Cassia obtusifolia MetE as a cytosolic target for potassium isolespedezate, a leaf-opening factor of Cassia plants: target exploration by a compact molecular-probe strategy.

Affinity chromatography by using ligand-immobilized bead technology is generally the first choice for target exploration of a bioactive ligand. However, when a ligand has comparatively low affinity against its target, serious difficulties will be raised in affinity-based target detection. We report here that the use of compact molecular probes (CMP) will be advantageous in such cases; it enables the retention of moderate affinity between the ligand and its target in contrast to immobilizing the ligand on affinity beads that will cause a serious drop in affinity to preclude target detection. In the CMP strategy, a CMP containing an azide handle is used for an initial affinity-based labeling of target, and subsequent tagging by CuAAC with a large FLAG tag will give a tagged target protein. By using the CMP strategy, we succeeded in the identification of Cassia obtusifolia MetE as a cytosolic target protein of potassium isolespedezate (1), a moderately bioactive ligand.

Aluminum-induced cell wall peroxidase activity and lignin synthesis are differentially regulated by jasmonate and nitric oxide.

Cassia tora is an annual legume and cultivated as a traditional medicinal herb for multiple therapies including regulation of blood pressure and blood lipid. Because of naturally occurring acidic soils in southeastern China, this plant species may possess strategies for tolerance to low pH and aluminum toxicity. In the search for the regulatory basis of biochemical response to Al, cell wall-bound peroxidases, including lignin-generated peroxidases and NADH oxidases, were investigated in the root tips of C. tora. Activities of both types of peroxidases significantly increased with Al concentrations. Analysis with native PAGE also demonstrated the strong induction of cell wall peroxidases by Al. The Al-induced increasing activities of peroxidases were closely correlated with lignin accumulation and H 2O 2 production. The biochemical effect of exogenous nitric oxide (NO) and methyl jasmonic acid (MJ) was examined to investigate signal properties and lignin synthesis under Al stress. Application of MJ at 10 microM promoted root sensitivity to Al by activating apoplastic peroxidase activity and accumulating H 2O 2 and lignin, whereas the opposite action was found for NO. The sensitivity of apoplastic peroxidases under Al stress was associated with the cross-talk of MJ and NO signals. The analysis reveals that the activity of lipoxygenase (an enzyme for MJ biosynthesis), with its transcripts increased in Al-exposed roots, was depressed by NO exposure. The effect of MJ on intracellular NO production was also investigated. It is shown that NO staining with 4,5-diaminofluorescein diacetate fluorescence was intensified by Al but was suppressed by MJ. These results suggest that NO and MJ may interplay in signaling the cell wall peroxidase activity and lignin synthesis in the roots exposed to Al.

Phytotoxicity of Phytolacca americana leaf extracts on the growth, and physiological response of Cassia mimosoides.

We examined the allelochemical effects of control soil, native soil (treated soil), and leaf extracts of Phytolacca americana (pokeweed) on the germination rate and seedling growth of Cassia mimosoides var. nomame. We also studied the resulting changes in root-tip ultrastructure and peroxidase isozyme biochemistry. P. americana leaf extract inhibited seed germination, seedling growth, and biomass when compared to control and treated soil. Root and shoot growth in treated soil was stimulated relative to control soil, but root growth was inhibited by 50% in the leaf extract treatment. Biomass of C. mimosoides seedlings grown on leaf extract was reduced sevenfold when compared to the control seedlings. The amounts of total phenolic compounds in the leaf extract, treated soil, and control soil were 0.77, 0.14, and 0.03 mg l(-1), respectively. The root tips of C. mimsoides treated with leaf extracts of P. americana showed amyloplasts and large central vacuoles with electron-dense deposits inside them when compared to control root tips. The activity of guaiacol peroxidase (GuPOX) in whole plant, roots, and shoots of C. mimosoides increased as leaf extract increased; maximum activity was observed in extract concentrations of 75% and higher. Root GuPOX activity was three times higher than in shoots. Therefore, we conclude that inhibition of C. mimosoides growth is related to the phenolic compounds in the P. americana leaf extract and the ultrastructure changes in root-tip cells and increased GuPOX activity is a response to these allelochemicals.

The emergence of embryos from hard seeds is related to the structure of the cell walls of the micropylar endosperm, and not to endo-beta-mannanase activity.

