cDNA cloning, expression, and antifungal activity of chitinase from Ficus microcarpa latex: difference in antifungal action of chitinase with and without chitin-binding domain.

MAIN CONCLUSION: A chitin-binding domain could contribute to the antifungal ability of chitinase through its affinity to the fungal lateral wall by hydrophobic interactions. Complementary DNA encoding the antifungal chitinase of gazyumaru (Ficus microcarpa), designated GlxChiB, was cloned and expressed in Escherichia coli cells. The results of cDNA cloning showed that the precursor of GlxChiB has an N-terminal endoplasmic reticulum targeting signal and C-terminal vacuolar targeting signal, whereas mature GlxChiB is composed of an N-terminal carbohydrate-binding module family-18 domain (CBM18) and a C-terminal glycoside hydrolase family-19 domain (GH19) with a short linker. To clarify the role of the CBM18 domain in the antifungal activity of chitinase, the recombinant GlxChiB (wild type) and its catalytic domain (CatD) were used in quantitative antifungal assays under different ionic strengths and microscopic observations against the fungus Trichoderma viride. The antifungal activity of the wild type was stronger than that of CatD under all ionic strength conditions used in this assay; however, the antifungal activity of CatD became weaker with increasing ionic strength, whereas that of the wild type was maintained. The results at high ionic strength further verified the contribution of the CBM18 domain to the antifungal ability of GlxChiB. The microscopic observations clearly showed that the wild type acted on both the tips and the lateral wall of fungal hyphae, while CatD acted only on the tips. These results suggest that the CBM18 domain could contribute to the antifungal ability of chitinase through its affinity to the fungal lateral wall by hydrophobic interactions.

Convergent evolution of the UbiA prenyltransferase family underlies the independent acquisition of furanocoumarins in plants.

Furanocoumarins (FCs) are plant-specialized metabolites with potent allelochemical properties. The distribution of FCs is scattered with a chemotaxonomical tendency towards four distant families with highly similar FC pathways. The mechanism by which this pathway emerged and spread in plants has not been elucidated. Furanocoumarin biosynthesis was investigated in Ficus carica (fig, Moraceae), focusing on the first committed reaction catalysed by an umbelliferone dimethylallyltransferase (UDT). Comparative RNA-seq analysis among latexes of different fig organs led to the identification of a UDT. The phylogenetic relationship of this UDT to previously reported Apiaceae UDTs was evaluated. The expression pattern of F. carica prenyltransferase 1 (FcPT1) was related to the FC contents in different latexes. Enzymatic characterization demonstrated that one of the main functions of FcPT1 is UDT activity. Phylogenetic analysis suggested that FcPT1 and Apiaceae UDTs are derived from distinct ancestors, although they both belong to the UbiA superfamily. These findings are supported by significant differences in the related gene structures. This report describes the identification of FcPT1 involved in FC biosynthesis in fig and provides new insights into multiple origins of the FC pathway and, more broadly, into the adaptation of plants to their environments.

Comparative multi-omics analysis reveals diverse latex-based defense strategies against pests among latex-producing organs of the fig tree (Ficus carica).

MAIN CONCLUSION: Latexes in immature fruit, young petioles and lignified trunks of fig trees protect the plant using toxic proteins and metabolites in various organ-dependent ways. Latexes from plants contain high amounts of toxic proteins and metabolites, which attack microbes and herbivores after exudation at pest-induced wound sites. The protein and metabolite constituents of latexes are highly variable, depending on the plant species and organ. To determine the diversity of latex-based defense strategies in fig tree (Ficus carica) organs, we conducted comparative proteomic, transcriptomic and metabolomic analyses on latexes isolated from immature fruit, young petioles and lignified trunks of F. carica after constructing a unigene sequence library using RNA-seq data. Trypsin inhibitors were the most abundant proteins in petiole latex, while cysteine proteases ("ficins") were the most abundant in immature fruit and trunk latexes. Galloylglycerol, a possible defense-related metabolite, appeared to be highly accumulated in all three latexes. The expression levels of pathogenesis-related proteins were highest in the latex of trunk, suggesting that this latex had adapted a defensive role against microbe attacks. Although young petioles and immature fruit are both unlignified soft organs, and potential food for herbivorous insects, unigenes for the sesquiterpenoid pathway, which likely produces defense-associated volatiles, and the phenylpropanoid pathway, which produces toxic furanocoumarins, were expressed less in immature fruit latex. This difference may indicate that while petioles and fruit protect the plant from attack by herbivores, the fruit must also attract insect pollinators at younger stages and animals after ripening. We also suggest possible candidate transcription factors and signal transduction proteins that are involved in the differential expression of the unigenes.

