Effects of mechanical wounding on Carica papaya cysteine endopeptidases accumulation and activity

The mechanical wounding impact on the Carica papaya latex protein pattern was investigated by analyzing three latexes. A first one commercially available, a second harvested from unripe but fully grown fruits, both obtained from regularly tapped fruits. A third one was collected from similar fruits but wounded for the first time. The results demonstrated both quantitative and qualitative changes in the protein content and in the enzymatic activity. Repeated wounding results in either, accumulation or activation (or both of them) of papain, chymopapain and caricain. Furthermore, new cysteine protease activity was found to transiently accumulate in the latex collected from newly wounded fruits. The possible implication of this enzymatic material in the papaya cysteine endopeptidases pro-forms activation is discussed.     

Chymopapain. Chromatographic purification and immunological characterization.

Chymopapain (EC 3.4.22.6) was purified from commercially available spray-dried latex of papaya (Carica papaya) fruit by (NH4)2SO4 fractionation and fast protein chromatography on the Mono S cation-exchange column. Multiple forms of chymopapain separated chromatographically were shown to be immunologically identical. A major form was isolated and found to be homogeneous by several criteria, and fully active, and its N-terminal amino acid was identified as tyrosine. Latex from fresh unripe papaya fruit contained predominantly one form of chymopapain, and it is concluded that chymopapain is a single enzyme distinct from the other cysteine proteinases of C. papaya latex.     

Purification and characterization of a wound-inducible thaumatin-like protein from the latex of Carica papaya

A 22.137 kDa protein constituent of fresh latex was isolated both from the latex of regularly damaged papaya trees and from a commercially available papain preparation. The protein was purified up to apparent homogeneity and was shown to be absent in the latex of papaya trees that had never been previously mechanically injured. This suggests that the protein belongs to pathogenesis-related protein family, as expected for several other protein constituents of papaya latex. The protein was identified as a thaumatin-like protein (class 5 of the pathogenesis-related proteins) on the basis of its partial amino acid sequence. By sequence analysis of the Carica genome, three different forms of thaumatin-like protein were identified, where the latex constituent belongs to a well-known form, allowing the molecular modeling of its spatial structure. The papaya latex thaumatin-like protein was further characterized. The protein appears to be stable in the pH interval from 2 to 10 and resistant to chemical denaturation by guanidium chloride, with a of 15.2 kcal/mol and to proteolysis by the four papaya cysteine proteinases. The physiological role of this protein is discussed.     

Label-free quantitative proteomics reveals differentially regulated proteins in the latex of sticky diseased Carica papaya L. plants

Papaya meleira virus (PMeV) is so far the only described laticifer-infecting virus, the causal agent of papaya (Carica papaya L.) sticky disease. The effects of PMeV on the laticifers' regulatory network were addressed here through the proteomic analysis of papaya latex. Using both 1-DE- and 1D-LC-ESI-MS/MS, 160 unique papaya latex proteins were identified, representing 122 new proteins in the latex of this plant. Quantitative analysis by normalized spectral counting revealed 10 down-regulated proteins in the latex of diseased plants, 9 cysteine proteases (chymopapain) and 1 latex serine proteinase inhibitor. A repression of papaya latex proteolytic activity during PMeV infection was hypothesized. This was further confirmed by enzymatic assays that showed a reduction of cysteine-protease-associated proteolytic activity in the diseased papaya latex. These findings are discussed in the context of plant responses against pathogens and may greatly contribute to understand the roles of laticifers in plant stress responses.     

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.     

伤处理和乙烯对桃ACC氧化酶基因表达的影响

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A thaumatin-like gene in nonclimacteric pepper fruits used as molecular marker in probing disease resistance,ripening, and sugar accumulation

During pepper (Capsicum annuum) fruit ripening, the ripe fruit interaction with the anthracnose fungus, Colletotrichum gloeosporioides, is generally incompatible. However, the unripe fruit can interact compatibly with the fungus. A gene, designated PepTLP (for pepper thaumatin-like protein), was isolated and characterized by using mRNA differential display. The PepTLP gene encodes a protein homologous to other thaumatin-like proteins and contains 16 conserved cysteine residues and the consensus pattern of thaumatin. PepTLP gene expression is developmentally regulated during ripening. The accumulation of PepTLP mRNA and PepTLP protein in the incompatible interaction was higher than that in the compatible one. Furthermore, PepTLP gene expression was stimulated by both jasmonic acid treatment and wounding during ripening, but by wounding only in the unripe fruit. Immunolocalization studies showed that it is localized to the intercellular spaces among cortical cells. The expression of the PepTLP gene upon fungal infection was a rise from the early-breaker fruit. The development of anthracnose became significantly prevented with beginning of fruit ripening, and the sum total of sugar accumulation increased. The results suggest that the PepTLP gene can be used as a molecular marker in probing for disease resistance, ripening, and sugar accumulation in nonclimacteric pepper fruits.     

