Plant Latex Research Articles

Comprehensive proteome analysis of lettuce latex using multidimensional protein-identification technology

Commercially, lettuce ( Lactuca sativa) is one of the most important leafy vegetables. Lettuce produces a milky latex of variable chemical compositions within its laticifers. As a step toward understanding the main physiological roles of this latex in higher plants, we embarked on its proteomic analysis. We investigated 587 latex proteins that were identified from the lettuce latex using multidimensional protein-identification technology. A bioinformatics analysis showed that the most frequently encountered proteins in the latex were organellar proteins from plastids and mitochondria, followed by nucleic and cytoplasmic proteins. Functional classification of the identified proteins showed that proteins related to metabolism, cell rescue, defense, and virulence were the most abundant in lettuce latex. Furthermore, numerous resistance proteins of lettuce and viral proteins were present in the latex suggesting for the first time a possible function of the lettuce latex in defense or pathogenesis. To the knowledge of the authors, this is the first large-scale proteome analysis of lettuce latex.

Thrombin like activity of Asclepias curassavica L. latex: Action of cysteine proteases

Aim of the study To validate the scientific basis of plant latex to stop bleeding on fresh cuts. Cysteine protease(s) from Asclepias curassavica (Asclepiadaceae) plant latex was assessed for pro-coagulant and thrombin like activities. Materials and methods A waxy material from the latex of Asclepias curassavica latex was removed by freezing and thawing. The resulted latex enzyme fraction was assayed for proteolytic activity using denatured casein as substrate. Its coagulant activity and thrombin like activity were determined using citrated plasma and pure fibrinogen, respectively. Inhibition studies were performed using specific protease inhibitors to know the type of protease. Results The latex enzyme fraction exhibited strong proteolytic activity when compared to trypsin and exerted pro-coagulant action by reducing plasma clotting time from 195 to 58 s whereas trypsin reduced clotting time marginally from 195 to 155 s. The pro-coagulant activity of this enzyme fraction was exerted by selectively hydrolyzing Aα and Bβ subunits of fibrinogen to form fibrin clot when pure fibrinogen was used as substrate as assessed by fibrinogen–agarose plate method and fibrinogen polymerization assay. Trypsin failed to induce any fibrin clot under similar conditions. The electrophoretic pattern of latex enzyme fraction-induced fibrin clot was very much similar to that of thrombin-induced fibrin clot and mimic thrombin like action. The proteolytic activity including thrombin like activity of Asclepias curassavica latex enzyme fraction was completely inhibited by iodoaceticacid (IAA). Conclusion Cysteine proteases from Asclepias curassavica latex exhibited strong pro-coagulant action and were found to be specific in its action (Thrombin like). This could be the basis for the use of plant latex in pharmacological applications that justify their use as folk medicine.

Effects of the Papaya meleira virus on papaya latex structure and composition

Spontaneous latex exudation is the main symptom of papaya sticky (meleira) disease caused by the Papaya meleira virus (PMeV), a double-stranded RNA (dsRNA) virus. This paper describes different effects of PMeV on papaya latex. Latex samples were subjected to different histochemical tests to evaluate their chemical composition. Additionally, the integrity of the latex particles was assessed by transmission and scanning electron microscopy analysis. Biochemical and micro- and macro-element measurements were performed. PMeV dsRNA extraction was performed to evaluate the interaction of the virus with the latex particles. Sticky diseased latex was positive for alkaloid biosynthesis and showed an accumulation of calcium oxalate crystals. PMeV also increased H(2)O(2) synthesis within sticky diseased laticifers. The protein, sugar and water levels were altered, probably due to chemical changes. The morphology of the latex particles was further altered; PMeV particles seemed to be bound to the latex particles. The alkaloid and H(2)O(2) biosynthesis in the papaya laticifers indicate a papaya defense response against PMeV. However, such efforts failed, as the virus affected the plant latex. The effects described here suggest some advantages of the infection process, including facilitating the movement of the virus within the papaya plant.

Enzyme induction as a possible mechanism for latex-mediated insect resistance in romaine lettuce.

