Towards Understanding the Involvement of H+-ATPase in Programmed Cell Death of Psammosilene tunicoides after Oxalic Acid Application.

Psammosilene tunicoides is a unique perennial medicinal plant species native to the Southwestern regions of China. Its wild population is rare and endangered due to over-excessive collection and extended growth (4-5 years). This research shows that H+-ATPase activity was a key factor for oxalate-inducing programmed cell death (PCD) of P. tunicoides suspension cells. Oxalic acid (OA) is an effective abiotic elicitor that enhances a plant cell's resistance to environmental stress. However, the role of OA in this process remains to be mechanistically unveiled. The present study evaluated the role of OA-induced cell death using an inverted fluorescence microscope after staining with Evans blue, FDA, PI, and Rd123. OA-stimulated changes in K+ and Ca2+ trans-membrane flows using a patch-clamp method, together with OA modulation of H+-ATPase activity, were further examined. OA treatment increased cell death rate in a dosage-and duration-dependent manner. OA significantly decreased the mitochondria activity and damaged its electron transport chain. The OA treatment also decreased intracellular pH, while the FC increased the pH value. Simultaneously, NH4Cl caused intracellular acidification. The OA treatment independently resulted in 90% and the FC led to 25% cell death rates. Consistently, the combined treatments caused a 31% cell death rate. Furthermore, treatment with EGTA caused a similar change in intracellular pH value to the La3+ and OA application. Combined results suggest that OA-caused cell death could be attributed to intracellular acidification and the involvement of OA in the influx of extracellular Ca2+, thereby leading to membrane depolarization. Here we explore the resistance mechanism of P. tunicoides cells against various stresses endowed by OA treatment.

[Accurate identification of Psammosilene tunicoides and its confused species by systematic identification method].

OBJECTIVE: To develop an effective identification method for accurately discriminating Psammosilene tunicoides and its confused species by the combined method of microscopic identification and molecular identification, so-called systematic identification of Chinese materia medica (SICMM). METHOD: P. tunicoides and its confused species were accurately discriminated by SICMM method, which was established by comprehensively use of microscopic identification and DNA identification method. The DNA identification included the following analysis: the BLAST alignment, specific bases and N-J phylogenetic tree analysis. RESULT: The cluster crystals were not observed in P. tunicoides, but great deals of them were found in Silene viscidula. Further more, big differences of ITS sequence were observed and analyzed between P. tunicoides and its confused specie of S. viscidula. CONCLUSION: The system method is a scientific and accurate method for the identification of P. tunicoides and its counterfeit species.

Effect of temperature on the progamic phase in high-mountain plants.

Progamic processes are particularly temperature-sensitive and, in lowland plants, are usually drastically reduced below 10 °C and above 30 °C. Little is known about how effectively sexual processes of mountain plants function under the large temperature fluctuations at higher altitudes. The present study examines duration and thermal thresholds for progamic processes in six common plant species (Cerastium uniflorum, Gentianella germanica, Ranunculus alpestris, R. glacialis, Saxifraga bryoides, S. caesia) from different altitudinal zones in the European Alps. Whole plants were collected from natural sites shortly before anthesis and kept in a climate chamber until further processing. Flowers with receptive stigmas were hand-pollinated with allopollen and exposed to controlled temperatures between -2 and 40 °C. Pollen performance (adhesion to the stigma, germination, tube growth, fertilisation) was quantitatively analysed, using the aniline blue fluorescence method. Pollen adhesion was possible from -2 to 40 °C. Pollen germination and tube growth occurred from around 0 to 35 °C in most species. Fertilisation was observed from 5 to 30-32 °C (0-35 °C in G. germanica). The progamic phase was shortest in G. germanica (2 h at 30 °C, 12 h at 5 °C, 24 h at 0 °C), followed by R. glacialis (first fertilisation after 2 h at 30 °C, 18 h at 5 °C). In the remaining species, first fertilisation usually occurred after 4-6 h at 30 °C and after 24-30 h at 5 °C. Thus, mountain plants show remarkably flexible pollen performance over a wide temperature range and a short progamic phase, which may be essential for successful reproduction in the stochastic high-mountain climate.

[Localization and dynamic change of saponin in root tuber of cultivated Pseudostellaria heterophylla].