Papain protects papaya trees from herbivorous insects: role of cysteine proteases in latex

Many plants contain latex that exudes when leaves are damaged, and a number of proteins and enzymes have been found in it. The roles of those latex proteins and enzymes are as yet poorly understood. We found that papain, a cysteine protease in latex of the Papaya tree (Carica papaya, Caricaceae), is a crucial factor in the defense of the papaya tree against lepidopteran larvae such as oligophagous Samia ricini (Saturniidae) and two notorious polyphagous pests, Mamestra brassicae (Noctuidae) and Spodoptera litura (Noctuidae). Leaves of a number of laticiferous plants, including papaya and a wild fig, Ficus virgata (Moraceae), showed strong toxicity and growth inhibition against lepidopteran larvae, though no apparent toxic factors from these species have been reported. When the latex was washed off, the leaves of these lactiferous plants lost toxicity. Latexes of both papaya and the wild fig were rich in cysteine-protease activity. E-64, a cysteine protease-specific inhibitor, completely deprived the leaves of toxicity when painted on the surface of papaya and fig leaves. Cysteine proteases, such as papain, ficin, and bromelain, all showed toxicity. The results suggest that plant latex and the proteins in it, cysteine proteases in particular, provide plants with a general defense mechanism against herbivorous insects.     

Identification of a putative triacylglycerol lipase from papaya latex by functional proteomics

Latex from Caricaceae has been known since 1925 to contain strong lipase activity. However, attempts to purify and identify the enzyme were not successful, mainly because of the lack of solubility of the enzyme. Here, we describe the characterization of lipase activity of the latex of Vasconcellea heilbornii and the identification of a putative homologous lipase from Carica papaya. Triacylglycerol lipase activity was enriched 74-fold from crude latex of Vasconcellea heilbornii to a specific activity (SA) of 57 μmol·min(-1)·mg(-1) on long-chain triacylglycerol (olive oil). The extract was also active on trioctanoin (SA = 655 μmol·min(-1)·mg(-1) ), tributyrin (SA = 1107 μmol·min(-1)·mg(-1) ) and phosphatidylcholine (SA = 923 μmol·min(-1)·mg(-1) ). The optimum pH ranged from 8.0 to 9.0. The protein content of the insoluble fraction of latex was analyzed by electrophoresis followed by mass spectrometry, and 28 different proteins were identified. The protein fraction was incubated with the lipase inhibitor [(14) C]tetrahydrolipstatin, and a 45 kDa protein radiolabeled by the inhibitor was identified as being a putative lipase. A C. papaya cDNA encoding a 55 kDa protein was further cloned, and its deduced sequence had 83.7% similarity with peptides from the 45 kDa protein, with a coverage of 25.6%. The protein encoded by this cDNA had 35% sequence identity and 51% similarity to castor bean acid lipase, suggesting that it is the lipase responsible for the important lipolytic activities detected in papaya latex.     

Domain construction of cherry-tomato lectin: relation to newly found 42-kDa protein

In the early stage of ripening of cherry-tomato fruits (Lycopersicon esculentum var. cherry), the lectin activity increased logarithmically and reached a plateau at day 10 after flowering. During purification of lectin from ripe and unripe fruits, a 42-kDa protein was found abundantly in unripe fruits. The protein cross-reacted with anti-cherry-tomato-lectin serum, retained chitin-binding ability, but showed no lectin activity. Comparative studies between the structure of the lectin and the 42-kDa protein were done. N-Terminal amino acid sequences of the lectin, peptides derived from the S-pyridylethylated lectin, and fragments generated by limited proteolysis of the native lectin showed that the lectin was comprised of three domains, Hyp-rich, Cys-rich, and Gln-rich, and the alignment of them was as this order from the N-terminus. Studies on the 42-kDa protein showed that it contained two of the three domains, Cys-rich and Gln-rich, but the amino acid sequence analysis showed that the protein should be a product of another gene.     

