Cathepsin B degrades RbcL during freezing-induced programmed cell death in Arabidopsis.

KEY MESSAGE: Cathepsin B plays an important role that degrades the Rubisco large subunit RbcL in freezing stress. Programmed cell death (PCD) has been well documented in both development and in response to environmental stresses in plants, however, PCD induced by freezing stress and its molecular mechanisms remain poorly understood. In the present study, we characterized freezing-induced PCD and explored its mechanisms in Arabidopsis. PCD induced by freezing stress was similar to that induced by other stresses and senescence in Arabidopsis plants with cold acclimation. Inhibitor treatment assays and immunoblotting indicated that cathepsin B mainly contributed to increased caspase-3-like activity during freezing-induced PCD. Cathepsin B was involved in freezing-induced PCD and degraded the large subunit, RbcL, of Rubisco. Our results demonstrate an essential regulatory mechanism of cathepsin B for Rubisco degradation in freezing-induced PCD, improving our understanding of freezing-induced cell death and nitrogen and carbohydrate remobilisation in plants.

BYPASS1-LIKE regulates lateral root initiation via exocytic vesicular trafficking-mediated PIN recycling in Arabidopsis.

Auxin and auxin-mediated signaling pathways are known to regulate lateral root development. Although exocytic vesicle trafficking plays an important role in recycling the PIN-FORMED (PIN) auxin efflux carriers and in polar auxin transport during lateral root formation, the mechanistic details of these processes are not well understood. Here, we demonstrate that BYPASS1-LIKE (B1L) regulates lateral root initiation via exocytic vesicular trafficking-mediated PIN recycling in Arabidopsis thaliana. b1l mutants contained significantly more lateral roots than the wild type, primarily due to increased lateral root primordium initiation. Furthermore, the auxin signal was stronger in stage I lateral root primordia of b1l than in those of the wild type. Treatment with exogenous auxin and an auxin transport inhibitor indicated that the lateral root phenotype of b1l could be attributed to higher auxin levels and that B1L regulates auxin efflux. Indeed, compared to the wild type, C-terminally green fluorescent protein-tagged PIN1 and PIN3 accumulated at higher levels in b1l lateral root primordia. B1L interacted with the exocyst, and b1l showed defective PIN exocytosis. These observations indicate that B1L interacts with the exocyst to regulate PIN-mediated polar auxin transport and lateral root initiation in Arabidopsis.

TRANSTHYRETIN-LIKE and BYPASS1-LIKE co-regulate growth and cold tolerance in Arabidopsis.

BACKGROUND: Cold stress inhibits normal physiological metabolism in plants, thereby seriously affecting plant development. Meanwhile, plants also actively adjust their metabolism and development to adapt to changing environments. Several cold tolerance regulators have been found to participate in the regulation of plant development. Previously, we reported that BYPASS1-LIKE (B1L), a DUF793 family protein, participates in the regulation of cold tolerance, at least partly through stabilizing C-REPEAT BINDING FACTORS (CBFs). In this study, we found that B1L interacts with TRANSTHYRETIN-LIKE (TTL) protein, which is involved in brassinosteroid (BR)-mediated plant growth and catalyses the synthesis of S-allantoin, and both proteins participate in modulating plant growth and cold tolerance. RESULTS: The results obtained with yeast two hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that B1L directly interacted with TTL. Similar to the ttl-1 and ttl-2 mutants, the b1l mutant displayed a longer hypocotyl and greater fresh weight than wild type, whereas B1L-overexpressing lines exhibited a shorter hypocotyl and reduced fresh weight. Moreover, ttl-1 displayed freezing tolerance to cold treatment compared with WT, whereas the b1l mutant and TTL-overexpressing lines were freezing-sensitive. The b1l ttl double mutant had a developmental phenotype and freezing tolerance that were highly similar to those of ttl-1 compared to b1l, indicating that TTL is important for B1L function. Although low concentrations of brassinolide (0.1 or 1 nM) displayed similarly promoted hypocotyl elongation of WT and b1l under normal temperature, it showed less effect to the hypocotyl elongation of b1l than to that of WT under cold conditions. In addition, the b1l mutant also contained less amount of allantoin than Col-0. CONCLUSION: Our results indicate that B1L and TTL co-regulate development and cold tolerance in Arabidopsis, and BR and allantoin may participate in these processes through B1L and TTL.

Nocardia mangyaensis sp. nov., a novel actinomycete isolated from crude-oil-contaminated soil.