Enzymatic hydrolysis of PET: functional roles of three Ca2+ ions bound to a cutinase-like enzyme, Cut190*, and its engineering for improved activity.

Cut190 from Saccharomonospora viridis AHK190 (Cut190) is the only cutinase that exhibits inactive (Ca2+-free) and active (Ca2+-bound) states, although other homologous cutinases always maintain the active states (Ca2+-free and bound). The X-ray crystallography of the S176A mutant of Cut190* (Cut190_S226P/R228S) showed that three Ca2+ ions were bound at sites 1-3 of the mutant. We analyzed the roles of three Ca2+ ions by mutation and concluded that they play different roles in Cut190* for activation (sites 1 and 3) and structural and thermal stabilization (sites 2 and 3). Based on these analyses, we elucidated the mechanism for the conformational change from the Ca2+-free inactive state to the Ca2+-bound active state, proposing the novel Ca2+ effect on structural dynamics of protein. The introduction of a disulfide bond at Asp250 and Glu296 in site 2 remarkably increased the melting temperatures of the mutant enzymes by more than 20-30 °C (while Ca2+-bound) and 4-14 °C (while Ca2+-free), indicating that a disulfide bond mimics the Ca2+ effect. Replacement of surface asparagine and glutamine with aspartic acid, glutamic acid, or histidine increased the melting temperatures. Engineered mutant enzymes were evaluated by an increase in melting temperatures and kinetic values, based on the hydrolysis of poly(butylene succinate-co-adipate) and microfiber polyethylene terephthalate (PET). A combined mutation, Q138A/D250C-E296C/Q123H/N202H, resulted in the highest thermostability, leading to the maximum degradation of PET film (more than 30%; approximately threefold at 70 °C, compared with that of Cut190* at 63 °C).

Ferritin 2 domain-containing protein found in lacquer tree (Toxicodendron vernicifluum) sap has negative effects on laccase and peroxidase reactions.

Lacquer tree sap, a raw material of traditional paints in East Asia, is hardened through laccase-catalyzed oxidation and the following polymerization of phenolic compound urushiol. In the sap's water-insoluble fraction, we found two plantacyanins and a ferritin 2 domain-containing protein (TvFe2D, a homolog of Arabidopsis AT1G47980 and AT3G62730). The recombinant TvFe2D protein suppressed the accumulation of laccase-catalyzed oxidation products of a model substrate syringaldazine without decreasing oxygen consumption, the second substrate of laccase. The suppression was also observed when another substrate guaiacol or another oxidizing enzyme peroxidase was used. The functional domain of the suppression was the C-terminal half, downstream of the ferritin 2 domain. The results suggest that this protein may be involved in regulating the sap polymerization/hardening. We also discuss the possibility that homologous proteins of TvFe2D in other plants might be involved in the laccase- or peroxidase-mediated polymerization of phenolic compounds, such as lignin and flavonoids.

Polyvinyl alcohol and polyethylene glycol form polymer bodies in the periplasm of Sphingomonads that are able to assimilate them.

Scanning electron microscopy (SEM) shows remarkable morphological surface changes in Sphingopyxis sp. 113P3 cells grown in polyvinyl alcohol (PVA) but not in Luria-Bertani medium (LB) (Hu et al. in Arch Microbiol 188: 235-241, 2007). However, transmission electron microscopy showed no surface changes in PVA-grown cells and revealed the presence of polymer bodies in the periplasm of PVA-grown cells, which were not observed in LB-grown cells. The presence of polymer bodies was supported by low-vacuum SEM observation of PVA- and LB-grown cells of strain 113P3, and the presence of similar polymer bodies was also found when Sphingopyxis macrogoltabida 103 and S. terrae were grown in polyethylene glycol (PEG). The extraction of PVA and PEG from the periplasmic fraction of cells using a modified Anraku and Heppel method and their analysis by MALDI-TOF mass spectrometry strongly suggested that the polymer bodies are composed of PVA and PEG, respectively, in Sphingopyxis sp. 113P3 (PVA degrader) and Sphingopyxis macrogoltabida 103 or S. terrae (PEG degraders). PEG-grown S. macrogoltabida 103 and S. terrae showed higher transport of (14)C-PEG 4000 than LB-grown cells. Recombinant PegB (TonB-dependent receptor-like protein consisting of a barrel structure) interacted with PEG 200, 4000 and 20000, suggesting that the barrel protein in the outer membrane contributes to the transport of PEG into the periplasm.

Two chitinase-like proteins abundantly accumulated in latex of mulberry show insecticidal activity.