Identification of a new phospholipase D in Carica papaya latex

Phospholipase D (PLD) is a lipolytic enzyme involved in signal transduction, vesicle trafficking and membrane metabolism. It catalyzes the hydrolysis and transphosphatidylation of glycerophospholipids at the terminal phosphodiester bond. The presence of a PLD in the latex of Carica papaya (CpPLD1) was demonstrated by transphosphatidylation of phosphatidylcholine (PtdCho) in the presence of 2% ethanol. Although the protein could not be purified to homogeneity due to its presence in high molecular mass aggregates, a protein band was separated by SDS-PAGE after SDS/chloroform-methanol/TCA-acetone extraction of the latex insoluble fraction. This material was digested with trypsin and the amino acid sequences of the tryptic peptides were determined by micro-LC/ESI/MS/MS. These sequences were used to identify a partial cDNA (723 bp) from expressed sequence tags (ESTs) of C. papaya. Based upon EST sequences, a full-length gene was identified in the genome of C. papaya, with an open reading frame of 2424 bp encoding a protein of 808 amino acid residues, with a theoretical molecular mass of 92.05 kDa. From sequence analysis, CpPLD1 was identified as a PLD belonging to the plant phosphatidylcholine phosphatidohydrolase family.     

Characterization of typo-, regio-, and stereo-selectivities of babaco latex lipase in aqueous and organic media

The unripe fruit of babaco (Vasconcellea × heilbornii; syn. Carica pentagona) contains a latex, similar to that in Carica papaya, which exhibits lipolytic activity. Herein, the regioselectivity, stereoselectivity and typoselectivity in both hydrolysis and acyltransfer reactions of babaco latex lipases were studied and compared to those of Carica papaya latex. In hydrolysis, both biocatalysts are 1,3-regioselective with ratios for 1,2-2,3-diacylglycerols/1,3-diacylglycerol of 6.5 and 21 for babaco and papaya, respectively. In contrast, papaya latex had a slight sn-3 stereopreference. Babaco latex displayed a higher activity on triacylglycerols with short chain and unsaturated fatty acids     

Purification and characterization of the cysteine proteinases in the latex of Vasconcellea spp

Latex of all Vasconcellea species analyzed to date exhibits higher proteolytic amidase activities, generally attributed to cysteine proteinases, than the latex of Carica papaya. In the present study, we show that this higher activity is correlated with a higher concentration of enzymes in the latex of Vasconcellea fruits, but in addition also results from the presence of other cysteine proteinases or isoforms. In contrast to the cysteine proteinases present in papaya latex, which have been extensively studied, very little is known about the cysteine proteinases of Vasconcellea spp. In this investigation, several cDNA sequences coding for cysteine proteinases in Vasconcellea x heilbornii and Vasconcellea stipulata were determined using primers based on conserved sequences. In silico translation showed that they hold the characteristic features of all known papain-class cysteine proteinases, and a phylogenetic analysis revealed the existence of several papain and chymopapain homologues in these species. Ion-exchange chromatography and gel filtration procedures were applied on latex of V. x heilbornii in order to characterize its cysteine proteinases at the protein level. Five major protein fractions (VXH-I-VXH-V) revealing very high amidase activities (between 7.5 and 23.3 nkat x mg protein(-1)) were isolated. After further purification, three of them were N-terminally sequenced. The observed microheterogeneity in the N-terminal and cDNA sequences reveals the presence of several distinct cysteine proteinase isoforms in the latex of Vasconcellea spp.     

Papain reduces gastric acid secretion induced by histamine and other secretagogues in anesthetized rats

We studied the effect of papain on rats' gastric acid secretion and found that: 1. Feeding of latex of unripe papaya fruit significantly reduced gastric acid secretion induced by methacholine; 2. Feeding of crystalline papain in doses of 3.2 mg/kg reduced gastric acid secretion induced by histamine, methacholine and tetragastrin; 3. The reduction of gastric acid secretion was observed as early as 2 hours after papain feeding, lasted up to 48 hours, and waned within 96 hours; 4. Intraperitoneal injection of papain had no effect on acid secretion. These results led us to believe tha the effect of papain on gastric acid secretion is a local one acting directly on the gastric mucosa, and this local effect of a single dose of papain is reversible, causing no permanent damage to the mucosa.     

The effect of unripe fruits on ripe fruit removal by birds in Pistacia terebinthus: flag or handicap?