Plant latex is a known storehouse of various secondary metabolites with demonstrated negative impact on insect fitness. A romaine lettuce cultivar, "Valmaine", possesses a high level of latex-mediated resistance against the banded cucumber beetle, Diabrotica balteata LeConte (Coleoptera: Chrysomelidae), compared to a closely related cultivar "Tall Guzmaine". Latex from damaged Valmaine plants was much more deterrent to adult D. balteata feeding than latex from undamaged plants when applied to the surface of artificial diet under choice conditions; no such difference was found in choice tests with latex from damaged and undamaged Tall Guzmaine plants. The intensities of whiteness and browning were significantly higher in Valmaine latex than in Tall Guzmaine latex. The activities of three enzymes (phenylalanine ammonia lyase, polyphenol oxidase, and peroxidase) significantly increased over time in latex from damaged Valmaine plants (i.e., 1, 3, and 6 days after feeding initiation), but they remained the same in Tall Guzmaine latex. The constitutive levels of phenylalanine ammonia lyase and polyphenol oxidase also were significantly higher in Valmaine latex than in Tall Guzmaine latex. These studies suggest that Valmaine latex chemistry may change after plant damage due to increased activity of inducible enzymes and that inducible resistance appears to act synergistically with constitutive resistance against D. balteata.

Supplemental host range ofAraujia mosaic virus, a potential biological control agent of moth plant in New Zealand

Araujia mosaic virus (ArjMV) has previously been confirmed to have a narrow host range and is considered a potential biological control agent for moth plant (Araujia hortorum), a troublesome environmental weed in northern regions of New Zealand. In this study, the virus was tested on 14 plant species, including species of importance to New Zealand to explore its possible use as a biocontrol agent for moth plant. Mosaic symptoms and occasional distortion were observed on leaves of the three Araujia species tested. Mosaic symptoms were seen on Morrenia odorata and Oxypetalum caeruleum, while mosaic symptoms, chlorotic spots and leaf distortion were observed on all three of the Gomphocarpus species tested. No symptoms were observed on Asclepias curassavica, Catharanthus roseus, Hoya carnosa, Mandevilla × amabilis, Nerium oleander and Vinca major. Because ArjMV infected the three Araujia species causing stunting, this virus would significantly reduce flowering and seed production in these species. The monarch butterfly (Danaus plexippus) relies on Gomphocarpus fruticosus for successful development and is highly valued in New Zealand. It may be possible to genetically modify ArjMV to prevent transmission by insects but it is not an option because genetically modified organisms are not widely supported by the New Zealand public. The research efforts are now focusing on other potential agents for biocontrol of moth plant in New Zealand.

A sensitive period for larval gustatory learning influences subsequent oviposition choice by the cabbage looper moth

We examined the effects of larval feeding experience with a deterrent plant latex on subsequent adult oviposition behaviour in the cabbage looper moth, Trichoplusia ni. Larvae were exposed to the latex of Hoodia gordonii at 100 ppm in an artificial diet. This plant material strongly deters oviposition in naive moths in laboratory choice tests. Larvae that were exposed to the latex either for their entire final instar or throughout larval development showed no oviposition deterrence. This effect persisted when larval exposure to H. gordonii latex was suspended for the final 2 days immediately prior to pupation. However, when exposure to latex was suspended for 1 or more days in the beginning of the final instar or for 3 or more days prior to pupation, oviposition behaviour was deterred in subsequent adult moths (i.e. the adults behaved as naive moths). Our observation that adult oviposition choice can be influenced by a change in diet in the final larval instar indicates the presence of a sensitive period whereby larval feeding memory can be lost or maintained in adulthood.

Cysteine proteases from the Asclepiadaceae plants latex exhibited thrombin and plasmin like activities

In the present study we evaluated the presence of cysteine protease from the latex of four plants Asclepias curassavica L., Calotropis gigantea R.Br., Pergularia extensa R.Br. and Cynanchum puciflorum R.Br. belongs to the family Asclepiadaceae. Cysteine proteases from these plants latex exhibited both thrombin and plasmin like activities. Latex enzyme fraction in a concentration dependent manner induced the formation of clot in citrated blood plasma. Direct incubation of fibrinogen with latex enzyme fraction resulted in the formation of fibrin clot similar to thrombin enzyme. However prolonged incubation resulted in degradation of the formed fibrin clot suggesting plasmin like activity. Latex enzyme fraction preferentially hydrolyzed Aalpha and Bbeta chains of fibrinogen to form fibrin clot. Latex enzyme fraction also hydrolyzed the subunits of fully cross linked fibrin efficiently, the order of hydrolysis was alpha-polymer > alpha-chains > beta-chain and gamma-gamma dimer. Cysteine proteases from all the four Asclepiadaceae plants latex exhibited similar action on fibrinogen and fibrin. This study scientifically validate the use of plant latex in stop bleeding and wound healing by traditional healers all over the world.