BACKGROUND: Plant latex is the cytoplasm of highly specialized cells known as laticifers, and is thought to have a critical role in defense against herbivorous insects. Proteins abundantly accumulated in latex might therefore be involved in the defense system. RESULTS: We purified latex abundant protein a and b (LA-a and LA-b) from mulberry (Morus sp.) and analyzed their properties. LA-a and LA-b have molecular masses of approximately 50 and 46 kDa, respectively, and are abundant in the soluble fraction of latex. Western blotting analysis suggested that they share sequence similarity with each other. The sequences of LA-a and LA-b, as determined by Edman degradation, showed chitin-binding domains of plant chitinases at the N termini. These proteins showed small but significant chitinase and chitosanase activities. Lectin RCA120 indicated that, unlike common plant chitinases, LA-a and LA-b are glycosylated. LA-a and LA-b showed insecticidal activities when fed to larvae of the model insect Drosophila melanogaster. CONCLUSIONS: Our results suggest that the two LA proteins have a crucial role in defense against herbivorous insects, possibly by hydrolyzing their chitin.

Cloning and Aggregation Characterization of Rubber Elongation Factor and Small Rubber Particle Protein from Ficus carica.

Rubber elongation factor (REF) and small rubber particle protein (SRPP) are major latex proteins harvested from Hevea brasiliensis (the rubber tree; HbREF and HbSRPP, respectively). Their amino acid sequences exhibit high homology with each other. In the present study, we cloned two cDNAs encoding REF/SRPP-family proteins (FcREF/SRPP-1 and -2) from the laticifers of Ficus carica (fig tree). The amino acid sequences of these proteins showed high homology not only with each other but also with HbREF and HbSRPP. Recombinant FcREF/SRPP-1 and -2 were expressed in E. coli, and their aggregation properties were examined using a Congo red binding assay, agarose gel electrophoresis, and transmission electron microscopy. FcREF/SRPP-1 formed fibrils when incubated in PBS, and grew to micrometer-sized amorphous aggregates that precipitated rapidly. These aggregation properties of FcREF/SRPP-1 are quite similar to those of HbREF, although the growth rate and size of FcREF/SRPP-1 aggregates were inferior to those of HbREF. FcREF/SRPP-2 also formed aggregates during the incubation, but they did not precipitate, as has been reported for HbSRPP. Our results suggest that FcREF/SRPP-1 and -2 correspond to HbREF and HbSRPP, respectively. These aggregation properties could provide useful benchmarks for classifying REF/SRPP-family proteins as REF or SRPP.

Transcriptome and proteome analyses provide insight into laticifer's defense of Euphorbia tirucalli against pests.

The cytoplasm of laticifers, which are plant cells specialized for rubber production and defense against microbes and herbivores, is a latex. Although laticifers share common functions, the protein constituents of latexes are highly variable among plant species and even among organs. In this study, transcriptomic and proteomic analyses of Euphorbia tirucalli's (Euphorbiaceae) latex were conducted to determine the molecular basis of the laticifer's functions in this plant. The hybrid de novo assembly of Illumina mRNA-seq and expressed sequence tags obtained by Sanger's sequencing revealed 26,447 unigenes. A unigene similar to Arabidopsis embryo-specific protein 3 (AT5G62200), which is a PLAT domain-containing protein, and rubber elongation factor showed the highest expression levels. The proteome analysis, studied by liquid chromatography-mass spectrometry with the de novo assembled unigenes as the database, revealed 161 proteins in the latex, 107 of which were not detected in the stem. A gene ontology analysis indicated that the laticifer's proteome was enriched with proteins related to proteolysis, phosphatase, defense against various environmental stresses and lipid metabolisms. D-mannose-binding lectin, ricin (which lacked the N-terminal conserved ribosome-inactivating protein domain), chitinase and peroxidase were highly accumulated, as confirmed by two-dimensional polyacrylamide gel electrophoresis. Thus, the lectins and chitinase may be the major defensive proteins against pests, and the other defense-related proteins and transcripts detected in latex may work in coordination with them. Highly expressing unigenes with unknown functions are candidate novel defense- or rubber production-related genes.

Whole-mount silver staining of Arabidopsis and maize: a new method for staining secondary wall thickenings in tracheary elements.

Secondary wall thickenings in tracheary elements were specifically stained by incubation of Arabidopsis and maize in Silver Stain Plus (Bio-Rad) staining solution, after pretreatment with SDS and ethanol solution. Scanning electron microscopic analysis of sections of celery revealed that silver particles were deposited on the secondary wall thickenings, indicating that the staining was due to the deposition of silver through the interaction of the stain with lignin. This method is more sensitive than the acidified phloroglucinol method.