The shrub Pistacia terebinthus produces crowded infructescences with up to several hundred fruits, which are bright red when unripe and turn green when ripe. Most fruits contain an empty seed and never reach maturity. More ripe fruits were removed by birds from experimental bicolored fruit displays (consisting of infructescences with ten ripe fruits and stripped of unripe fruits, paired with infructescences with only unripe fruits) than from monocolored ones (single infructescences with ten ripe fruits and stripped of unripe fruits). Thus, the presence of unripe fruits seems to increase the conspicuousness or attractiveness of fruit displays to fruit-eating birds. A second experiment compared ripe fruit removal from experimental infructescences having only ripe fruits, with that from control infructescences containing both ripe fruits and natural numbers of unripe fruits, all on P. terebinthus plants. Unlike the first experiment, each bicolored display in this case consisted of a single infructescence with both unripe and ripe fruits. A higher proportion of ripe fruits was removed by birds from infructescences free of unripe fruits. This result suggests that the presence of unripe fruits reduces the accessibility of ripe fruits for fruit-eating birds. This is further supported by field observations of bird foraging behavior.     

Anti-inflammatory and anti-arthritic activities of silymarin acting through inhibition of 5-lipoxygenase.

Silymarin, a mixture of flavonolignans, comprised mainly of three isomers, silybin, silydianin and silychristin isolated from the fruits of Silybum marianum, is currently in therapeutic use as a hepatoprotective agent. Silymarin on evaluation exhibited significant antiinflammatory and antiarthritic activities in the papaya latex induced model of inflammation and mycobacterial adjuvant induced arthritis in rats. Results of the study indicate its action through inhibition of 5-lipoxygenase for antiinflammatory and antiarthritic activities.     

The thiol proteinases from the latex of Carica papaya L. III. The primary structure of chymopapain

The amino-acid sequence of chymopapain is presented. It was isolated from the latex of the fruits from the tropical species Carica papaya L. and is, besides papain and papaya proteinase omega, the third thiol proteinase from this source. The primary structure contains 218 amino-acid residues. It was deduced from sequence analysis of the native enzyme and of peptides obtained by tryptic, chymotryptic, peptic, thermolysinolytic and mild acidic hydrolysis. Out of a total of eight cysteine residues, six are involved in the formation of three disulfide bonds, the location of which has been established with the help of peptic and thermolysinolytic peptides and fragments, obtained by mild acidic hydrolysis. Chymopapain shares 126 identical amino-acid residues (58%) with papain and 141 (65%) with papaya proteinase omega, including the three disulfide bridges and the free cysteine in position 25, required for activity. Except some amino-acid residues in the substrate-binding site, all residues involved in the catalytic mechanism are conserved. The homology between papaya proteinases is discussed.     

Isolation of an osmotin-like protein gene from strawberry and analysis of the response of this gene to abiotic stresses

A strawberry genomic clone containing an osmotin-like protein (OLP) gene, designated FaOLP2, was isolated and sequenced. FaOLP2 is predicted to encode a precursor protein of 229 amino acid residues, and its sequence shares high degrees of homology with a number of other OLPs. Genomic DNA hybridization analysis indicated that FaOLP2 represents a multi-gene family. The expression of FaOP2 in different strawberry organs was analyzed using real-time PCR. The results showed that FaOLP2 expressed at different levels in leaves, crowns, roots, green fruits and ripe red fruits. In addition, the expression of FaOLP2 under different abiotic stresses was analyzed at different time points. All of the three tested abiotic stimuli, abscisic acid, salicylic acid and mechanical wounding, triggered a significant induction of FaOLP2 within 2-6h post-treatment. Moreover, FaOLP2 was more prominently induced by salicylic acid than by abscisic acid or mechanical wounding. The positive responses of FaOLP2 to the three abiotic stimuli suggested that strawberry FaOLP2 may help to protect against osmotic-related environmental stresses and that it may also be involved in plant defense system against pathogens.     

Localization of benzyl glucosinolate and thioglucosidase in Carica papaya fruit

Macerated papaya seeds and pulp contained benzyl isothiocyanate, produced by the enzymatic hydrolysis of benzyl glucosinolate by thioglucosidase. The substrate and enzyme were localized in different areas. In mature papaya seeds, thioglucosidase was found in sarcotestae but not in endosperms, while the reverse was true for benzyl glucosinolate, which constituted more than 6% (w/w) of the endosperms. Both the enzyme and substrate were present in embryos and the amount of the latter was 3·9% (w/w). In immature papaya pulp, benzyl glucosinolate was localized principally, if not exclusively, in the latex, ranging from 7·3 to 11·6% of the dry wt of latex fluid. No thioglucosidase activity was found in papaya latex. The possible significance of the localization of this enzyme-substrate system and aspects concerning functions of papaya latex are discussed.