbia peroxidase to a high-activity form are Asp51, Val54, Gly56, Asp58 and Ser60. (Medda et al. 2003).

384 F. Pintus et al.

Perhaps, the most remarkable property of ELP, is the context of latex, that is, whether a CaM-like that it contains two distinct calmodulin (CaM)- protein was constitutively expressed in this tissue. binding sites. It is worth recalling that CaM is a Indeed, the cDNA encoding for an E. characias CaM small, highly acidic cytosolic protein, with a MW of was found and sequenced, and its protein product was 16.7–18.8 kDa, involved in the response to fluctua- later detected in the latex (Mura et al. 2005). E. chara- tions of the intracellular concentration of Ca2+ and cias latex CaM (ELCaM) cDNA (785 bp) contains in regulating the activities of multiple proteins. an open reading frame (ORF) of 447 bp which can Analysis of the predicted amino acid sequence of be translated into a protein sequence of 149 amino ELP for putative CaM-binding sites was made with acids (Figure 3). The calculated molecular mass for the tools provided by the web-based Calmodulin the predicted protein is 16.8 kDa, with a pI of 4.1. EF- Target Database (http://calcium.uhnres.utoronto.ca/ hand domains are located at positions 21–32, 57–68, ctdb). In this method, sequences are analysed for 95–105, 130–141 with their clusters of particularly features such as hydropathy, α-helical propensity, well-conserved, non-contiguous residues. Not residue charge, and hydrophobic residue content, surprisingly, the ELCaM amino acid sequence shows and a normalised score (0–9) is attributed based a very high degree of identity (91–100%) and on these criteria. Our search revealed the presence of similarity (99–100%) to CaMs isolated from several a putative CaM-binding domain between residues other higher plants, including Prunus avium, Elaeis 26 and 39 of ELP, a 14 amino acid sequence guineensis, Medicago truncatula, Pisum sativum, Phaseo- (IQKELKKLFKKDVE) with the characteristics of lus vulgaris and Nicotiana tabacum. an IQ-like motif. In addition, a related motif for Figure 3. Nucleotide and deduced amino acid sequence of the E. characias latex calmodulin (ELCaM). The four EF-hand domains are underlined, and the Ca 2+-binding amino acid residues are boxed. CaM-binding, termed 1–8–14, was spotted between Catalase residues 79 and 92 (LSLRKQAFKIVNDL) (Mura et al. 2005). The IQ motif and related sequences are Catalase (EC 1.11.1.6) is a tetrameric haem- present, often in multiple copies, in diverse families containing enzyme promoting the dismutation of of CaM-binding proteins, such as myosins, neuro- hydrogen peroxide to water and molecular oxygen → modulin, neurogranin and brain-specific polypep- (2H2O2 2H2O + O2). It is one of the most impor- tide PEP-19, and have been shown to bind CaM tant plant H2O2-scavenging enzymes due to its both in the presence and in the absence of calcium, capacity for degrading H2O2, produced mainly in depending on the occurrence of particular residues the peroxisomes by oxidation of photorespiratory in the sequence. The 1–8–14 motif makes a subclass glycolate and in the glyoxysomes by β-oxidation of of the larger 1–14 motif family, a group of sequences fatty acids, without consuming cellular reducing characterised by the presence of two or more bulky equivalents. Catalase is also an important compo- hydrophobic residues spaced by a variable number nent of the system involved in degradation of H2O2 of amino acids (see Mura et al. 2005 and references generated in excess by biotic and abiotic stresses therein). These sequences bind to CaM primarily in (Willekens et al. 1995, 1997). the presence of calcium. In plants, catalase is present as multiple isoforms, Calmodulin plays a pivotal role in physiological encoded by a small gene family. Catalase gene processes, as shown by its highly conserved primary expression is well characterised in the monocotyle- structure in all living organisms. CaMs possess four donous Zea mays (Abler & Scandalios 1993; Guan & functional Ca2+-binding domains called EF-hand, Scandalios 1993; Guan et al. 1996) and in the dicot- numbered I through IV, beginning from the amino- yledonous Arabidopsis thaliana and Nicotiana plum- (N) termini of the protein. Between EF-hands I–II baginifolia (Willekens et al. 1994; Frugoli et al. and III–IV is located a solvent exposed α-helical 1996). Each of these contains three active genes region. The binding of Ca2+ determines structural encoding catalase, namely Cat1, Cat2 and Cat3. The modifications of this protein portion, converting differential expression of these catalase genes in apo-CaM in its active form, a more flexible structure different tissues and during plant development, as able to bind to the target protein(s). In this region, well as the differential regulation of each gene by some hydrophobic residues (Leu, Ile and Val) light, suggest the association of each specific gene responsible for the interaction between CaM and product to a particular H2O2-producing process. N. CaM-binding proteins are particularly conserved. plumbaginifolia Cat1 is highly expressed in light- Also in plants, CaM plays a significant role whose grown leaves and is positively regulated by light, importance is increasingly understood and appreci- suggesting that Cat1 might be involved in scavenging ated (Zielinski 1998; Sathyanarayanan & Poovaiah the H2O2 generated during photorespiration. N. 2004; Ma & Berkowitz 2007). plumbaginifolia Cat2 is the predominant catalase Following the finding of ELP as a CaM-binding transcript in stems and vascular tissues and its protein, the next logical step has been to verify expression is not affected by light (Willekens et al. whether this property had a functional significance in 1994). The rapid induction of Cat2 by ozone, SO2