Post-harvest storage effects on guayule latex, rubber, and resin contents and yields

Guayule is a new crop being commercialized for hypoallergenic latex production. Because natural processes that occur in the plant following harvest, notably dehydration, result in rapid loss of latex and immediate processing of guayule shrub for latex on a commercial scale is not feasible, storage conditions that maintain latex concentration and yield need to be established. The objective of this study was to determine the effects of different storage conditions on the extractable latex, total rubber, resin, and guayulin A and B contents, and extractable latex, total rubber, and resin yields in harvested guayule shrub. The experiment was established using plants transplanted into the field at the University of Arizona Maricopa Agricultural Center, Maricopa, AZ, USA, on 22 March 2001. A randomized complete block design with four replications was used. Two germplasm lines (11591 and AZ-2) were used for this experiment. Twenty plants of each line were harvested six times (November 2002, March 2003, July 2003, November 2003, March 2004, and July 2004) from each field plot. Two plants of each line were randomly assigned to each of 10 storage treatment combinations reflecting wet, dry, or wet alternated with dry conditions prior to chipping for latex extraction. Extractable latex content, total rubber content, resin content, and guayulin A and B contents were determined after storage and compared with freshly harvested shrub. Plant biomass, latex yield, rubber yield, and resin yield were also determined and compared with fresh harvested shrub. AZ-2 was significantly lower in latex, rubber, and guayulin A content than 11591, and significantly higher in biomass, latex yield, rubber yield, resin content, resin yield, and guayulin B content. The results from this study show that moist storage of harvested shrub prior to dry chipping allows a higher yield of latex. Storing the shrub under moist conditions may allow more flexible harvesting and processing schedules, by limiting post-harvest latex losses and increasing the time interval between harvesting and processing.

Plant population, planting date, and germplasm effects on guayule latex, rubber, and resin yields

Guayule ( Parthenium argentatum Gray) is a perennial shrub native to the Chihuahuan Desert. While guayule traditionally has been cultivated for rubber, more recently it is being cultivated for its hypoallergenic latex. Other uses including termite resistant wood products and an energy source have also been identified. However, the effects of various agronomic practices, such as planting and harvesting dates, plant spacing, cutting height and frequency, irrigation frequency, and herbicide application, on latex concentration and yield of newly developed germplasm have not been reported. The objectives of this study were to determine the yield and concentration of latex, rubber, and resin of four guayule lines planted at two populations and two planting dates. Four guayule lines (AZ-1, AZ-3, AZ-5, and 11591) were transplanted at two dates (28 November 2000 and 7 June 2001) and two plant populations (27,000 and 54,000 plants ha −1). Treatments were replicated four times. Each treatment plot was subdivided into six subplots for harvesting at 6-month intervals beginning 1 year after transplanting. Results showed that transplanting date did not affect plant size or latex concentration or yield consistently. Instead, it appeared that the time of harvest (fall vs. spring) was more important. The sixth (last harvest) in the fall planting date and the fifth harvest date in the spring planting date were the optimum for plant biomass and latex, rubber, and resin concentrations and yields. The lines AZ-1 and AZ-3 were larger, whereas AZ-5 had higher latex and rubber concentrations than the control, 11591. The greater plant population (54,000 plants ha −1) had higher biomass, rubber, and resin yields than the lower population (27,000 plants ha −1) at the early harvest dates, but not at the later harvest dates (5 and 6). More studies must to be conducted to determine the optimum plant population and transplanting date for other newly developed guayule germplasm lines.

Screening method for detecting proteases in plant latices

Screening method for detecting proteases in plant latices

Occurrence and Properties of Proteases in Plant Latices

Proteases appear to play key roles in the regulation of biological processes in plants, such as the recognition of pathogens and pests and the induction of effective defence responses. On the other side these enzymes are able to activate protease-activated receptors (PARs) and in that way to act as agents with pharmacological and toxicological significance. An important source of plant proteases used in traditional medicine and industry is latex. Over 110 latices of different plant families are known to contain at least one proteolytic enzyme. Most of them belong to the cysteine or serine endopeptidases family and only one to the aspartatic endopeptidases family. This review focuses on the characterization of proteases found in latices of several plant families (Apocynaceae, Asclepiadaceae, Asteraceae, Caricaceae, Convolvulaceae, Euphorbiaceae, Moraceae), and summarizes the known chemical and biological properties of the isolated proteases as well as their importance in pharmacology and toxicology.

Purification, crystallization and preliminary X-ray analysis of CMS1MS2: a cysteine proteinase from Carica candamarcensis latex.