Anatomical, histochemical and phytochemical methods were used to investigate the structure, the localization and content changes of total saponin of the root tuber of Pseudostellaria heterophylla on different developmental stages. Results showed that the primary structure and secondary structure of P. heterophylla adventitious root were similar to that of other herbaceous dicotyledons. 80% of mature root tuber were secondary xylem which consisted of most parenchyma cells and less vessels. The secondary phloem were composed of parenchyma cells too. The results of histochemical methods showed that saponin distributed in pericycle and parenchyma cells of primary phloem in the primary structure of root. In secondary structure and mature root tuber, saponin distributed in periderm and secondary vascular structure except cork layer and vessel. Besides, the colors of the secondary phloem were darker. The results of phytochemical test showed that the content of saponin in skin areas was higher than that in xylems in February and July. The result was consistent with that of histochemistry. The content of saponin in the head of the root was higher than that in the end of the root which was in turn higher than that in the middle part of the root. In the developmental process of root tuber, the content of saponin showed a dynamic tendency of high-low-high. This characteristics were related to the development of root system.

[Anther development and sperm isolation of Pseudostellaria heterophylla (Miq.)].

Anther wall is general and tapetum is glandular. The process of meiosis of microspore mother cells is simultaneous and the tetrads are tetrahedral. The mature pollen of Pseudostellaria heterophylla (Miq.) is tree-celled. There are 22-30 germ pores on the pollen wall. Many pollen grains could burst in 10% mannitol or 15% sucrose solution and release a pair of sperm cells which could keep alive for 25-50 min by FDA fluorescence. Using micromanipulator the released sperm cells could be collected. When pollen grains were put into a solution containing 0.03% CaCl2, 0.01% H3BO3, 0.01% KH2PO4 and 20% PEG for 2-5 min, they would germinate and the pollen tubes would reach 815 microm at 2h after cultured. A pair of sperms would enter into pollen tube when it grew to 500-600 microm. The fluorescence of both sperms would be observed clearly in pollen tube after DAPI staining. When the pollen tubes were burst in a bursting solution, a pair of sperms would be released from pollen tube.

Subcellular distribution of the V-ATPase complex in plant cells, and in vivo localisation of the 100 kDa subunit VHA-a within the complex.

BACKGROUND: Vacuolar H+-ATPases are large protein complexes of more than 700 kDa that acidify endomembrane compartments and are part of the secretory system of eukaryotic cells. They are built from 14 different (VHA)-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques (FRET). Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i) a less conserved cytoplasmically orientated N-terminus and (ii) a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed. RESULTS: To elucidate the presence and function of VHA-a in the plant complex, three approaches were undertaken: (i) co-immunoprecipitation with antibodies directed to epitopes in the N- and C-terminal part of VHA-a, respectively, (ii) immunocytochemistry approach including co-localisation studies with known plant endomembrane markers, and (iii) in vivo-FRET between subunits fused to variants of green fluorescence protein (CFP, YFP) in transfected cells. CONCLUSIONS: All three sets of results show that V-ATPase contains VHA-a protein that interacts in a specific manner with other subunits. The genomes of plants encode three genes of the 95 kDa subunit (VHA-a) of the vacuolar type H+-ATPase. Immuno-localisation of VHA-a shows that the recognized subunit is exclusively located on the endoplasmic reticulum. This result is in agreement with the hypothesis that the different isoforms of VHA-a may localize on distinct endomembrane compartments, as it was shown for its yeast counterpart Vph1.

Presence and behaviour of B-chromosomes in Acanthophyllum laxiusculum (Caryophyllaceae).

Two karyotypic variants were recognized in populations of Acanthophyllum laxiusculum Schiman-Czeika. Variant A had 2N = 30 and variant B showed the presence of 0 to 3 B-chromosomes in addition to 2N = 30 chromosomes. Analysis of chromosome behaviour at meiosis showed that the presence of B-chromosomes increases chiasma frequency in A-chromosomes; this effect was higher for plants with odd numbers of B-chromosomes compared with plants with even numbers of B-chromosomes. Comparisons of variants A and B, suggests that B-chromosomes have an effect pollen stainability and seed production. It seems that the presence of B-chromosomes may increase pollen stainability and seed production in variant B.