Euphorbia latex biochemistry 385

Figure 3. Nucleotide and deduced amino acid sequence of the E. characias latex calmodulin (ELCaM). The four EF-hand domains are underlined, and the Ca2+-binding amino acid residues are boxed. and UV-B suggests an important role in the scaveng- ECat shows a very high identity (84%) and similar- ing of H2O2 produced during stress conditions ity (91%) with other plant catalases (Manihot (Scandalios 1990). Cat3 mRNA levels are high in esculenta, Prunus persica, Helianthus annuus and A. seeds as well as in mature and senescing petals, thaliana) (Mura et al. 2007). Recent studies report suggesting a specific role in glyoxysomal fatty acid that Ca2+/CaM may bind to plant catalases and degradation (Willekens et al. 1994). Based on the activate them, as found, for example, in Arabidopsis expression profiles of genes encoding catalase (Yang & Poovaiah 2002). The analysis of the ECat isozymes in well-known experimental models, plant amino acid sequence – obtained by means of a catalases can be divided into three different classes. bioinformatics programme, as discussed above for Class I is characterised by being highly expressed in peroxidase and CaM – revealed the presence of a photosynthetic tissues; N. plumbaginifolia Cat1 and CaM-binding domain stretching 14 residues with Z. mays Cat2 can be hosted in this class. Class II the characteristic of an IQ-like motif at position catalases, including N. plumbaginifolia Cat2 and Z. 300–313 (LQEIGRLVLNRNID). In addition, mays Cat3, are highly abundant in vascular tissues. three related motifs for CaM-binding belonging to