Cysteine proteinases from the latex of plants of the family Caricaceae are widely used industrially as well as in pharmaceutical preparations. In the present work, a 23 kDa cysteine proteinase from Carica candamarcensis latex (designated CMS1MS2) was purified for crystallization using three chromatography steps. The enzyme shows about fourfold higher activity than papain with BAPNA as substrate. Crystals suitable for X-ray diffraction experiments were obtained by the hanging-drop method in the presence of PEG and ammonium sulfate as precipitants. The crystals are monoclinic (space group P2(1)), with unit-cell parameters a = 53.26, b = 75.71, c = 53.23 A, beta = 96.81 degrees , and diffract X-rays to 1.8 A resolution.

A Stable Serine Protease, Wrightin, from the Latex of the Plant Wrightia tinctoria (Roxb.) R. Br.: Purification and Biochemical Properties

Today proteases have become an integral part of the food and feed industry, and plant latex could be a potential source of novel proteases with unique substrate specificities and biochemical properties. A new protease named "wrightin" is purified from the latex of the plant Wrightia tinctoria (Family Apocynaceae) by cation-exchange chromatography. The enzyme is a monomer having a molecular mass of 57.9 kDa (MALDI-TOF), an isoelectric point of 6.0, and an extinction coefficient (epsilon1%280) of 36.4. Optimum activity is achieved at a pH of 7.5-10 and a temperature of 70 degrees C. Wrightin hydrolyzes denatured natural substrates such as casein, azoalbumin, and hemoglobin with high specific activity; for example, the Km value is 50 microM for casein as substrate. Wrightin showed weak amidolytic activity toward L-Ala-Ala-p-nitroanilide but completely failed to hydrolyze N-alpha-benzoyl- DL-arginine-p-nitroanilide (BAPNA), a preferred substrate for trypsin-like enzymes. Complete inhibition of enzyme activity by serine protease inhibitors such as PMSF and DFP indicates that the enzyme belongs to the serine protease class. The enzyme was not inhibited by SBTI and resists autodigestion. Wrightin is remarkably thermostable, retaining complete activity at 70 degrees C after 60 min of incubation and 74% of activity after 30 min of incubation at 80 degrees. Besides, the enzyme is very stable over a broad range of pH from 5.0 to 11.5 and remains active in the presence of various denaturants, surfactants, organic solvents, and metal ions. Thus, wrightin might be a potential candidate for various applications in the food and biotechnological industries, especially in operations requiring high temperatures.

Developing novel anthelmintics from plant cysteine proteinases

Intestinal helminth infections of livestock and humans are predominantly controlled by treatment with three classes of synthetic drugs, but some livestock nematodes have now developed resistance to all three classes and there are signs that human hookworms are becoming less responsive to the two classes (benzimidazoles and the nicotinic acetylcholine agonists) that are licensed for treatment of humans. New anthelmintics are urgently needed, and whilst development of new synthetic drugs is ongoing, it is slow and there are no signs yet that novel compounds operating through different modes of action, will be available on the market in the current decade. The development of naturally-occurring compounds as medicines for human use and for treatment of animals is fraught with problems. In this paper we review the current status of cysteine proteinases from fruits and protective plant latices as novel anthelmintics, we consider some of the problems inherent in taking laboratory findings and those derived from folk-medicine to the market and we suggest that there is a wealth of new compounds still to be discovered that could be harvested to benefit humans and livestock.

First complete chromosomal organization of a protozoan plant parasite ( Phytomonas spp.)

Phytomonas spp. are members of the family Trypanosomatidae that parasitize plants and may cause lethal diseases in crops such as Coffee Phloem necrosis, Hartrot in coconut, and Marchitez sorpresiva in oil palm. In this study, the molecular karyotype of 6 isolates from latex plants has been entirely elucidated by pulsed-field gel electrophoresis and DNA hybridization. Twenty-one chromosomal linkage groups constituting heterologous chromosomes and sizing between 0.3 and 3 Mb could be physically defined by the use of 75 DNA markers (sequence-tagged sites and genes). From these data, the genome size can be estimated at 25.5 (± 2) Mb. The physical linkage groups were consistently conserved in all strains examined. Moreover, the finding of several pairs of different-sized homologous chromosomes strongly suggest diploidy for this organism. The definition of the complete molecular karyotype of Phytomonas represents an essential primary step toward sequencing the genome of this parasite of economical importance.

Crotogossamide, a Cyclic Nonapeptide from the Latex of Croton gossypifolius

A new cyclic nonapeptide, crotogossamide (1), was isolated from the latex of Croton gossypifolius. Its structure was elucidated by use of 1D and 2D NMR and MS and by hydrolysis followed by GC-MS analysis as cyclo(-Gly(1)-Ala(2)-Ser(3)-Gly(4)-Leu(5)-Asn(6)-Gly(7)-Ile(8)-Phe(9)-). This is the first report of a cyclic peptide from a Croton species. The known flavonoids kaempferol 3-O-rhamnopyranoside, quercitrin, and myricitrin were also isolated from the plant latex.