BACKGROUND AND AIMS: Seeds of carob, Chinese senna, date and fenugreek are hard due to thickened endosperm cell walls containing mannan polymers. How the radicle is able penetrate these thickened walls to complete seed germination is not clearly understood. The objective of this study was to determine if radicle emergence is related to the production of endo-beta-mannanase to weaken the mannan-rich cell walls of the surrounding endosperm region, and/or if the endosperm structure itself is such that it is weaker in the region through which the radicle must penetrate. METHODS: Activity of endo-beta-mannanase in the endosperm and embryo was measured using a gel assay during and following germination, and the structure of the endosperm in juxtaposition to the radicle, and surrounding the cotyledons was determined using fixation, sectioning and light microscopy. KEY RESULTS: The activity of endo-beta-mannanase, the major enzyme responsible for galactomannan cell wall weakening increased in activity only after emergence of the radicle from the seed. Thickened cell walls were present in the lateral endosperm in the hard-seeded species studied, but there was little to no thickening in the micropylar endosperm except in date seeds. In this species, a ring of thin cells was visible in the micropylar endosperm and surrounding an operculum which was pushed open by the expanding radicle to complete germination. CONCLUSIONS: The micropylar endosperm presents a lower physical constraint to the completion of germination than the lateral endosperm, and hence its structure is predisposed to permit radicle protrusion.

Nitric oxide reduces aluminum toxicity by preventing oxidative stress in the roots of Cassia tora L.

Nitric oxide (NO) as a key signaling molecule has been involved in mediation of various biotic and abiotic stress-induced physiological responses in plants. In the present study, we investigated the effect of NO on Cassia tora L. plants exposed to aluminum (Al). Plants pre-treated for 12 h with 0.4 mM sodium nitroprusside (SNP), an NO donor, and subsequently exposed to 10 microM Al treatment for 24 h exhibited significantly greater root elongation as compared with the plants without SNP treatment. The NO-promoted root elongation was correlated with a decrease in Al accumulation in root apexes. Furthermore, oxidative stress associated with Al treatment increased lipid peroxidation and reactive oxygen species, and the activation of lipoxygenase and antioxidant enzymes was reduced by NO. Such effects were confirmed by the histochemical staining for the detection of peroxidation of lipids and loss of membrane integrity in roots. The ameliorating effect of NO was specific, because the NO scavenger cPTIO [2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylinidazoline-1-oxyl-3-oxide] completely reversed the effect of NO on root growth in the presence of Al. These results indicate that NO plays an important role in protecting the plant against Al-induced oxidative stress.

Molecular characterization of root-specific chalcone synthases from Cassia alata.

Three cDNAs encoding very similar but unique isoforms of chalcone synthase (EC 2.3.1.74) were isolated from a cDNA library prepared from RNA from root tissue of the Thai medicinal plant Cassia alata L. (ringworm bush, Leguminosae). Gene transcript for these three type-III polyketide synthases was found to accumulate predominantly in roots. The heterologously expressed enzymes accepted acetyl-, n-butyryl-, isovaleryl-, n-hexanoyl-, benzoyl-, cinnamoyl-, and p-coumaroyl-CoA as starter molecules and together with the co-substrate malonyl-CoA, formed multiple products. With the exception of the assay in which acetyl-CoA was used as the starter molecule, all substrates yielded a phloroglucinol derivative resulting from three sequential condensations of acetate units derived from three malonyl-CoA decarboxylations. Every substrate tested also produced two pyrone derivatives, one resulting from two acetate unit condensations (a bis-noryangonin-type pyrone derailment product) and one resulting from three acetate unit condensations (a 4-coumaroyltriacetic acid lactone-type pyrone derailment). C. alata accumulates the flavonoids quercetin, naringenin and kaempferol in roots, suggesting that the in planta function of these enzymes is the biosynthesis of root flavonoids.

Purification and partial characterization of a peroxidase from plant cell cultures of Cassia didymobotrya and biotransformation studies.

An acidic peroxidase (EC 1.11.1.7) produced by cell suspension cultures of Cassia didymobotrya (wild senna) was purified from culture medium collected on the 29th day. The enzyme was shown to be a glycoprotein with a pI of 3.5, a molecular mass of approx. 43 kDa by SDS/PAGE and 50 kDa by gel filtration. The N-terminal sequence was very similar to those of other plant peroxidases. The peroxidase was characterized by a high specificity towards coniferyl alcohol and other natural phenolics such as guaiacol and ferulic and caffeic acids. These findings suggest that the enzyme is involved in lignification processes of the cell wall. Moreover, the enzyme was able to catalyse the oxidation of 4,3',4'-trihydroxychalcone and 4, 3',4'-trihydroxy-3-methoxychalcone to the corresponding 3, 3'-biflavanones, as mixtures of racemic and meso forms.