Class III, destroying glyoxysomal H2O2, is mainly the subclasses termed 1–8–14 and 1–16 are found expressed in seeds and young seedlings; this class between residues 64 and 77 (VHARGASAKG- includes Cat3 from N. plumbaginifolia and Cat1 from FFQV), 207 and 222 (VNTYTLINKAGKAHYV) maize (Willekens et al. 1995). and 408 and 423 (IPNAIISGGRRMKTVL). We cloned the cDNA encoding for a catalase in E. characias latex (ECat) (Mura et al. 2007) Copper amine oxidase (GeneBank accession number AAX88799). The deduced amino acid sequence consists of 493 resi- Copper/quinone-containing amine oxidases [amine: dues with a theoretical molecular weight of 56.8 oxygen oxidoreductase (deaminating) (copper kDa and a pI of 7.12. The amino acid sequence of containing]; EC 1.4.3.6) (Cu/TPQ AOs) are widely 386 F. Pintus et al. distributed throughout nature and are found in inserted in the consensus sequence Asn–Tyr–Asp of bacteria, yeasts, fungi, plants and mammals (Floris the polypeptide chain; the copper atom, buried and & Finazzi Agrò 2004). In plants, copper AOs are not directly accessible from the solvent, can be coor- involved in processes of yellowing, senescence, dinated by the imidazole groups of three conserved wound healing and cell wall biosynthesis, and play His residues (Padiglia et al. 2002). As recently an important role in cell growth by regulating the shown, ELAO and ELP are able to oxidise the intracellular di- and polyamine levels, while the alde- important plant intermediate tyramine, and could hyde products might have a key role in the biosyn- play a role in its metabolism (Mura et al. 2008). thesis of some alkaloids (Frébort & Adachi 1995). Well-studied examples of plant AOs include the Antiquitin enzymes isolated from the seedlings of dry pea (Pisum sativum) (Kumar et al. 1996), lentil (Lens Aldehyde dehydrogenases (ALDHs; EC 1.2.1.19) esculenta) (Agostinelli et al. 2005) and from E. chara- make up a superfamily of proteins that share a role cias latex (ELAO) (Padiglia et al. 1998). in the metabolism of endogenous and exogenous ELAO shows very similar spectroscopic and aldehydes, but that at the same time are greatly chemical features to those of other plant copper diversified from the functional point of view. Indeed, AOs. It is a soluble homodimeric protein, and each belonging to this group are proteins with an assigned subunit (MW ≅ 74 kDa) contains an active site with detoxification role related to the conversion of alde- a tightly bound CuII ion and an organic cofactor hydes into the corresponding organic acids using known as Topaquinone (TPQ), derived from the NAD(P)+. Other ALDHs seem to participate in the post-translational modification of a Tyr residue intermediary metabolism of amino acids and retinoic inserted in the polypeptide chain (Janes et al. 1990; acid, and in the protection from osmotic stress Tanizawa 1995). Due to the presence of TPQ, the through the generation of osmoprotectants, such as oxidised form of ELAO has a distinctive pink colour glycinebetaine (Ishitani et al. 1995). Over 1000 and shows, in addition to the protein absorbance different proteins are up to now ascribed to the ε −1 −1 maximum at 278 nm ( 278 = 378 mM cm ), a ALDH superfamily on the basis of nucleotide and broad absorption band in the visible region at 490 protein sequence homologies. Those showing a ε −1 −1 nm ( 490 = 6 mM cm ) (Padiglia et al. 1998). >40% identity are considered to belong to the same Similar to the other AOs, ELAO catalyses the family and, inside the same family, those sharing a oxidative deamination of primary amines to the 60% identity form a subfamily. Using these criteria, corresponding aldehydes, with the concomitant about 20 families of eukaryotic ALDHs are currently reduction of molecular oxygen to hydrogen perox- identified (Sophos et al. 2006; further information ide. The ping–pong catalytic mechanism can be can be retrieved from the ALDH Gene Superfamily divided into two half-reactions (Figure 4). The Database at www.uchsc.edu/sop/pharmscience/- substrate specificity of ELAO is much narrower than alcdbase/aldhcov.html). that found in lentil and pea seedling AOs, being A specific family of ALDH, namely ALDH7, is limited to diamines of critical molecular dimensions, particularly remarkable as its member proteins from and its activity for the best-known substrate animals and plants show an exceptional nucleotide putrescine (kc = 34) is about one-fourth than that of sequence homology. For example, the identity ≈ the same reference AOs (kc 155). between human ALDH7B1 and its counterpart in Figurecofactor.ammonia 4. Theand CatalyticThe UV–vishydrogen mechanism absorption peroxide. ofspectra Since E. cDNAcharacias Cu of Ithe readily resting latex reacts oxidisedamine with oxidase dioxygen,enzyme (ELAO). (I), it andis encodinglikely The of intermediate ping-pong that the formation catalytic species ofCumechanism CuII-aminoquinolI and semiquinolamine can be (II)divided and Cuintofor radicalI-semiquinolamine two half-reactions:is an obligate, an radical intermediary(a) Reductive (III) are AOstep shown;half just react (b)beforeion, Oxidative which the oxidative involves half-reaction,has half-reaction. the oxidation which involves ofbeen amine the to reoxidationaldehyde and of thethe formationenzymeisolated with of athe reduced simultaneous form of releasethe TPQ of dry pea attains 60% (Lee et al. 1994), which is from young leaves of E. characias and sequenced surprising considering the long evolutionary distance (GeneBank accession number AF171698) (Padiglia between the two organisms. This finding suggests et al. 2002). A single long ORF of 2068 bp encodes that the proteins of this family must play an impor- a protein composed of 653 amino acids with a tant role in cellular processes, so that their structure molecular mass of about 74 kDa. Alignments of the has remained substantially unchanged for a long Euphorbia AO cDNA nucleotide sequence with that stretch of the evolutionary history of life. Owing to of AO from lentil and dry pea seedlings revealed this feature, the name antiquitin (Atq) was assigned several conserved regions, especially in the C-termi- to this protein family to underscore its ancient nus, with a 90–97% homology. The near 5′-region origin. This family is further divided into three shows several insertions, deletions and a different subfamilies: ALDH7A, ALDH7B and ALDH7C. nucleotide sequence with ca. 60% homology. The The first includes proteins found in animals, the defined active site of Euphorbia AO, as deduced second in plants, while the third is restricted to from its cDNA sequence, is apparently very similar Drosophila melanogaster (Fong et al. 2006). to that of other well-known plant enzymes. In In plants, genes encoding for ALDH7 have been particular, the tyrosine residue post-translationally now characterised in a number of species, including modified in TPQ can be identified as Tyr392, Pisum sativum (Guerrero et al. 1990), Brassica napus Euphorbia latex biochemistry 387

