Characterization and cloning of the chlorophyll-degrading enzyme pheophorbidase from cotyledons of radish.

Enzymatic removal of the methoxycarbonyl group of pheophorbide (Pheid) a in chlorophyll degradation was investigated in cotyledons of radish (Raphanus sativus). The enzyme pheophorbidase (PPD) catalyzes the conversion of Pheid a to a precursor of pyropheophorbide (PyroPheid), C-13(2)-carboxylPyroPheid a, by demethylation, and then the precursor is decarboxylated nonenzymatically to yield PyroPheid a. PPD activity sharply increased with the progression of senescence in radish, suggesting de novo synthesis of PPD. The enzyme activity was separated into two peaks in anion-exchange and hydrophobic chromatography; the terms type 1 and type 2 were applied according to the order of elution of these enzymes in anion-exchange chromatography. PPD types 1 and 2 were purified 9,999- and 6,476-fold, with a yield of 0.703% and 2.73%, respectively. Among 12 substrates tested, both enzymes were extremely specific for Pheids of the dihydroporphyrin and tetrahydroporphyrin types, indicating that they are responsible for the formation of these PyroPheids. Both PPDs had molecular masses of 113,000 kD on gel filtration and showed three bands of 16.8, 15.9, and 11.8 kD by SDS-PAGE. The partial N-terminal amino acid sequences for these bands of PPD (type 2) were determined. Based on their N-terminal amino acid sequences, a full-length cDNA of PPD was cloned. The molecular structure of PPD, particularly the molecular mass and subunit structure, is discussed in relation to the results of SDS-PAGE.

Molecular cloning and characterization of a senescence-induced tau-class Glutathione S-transferase from barley leaves.

Glutathione S-transferases (GSTs) (EC 2.5.1.18) are multifunctional proteins involved in such diverse intracellular events as primary and secondary metabolism, signaling and stress metabolism. In this study, we found a senescence-induced tau-class GST (SIGST) in senescent leaves of barley (Hordeum vulgare L.). The SIGST was purified 19-fold to homogeneity from initial crude extracts by three steps of chromatography with a yield of 5%. The purified SIGST had a GSH-conjugating activity and peroxidase (POD) activity at the same level of 1.7 micromol min(-1) mg protein(-1), although restricted substrate selectivity could be seen in POD activity. Barley SIGST is a slightly acidic protein with a molecular weight of 49 k and is composed of two subunits. The enzyme exhibited a single pH optimum at pH 8.3. The K(m) values were 0.285 mM for GSH and 0.293 mM for 1-chloro-2,4-dinitrobenzene. In most respects, the barley enzyme resembles those that have been reported from other higher plants. The SIGST gene was cloned from cDNA of senescent barley leaves. DNA sequence analysis shows that the cloned SIGST had only one base different from the barley embryo GST, ECGST. The obtained sequence indicates that SIGST is classified into the plant-specific tau class. mRNA expression analysis showed that in addition to senescence, SIGST was strongly induced by treatment with a herbicide and low temperature. The responses to these stresses suggest that SIGST may be involved at least partly in the secondary metabolism as an antioxidant and enhancement of enzymatic activity during senescence.

Effects of scavengers for active oxygen species on cell death by cryptogein.

The hypersensitive reaction is a type of programmed cell death in plants. Cryptogein is a proteinaceous elicitor secreted from Phythophthora cryptogea. In one current model, active oxygen species (AOS) trigger programmed cell death in plants. In this study, we examined a variety of AOS scavengers to elucidate the function of AOS in the death program. Most of these AOS scavengers, including tiron, a scavenger for superoxide radical, catalase for hydrogen peroxide, and hydroquinone, sodium ascorbate and propyl gallate for free radicals, almost completely removed extracellular AOS. However, none of the reagents completely blocked the cell death process. Other reagents, such as histidine and dimethylfuran, scavengers for singlet oxygen, and diphenyleneiodonium chloride, an inhibitor of NADPH oxidase, showed significant toxicity in BY-2 cells. These results indicate that AOS produced in the extracellular space do not play a role in hypersensitive cell death.

Lipid-binding form is a key conformation to induce a programmed cell death initiated in tobacco BY-2 cells by a proteinaceous elicitor of cryptogein.

Cryptogein, a proteinaceous elicitor secreted by Phytophthora cryptogea, induces a remarkable hypersensitive cell death in tobacco cells. Two cryptogein mutants were analysed to characterize the induction mechanism of cell death; one was a newly synthesized mutant N93A whose 93rd Asn residue was changed to Ala, the other was K13V whose Lys at position 13 was replaced with Val. The effect of these mutations was evaluated in terms of extracellular alkalization, production of active oxygen species (AOS) and progression to death. The mutation N93A resulted in a reduction in activity to 71.0, 74.6 and 24.5% for original rates of extracellular alkalization, AOS production and cell death progression, respectively. In the case of the K13V mutation, these rates changed to 114, 3.38 and 7.40%, respectively. The lipid-binding activities of the mutants were analysed using fluorogenic lipid of dehydroergosterol. The results for N93A and K13V were 38.3 and 3.40% compared with the wild type, respectively. These findings indicate that the lipid-binding form was the only conformation to induce the production of AOS and programmed cell death in plants.

Chloroplasts in seeds and dark-grown seedlings of lotus.

In most higher plants, mature dry seeds have no chloroplasts but etioplasts. Here we show that in a hydrophyte, lotus (Nelumbo nucifera), young chloroplasts already exist in shoots of mature dry seeds and that they give rise to mature chloroplasts during germination, even in darkness. These shoots contain chlorophyll and chlorophyll-binding proteins CP1 and LHCP. The unique features of chloroplast formation in N. nucifera suggest a unique adaptive strategy for seedling development correlated with the plant's habitat.

A versatile assay for the accurate, time-resolved determination of cellular viability.

A convenient and versatile method for the accurate, time-resolved determination of cellular viability has been developed. The conventional viability indicator fluorescein diacetate (FDA), which is converted to the fluorescent compound fluorescein in living cells, was employed as a viability probe. Fluorescence emission from cells was measured using a spectrofluorimeter equipped with a magnetic stirrer. Using this assay cell suspensions exhibiting densities in the range 0.5 x 10(5) to 2.0 x 10(5) cells displayed a linear response when FDA concentrations less than 12 micro M were employed. To calibrate the method, viability standards were elaborated using different proportions of living and dead cells, and a correlation coefficient for the viability of tobacco BY-2 suspensions was calculated as 0.998. This viability assay was also found to be applicable to Chlamydomonas reinhardtii and Arabidopsis thaliana cultured cells. Using this cell viability assay, kinetic analyses of cell death could be performed. Using the proteinaceous elicitor from Phytophthora cryptogea, cryptogein, to induce cell death in tobacco cell suspensions, values for the maximum velocity of death induction rate (V(max)) and the LD50 (half-maximal velocity or k(1/2)) were calculated as 17.2 (% death/h) and 65 nM, respectively.