A Gram-stain-positive, aerobic, non-motile and mycolic-acid-containing strain, designated Y48T, was isolated from soil contaminated by crude oil located in the northern margin of the Qaidam Basin. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Y48T belongs to the genus Nocardia and is closely related to N. cummidelens DSM 44490T (99.0 % similarity), N. soli DSM 44488T (99.0 %), N. lasii 3C-HV12T (98.9 %), N. salmonicida NBRC 13393T (98.6 %), N. ignorata NBRC 108230T (98.6 %) and N. coubleae NBRC 108252T (98.6 %). The average nucleotide identity and DNA-DNA hybridization values between strain Y48T and the reference strains were 75.9-84.5 and 27.5-29.0 %, respectively, values that were below the thresholds for species delineation. Chemotaxonomic analysis indicated that the major fatty acids of strain Y48T were C16 : 0, summed feature 3 (C16 : 1ω6c/C16 : 1ω7c), C18 : 1ω9c and C18 : 0 10-methyl (TBSA). The respiratory quinone was MK-8(H4, ω-cycl). The polar lipid profile was composed of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside, two glycolipids and three unidentified lipids. The cell-wall hydrolysates contained meso-diaminopimelic acid, with ribose, arabinose, glucose and galactose as whole-cell sugars. A combination of 16S rRNA gene sequence analysis, and phenotypic and chemotaxonomic characterizations demonstrated that strain Y48T represents a novel species of the genus Nocardia, for which the name Nocardia mangyaensis sp. nov. is proposed. The type strain is Y48T (=JCM 32795T=CGMCC 4.7494T).

Effect of root exudates of intercropping Vicia faba and Arabis alpina on accumulation and sub-cellular distribution of lead and cadmium.

A field experiment in which a hyperaccumulator (Arabis alpina) was intercropped with winter crop (Vicia faba), was conducted to understand effect of the root exudates on the content and accumulated amounts, sub-cellular distribution of Cd and Pb of the intercropped plants during the ripening period of V. faba (120 d after sowing). The results showed that contents of soluble sugars exuded from the roots of intercropped A. alpina were 67.6% less than that of the monocropped plant, whereas contents of free amino acids was 57.9% greater. The total contents of organic acids exuded from roots of intercropped A. alpina and V. faba were 578.8% and 37.8% greater than that of monocropped plants, respectively. The contents of tartaric acid and malic acid exuded by roots of intercropped A. alpina were greater 31.9 times and 15.9 times than those of monocropped A. alpina, respectively. The contents and accumulated amounts of Cd and Pb in intercropped A. alpina were greater than those of monocropped A. alpina. The contents of Pb bound to organic matter in cell walls, cytoplasm and organelles of intercropped plants were greater than those of monocropped plants. These results demonstrate that increases in accumulated amounts of Pb and Cd caused by intercropping were closely related to migration of Cd and Pb in plants mediated by the composition and content of the root exudates.

A reference-grade genome of the xerophyte Ammopiptanthus mongolicus sheds light on its evolution history in legumes and drought-tolerance mechanisms.

Plants that grow in extreme environments represent unique sources of stress-resistance genes and mechanisms. Ammopiptanthus mongolicus (Leguminosae) is a xerophytic evergreen broadleaf shrub native to semi-arid and desert regions; however, its drought-tolerance mechanisms remain poorly understood. Here, we report the assembly of a reference-grade genome for A. mongolicus, describe its evolutionary history within the legume family, and examine its drought-tolerance mechanisms. The assembled genome is 843.07 Mb in length, with 98.7% of the sequences successfully anchored to the nine chromosomes of A. mongolicus. The genome is predicted to contain 47 611 protein-coding genes, and 70.71% of the genome is composed of repetitive sequences; these are dominated by transposable elements, particularly long-terminal-repeat retrotransposons. Evolutionary analyses revealed two whole-genome duplication (WGD) events at 130 and 58 million years ago (mya) that are shared by the genus Ammopiptanthus and other legumes, but no species-specific WGDs were found within this genus. Ancestral genome reconstruction revealed that the A. mongolicus genome has undergone fewer rearrangements than other genomes in the legume family, confirming its status as a "relict plant". Transcriptomic analyses demonstrated that genes involved in cuticular wax biosynthesis and transport are highly expressed, both under normal conditions and in response to polyethylene glycol-induced dehydration. Significant induction of genes related to ethylene biosynthesis and signaling was also observed in leaves under dehydration stress, suggesting that enhanced ethylene response and formation of thick waxy cuticles are two major mechanisms of drought tolerance in A. mongolicus. Ectopic expression of AmERF2, an ethylene response factor unique to A. mongolicus, can markedly increase the drought tolerance of transgenic Arabidopsis thaliana plants, demonstrating the potential for application of A. mongolicus genes in crop improvement.

Cloning, expression and characterization of trehalose-6-phosphate phosphatase from a psychrotrophic bacterium, Arthrobacter strain A3.