Figure 4. Catalytic mechanism of E. characias latex amine oxidase (ELAO). The ping-pong catalytic mechanism can be divided into two half-reactions: (a) Reductive half reaction, which involves the oxidation of amine to aldehyde and the formation of a reduced form of the TPQ cofactor. The UV–vis absorption spectra of the resting oxidised enzyme (I), and of intermediate species CuII-aminoquinol (II) and CuI-semiquinolamine radical (III) are shown; (b) Oxidative half-reaction, which involves the reoxidation of the enzyme with the simultaneous release of ammonia and hydrogen peroxide. Since CuI readily reacts with dioxygen, it is likely that the formation of CuI and semiquinolamine radical is an obligate, intermediary step just before the oxidative half-reaction.

(Storeher el al. 1995) and Sorghum bicolor (Buchanan ification. However, evidence – such as the implica- et al. 2005). In all cases, an increase of gene expres- tion of Atq in several human diseases – is emerging sion was observed in response to dehydration- and that its physiological functions might be more diver- salinity-induced stress. Accordingly, plants of A. sified and complex than was thought so far (Fong thaliana and N. tabacum expressing a soybean homo- et al. 2006). logue Atq gene were shown to display enhanced In E. characias, Atq cDNA was isolated and char- tolerance to drought and salinity, and also to oxida- acterised from latex (Mura et al. 2007). The cloned tive stress, suggesting that besides acting in osmoreg- cDNA encoding for this Atq (EAtq) contains an ulation, Atq may be also involved in adaptive ORF of 1527 bp which can be translated into a responses mediated by a physiologically relevant protein sequence of 508 amino acids (GenBank detoxication pathway in plants (Rodrigues et al. accession). The calculated theoretical molecular 2006). Although information on the role of Atq in mass for the predicted protein is 54.6 kDa , with a pI animals and humans is comparatively less abundant, of 5.54. The EAtq amino acid sequence shows a it is generally assumed that the function of the high degree of identity (72%) and similarity (82%) protein in these organisms is similar to that in plants, to Atqs isolated from several other higher plants that is, centred around osmoregulation and/or detox- (Mura et al. 2007). 388 F. Pintus et al.