Comparative Study on Plant Latex Proteases and their Involvement in Hemostasis: A Special Emphasis on Clot Inducing and Dissolving Properties

In the present study we compared the clot inducing and dissolving properties of Calotropis gigantea R. Br. (Asclepiadaceae), Synadenium grantii Hook. f. (Euphorbiaceae) and Wrightia tinctoria R. Br. (Apocynaceae) latex extracts. All the three latex extracts hydrolyzed casein, fibrinogen and crude fibrin dose-dependently. The proteolytic action on fibrinogen subunity was in the order of Aalpha > Bbeta > gamma. All extracts exhibited procoagulant activity as assayed by re-calcification time. However, thrombin like activity is restricted to C. gigantea. In addition, the extracts dose-dependently hydrolyzed blood and plasma clots. Furthermore, the hydrolyzing pattern of fibrin in the plasma clot was substantiated by SDS-PAGE. The extracts hydrolyzed all the subunits (alpha polymer, alpha-chains, gamma-gamma dimer and beta-chain) of fibrin efficiently. Both fibrinogenolytic and fibrinolytic activity potency of the extracts were in the order of C. gigantea > S. grantii > W. tinctoria. Among the three latices, C. gigantea is toxic with a minimum hemorrhagic dose (MHD) of > 75 microg, whereas S. grantii and W. tinctoria latex extracts were non-toxic and did not induce any hemorrhagic effect at the tested dose (> 200 microg). The proteolytic activity of C. gigantea latex extract on different substrates was inhibited by IAA. On the other hand, the proteolytic activities of S. grantii and W. tinctoria were inhibited by PMSF. Thus, this study provides the basis for the probable action of plant latex proteases to stop bleeding and effect wound healing as exploited in folk medicine.

Nematicidal effects of cysteine proteinases against sedentary plant parasitic nematodes

Cysteine proteinases from the fruit and latex of plants, such as papaya, pineapple and fig, have previously been shown to have substantial anthelmintic efficacy, in vitro and in vivo, against a range of animal parasitic nematodes. In this paper, we describe the in vitro effects of these plant extracts against 2 sedentary plant parasitic nematodes of the genera Meloidogyne and Globodera. All the plant extracts examined caused digestion of the cuticle and decreased the activity of the tested nematodes. The specific inhibitor of cysteine proteinases, E-64, blocked this activity completely, indicating that it was essentially mediated by cysteine proteinases. In vitro, plant cysteine proteinases are active against second-stage juveniles of M. incognita and M. javanica, and some cysteine proteinases also affect the second-stage juveniles of Globodera rostochiensis. It is not known yet whether these plant extracts will interfere with, or prevent invasion of, host plants.

植物乳液および乳液中のタンパク質・二次代謝物質は虫害抵抗性において決定的に重要である：パパイア乳液中のシステインプロテアーゼとクワ乳液中の糖類似アルカロイドの役割

植物乳液および乳液中のタンパク質・二次代謝物質は虫害抵抗性において決定的に重要である：パパイア乳液中のシステインプロテアーゼとクワ乳液中の糖類似アルカロイドの役割

Anthelmintic action of plant cysteine proteinases against the rodent stomach nematode,Protospirura muricola,in vitroandin vivo

Cysteine proteinases from the fruit and latex of plants, including papaya, pineapple and fig, were previously shown to have a rapid detrimental effect, in vitro, against the rodent gastrointestinal nematodes, Heligmosomoides polygyrus (which is found in the anterior small intestine) and Trichuris muris (which resides in the caecum). Proteinases in the crude latex of papaya also showed anthelmintic efficacy against both nematodes in vivo. In this paper, we describe the in vitro and in vivo effects of these plant extracts against the rodent nematode, Protospirura muricola, which is found in the stomach. As in earlier work, all the plant cysteine proteinases examined, with the exception of actinidain from the juice of kiwi fruit, caused rapid loss of motility and digestion of the cuticle, leading to death of the nematode in vitro. In vivo, in contrast to the efficacy against H. polygyrus and T. muris, papaya latex only showed efficacy against P. muricola adult female worms when the stomach acidity had been neutralized prior to administration of papaya latex. Therefore, collectively, our studies have demonstrated that, with the appropriate formulation, plant cysteine proteinases have efficacy against nematodes residing throughout the rodent gastrointestinal tract.