An enzymatic interactome in Euphorbia latex toxic products, or could use them as substrates for the synthesis of osmoprotectants within the laticifer Two tubing systems have evolved in plants that play vacuole, thus contributing to turgour control in a role in storing, moving and physically releasing laticifer cells. As mentioned above, the conservation secondary metabolites: the laticifers and the secre- of sufficient internal turgour pressure as a result of tory ducts. In the first case, the secondary metabo- osmotic water uptake, and its rapid recovery after lites are stored inside the living cell(s) which latex discharge upon puncture, is a key element of produce(s) them, while in secretory ducts they are laticifer function. Studies in Hevea have indicated stored in an extracellular space. It is generally agreed that potassium ions, sucrose, malate and amino that the ecological role of laticifers is to deter insect acids are the principal osmotically active solute herbivory and possibly discourage foraging by higher species in latex (Pickard 2008 and references animals. “To achieve these ends, laticifers normally therein), but secondary metabolites may also play a contain sequestered chemicals that discourage their role in this sense, and also in assuring protection herbivores, and maintain internal pressures high from osmotic stress. Glycinebetaine, for example, is enough to spew these chemicals onto or into herbi- a metabolite with osmoprotectant properties synthe- vores that puncture them,” wrote William Pickard sised by two enzymes: a ferredoxin-dependent in a recent review on the general physiology and choline monooxygenase which catalyses the oxida- ecophysiology of laticifers and secretory ducts (Pick- tion of choline into betaine aldheyde, and glycinebe- ard 2008). Besides insects, other organisms, includ- taine aldheyde dehydrogenase (BADH; EC 1.2.1.8) ing fungi and other microbial pathogens, may which converts betaine aldheyde into glycinebe- represent a potential threat to plant health and integ- taine. It represents a common compatible solute in rity, and the laticifers/latex system seems to be higher plants where it accumulates in response to equipped to counterfeit these attacks as well. In any drought and salinity, and to low and high tempera- cases, selected enzymes, such as chitinases against ture stress. Various other ALDHs (EC 1.2.1.19) are fungi and cysteine proteases against insects, are also present in plants, with different substrate speci- either directly involved in defence reactions and/or ficity (Brauner et al. 2003). Only the Atq gene has synthesise biologically active compounds (Graham been detected in E. characias latex so far, and no & Sticklen 1994; Konno et al. 2004). information is currently available even on the pres- The evidence presented above clearly indicates ence/absence of glycinebetaine itself, so it is the coexistence of multiple enzymatic activities within currently not possible to tell whether BADH is the latex-driving system of E. characias. In particular, present or not in this peculiar metabolic environ- two main players have been characterised so far in this ment. Future studies should be aimed at exploring experimental model, namely the H2O2-producing further the mechanism through which E. characias amine oxidase (ELAO) – a copper/quinone-contain- regulates laticifer osmotic pressure, and how it ing enzyme that catalyses the oxidative deamination protects itself from osmotic and other environmen- of diamines and polyamines to aldehyde and ammo- tal stresses, such as drought and salinity.

FigurebiogenicHthroughO to 5. oxidisethe amines Potentialaction a by second of Euphorbia interactionsELAO reducing can amine bebetween substrate oxidised oxidase enzymatic (XHto (ELAO; a carboxylic). Euphorbia systems complex acid in catalasea) E.by generates characias Euphorbia (ECat, H laticifers. complexOantiquitin, aldehyde A c) (EAq,variety promotes and complexammonia. of bioticthe conversion d). andScavenging abiotic of stresses Hof HO toO may wateris mediated elicit and the molecular bytransient a complex oxygen. increase network H ofO cytosolic of also enzymes, activates Ca 2+ including levels plasma by themembrane modulating Ca 2+/CaM-regulated Ca the2+ openingchannels, peroxidase of further plasma stimulating membraneand catalase. HCa OEuphorbia2+-permeable production. peroxidase cation Finally, channels. (ELP the aldehydes complex The oxidation b)produced utilizes of nia, concomitantly with a two-electron reduction of 2 2 Finally,2 since2 2 both ELP2 2 2 and2 2 ECat are involved2 2 in 2+ dioxygen to hydrogen peroxide; a Ca /CaM-regu- the regulation of a number of H2O2-signaling path- lated Class III secreted peroxidase (ELP) – the first ways related to defence against invading pathogens/ example to date of a peroxidase regulated by this environmental stresses, and in the control of H2O2 classic signal transduction mechanism – probably homeostasis in many better characterised plants, one involved in the activation of plant defence responses would expect them to have the same functions in and in the homeostasis of H2O2. The later finding that Euphorbia laticifers. A particular feature of ELP and, catalase and Atq genes are expressed in E. characias inferentially, of ECat is that they are CaM-binding laticifers further extends our view of latex biochem- proteins, which might call for their stance as impor- istry, and allows us to draw a more detailed map of tant nodes in the finely tuned cross-talk between some of the multi-enzymatic interactions that could calcium and H2O2 that is increasingly emerging as an potentially take place in this unusual environment. important characteristic of plant defence systems As shown in Figure 5, one could indeed hypothe- (Lamb & Dixon 1997; Demidchik et al. 2002; Yang sise that ELAO controls the level of mono-, di- and & Poovaiah 2002; Foreman et al. 2003; Mittler et al. polyamines, and presumably participates in cell wall 2004). It is well known that CaM is a cytoplasmatic lignification and, through the production of hydro- protein, and, in the ELCaM gene, a sequence that gen peroxide, in the defensive oxidative burst. The can be translated to a leader sequence with the char- oxidation of biogenic amines, on the other hand, acteristics of a secretion signal peptide is absent. may generate biologically active substances, specifi- Thus, its detection in the vacuolar system (latex) cally aldehydes. EAtq, therefore, could either catal- could be a particular event related to stress condi- yse the oxidation of these metabolic and potentially tions. One could assume that, following plant injury Euphorbia latex biochemistry 389

Figure 5. Potential interactions between enzymatic systems in E. characias laticifers. A variety of biotic and abiotic stresses may elicit the transient increase of cytosolic Ca2+ levels by modulating the opening of plasma membrane Ca2+-permeable cation channels. The oxidation

of biogenic amines by Euphorbia amine oxidase (ELAO; complex a) generates H2O2, aldehyde and ammonia. Scavenging of H2O2 is mediated by a complex network of enzymes, including the Ca2+/CaM-regulated peroxidase and catalase. Euphorbia peroxidase (ELP com-

plex b) utilizes H2O2 to oxidise a second reducing substrate (XH2). Euphorbia catalase (ECat, complex c) promotes the conversion of H2O2 2+ to water and molecular oxygen. H2O2 also activates plasma membrane Ca channels, further stimulating H2O2 production. Finally, the aldehydes produced through the action of ELAO can be oxidised to a carboxylic acid by Euphorbia antiquitin (EAtq, complex d). and tissue rupture, the latex and cytoplasm contents and by a grant from Fondazione Banco di Sardegna, mix, so that CaM interacts with its target proteins in Sassari (Italy). latex. Another possibility is suggested by the tyrosine-based sorting signal 139YEEF142 found in the ELCaM sequence. The YxxΦ (Φ, bulky hydro- References phobic residue) peptide has been described in vari- Abler ML, Scandalios JG. 1993. Isolation and characterization of ous proteins destined to be internalised by clathrin a genomic sequence encoding the maize Cat3 catalase gene. pathways (Ohno et al. 1995). Normally, YxxΦ is Plant Mol Biol 22: 1031–1038. recognised by the µ1 subunit of the heterotetrameric Agostinelli E, Belli F, Dalla Vedova L, Longu S, Mura A, Floris G. 2005. Catalytic properties and the role of copper adaptor protein AP1, and the derived complex is in bovine and lentil seedling copper/quinone-containing then internalised into the vacuole through clathrin- amine oxidases: Controversial opinions. Eur J Inorg Chem coated vesicles (Brett et al. 2002). 9: 1635–1641. In conclusion, although we have just begun to look Appendino G, Belloro E, Tron GC, Jakupovic J, Ballero M. 2000. into latex biochemistry in a more analytical way than Polycyclic diterpenoids from Euphorbia characias. Fitoterapia 71: 134–142. attempted before, the picture we obtain is already a Arif SAM, Hamilton RG, Yusof F, Chew NP, Loke YH, Nimkar stimulating one, and the perspectives far reaching. S, et al. 2004. Isolation and characterization of the early nodule-speciﬁc protein homologue (Hev b 13): An allergenic lipolytic esterase from Hevea brasiliensis latex. J Biol Chem Acknowledgements 279: 23933–23941. ˇ Brauner F, S[ocarn] ebela M, Snégaroff J, Pecoa[rn]ˇ P, Meunier J-C. 2003. This study was partially supported by PRIN 2006 Pea seedling aminoaldehyde dehydrogenase: Primary struc- (Progetti di Ricerca di Interesse Nazionale) funds ture and active site residues. Plant Physiol Biochem 41: 1–10. 390 F. Pintus et al.

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