A trehalose-6-phosphate phosphatase (TPP) gene, otsB, from a psychrotrophic bacterium, Arthrobacter strain A3, was identified. The product of this otsB gene is 266 amino acids in length with a calculated molecular weight of 27,873 Da. The protein was expressed in Escherichia coli and purified to apparent homogeneity. The purified recombinant TPP catalyzed the dephosphorylation of trehalose-6-phosphate to form trehalose and showed a broad optimum pH range from 5.0 to 7.5. This enzyme also showed an absolute requirement for Mg(2+) or Co(2+) for catalytic activity. The recombinant TPP had a maximum activity at 30 °C and maintained activity over a temperature range of 4-30 °C. TPP was generally heat-labile, losing 70 % of its activity when subjected to heat treatment at 50 °C for 6 min. Kinetic analysis of the Arthrobacter strain A3 TPP showed ~tenfold lower K (m) values when compared with values derived from other bacterial TPP enzymes. The highest k (cat)/K (m) value was 37.5 mM(-1) s(-1) (repeated three times), which is much higher than values published for mesophilic E. coli TPP, indicating that the Arthrobacter strain A3 TPP possessed excellent catalytic activity at low temperatures. Accordingly, these characteristics suggest that the TPP from the Arthrobacter strain A3 is a new cold-adapted enzyme. In addition, this is the first report characterizing the enzymatic properties of a TPP from a psychrotrophic organism.

Regulation of expression of trehalose-6-phosphate synthase during cold shock in Arthrobacter strain A3.

Trehalose is a chemical chaperone known to protect a variety of organisms against cold stress. Members of the genus Arthrobacter, which belongs to the Actinomycetales group, exhibit strong resistance to stress conditions, but exactly how trehalose synthesis is regulated in conditions of cold stress is still unknown. Here, we report that Arthrobacter strain A3, which was isolated from the alpine permafrost, has only two trehalose synthesis pathways (OtsA/B and TreS), while other Arthrobacter spp. have three. Mutants and immunoblot analyses indicate that trehalose is mainly synthesized via OtsA at low temperatures in Arthrobacter strain A3. Therefore, we have focused on the regulation of OtsA expression during cold shock. The results indicated that both low temperature and accumulation of trehalose can inhibit OtsA expression. The elongation factor Tu, which binds to OtsA, stabilizes the expression of OtsA in the cold.

Effect of inoculation with the endophyte Clavibacter sp. strain Enf12 on chilling tolerance in Chorispora bungeana.

Endophytic bacteria have been shown to increase resistance against biotic stress and tolerance to abiotic stress in many plants. The objective of this study was to evaluate the effect of an endophytic bacterium, Clavibacter sp. strain Enf12, in regenerated plantlets of Chorispora bungeana subjected to chilling stress (0°C). Aerial biomass and physiological markers for chilling stress, such as electrolyte leakage, lipid peroxidation, reactive oxygen species (ROS) accumulation, proline content and activities of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), guaiacol peroxidase (EC 1.11.1.7) and ascorbate peroxidase (EC 1.11.1.11), were assessed. We demonstrated that Clavibacter sp. strain Enf12 was capable of colonizing internal tissues of regenerated plantlets of C. bungeana and maintained stable population densities under both normal (20°C) and chilling (0°C) conditions. Inoculation enhanced plantlet growth under both conditions and significantly attenuated the chilling-induced electrolyte leakage, lipid peroxidation and ROS accumulation. The endophyte significantly increased the activities of antioxidant enzymes and proline content in C. bungeana plantlets under chilling stress. These findings suggest that Clavibacter sp. strain Enf12 inoculation stimulates the growth of C. bungeana plantlets and improves their tolerance to chilling stress through enhancing the antioxidant defense system.

Analysis of the composition and characteristics of culturable endophytic bacteria within subnival plants of the Tianshan Mountains, northwestern China.

This study first described the composition and characteristics of culturable endophytic bacteria isolated from wild alpine-subnival plant species growing under extreme environmental conditions (i.e., on the border of a glacier with frequently fluctuating and freezing temperatures, strong wind, and high ultraviolet radiation). Using a cultivation-dependent approach and 16S rRNA gene amplification techniques, 93 bacterial isolates showing different phenotypic properties were obtained from 20 different subnival plant species, of which gram-positive bacteria (61.5%), psychrotolerant bacteria (67.3%), and pigmented isolates (70.9%) accounted for a large proportion. All these characteristics of endophytes were closely related to the survival environment of their host plants and were in good agreement with microbes occurring in other cold environments. Phylogenetic analysis indicated that the endophytic isolates consisted of five phylogenetic groups comprising α-proteobacteria, γ-proteobacteria, the high G+C content gram-positive bacteria, the low G+C content gram-positive bacteria, and Flavobacterium-Bacteroides-Cytophaga. The largest generic diversity was found in the HGC group, while Clavibacter, Agreia, Rhodococcus, Sphingomonas, and Pseudomonas were the most prevalent genera. Of all isolates, 46.4% showed a high sequence similarity (98-100%) to strains discovered from other cold environments such as glaciers, tundra, and polar seas. Furthermore, 36.4% of the isolates produced Indole-3-acetic acid and 76.3% were able to solubilize mineral phosphate, which revealed that endophytic bacteria with multiple physiological functions were abundant and widespread in subnival plants. These results are essential for understanding the ecological roles of endophytes and as a foundation for further studying the interactions with plants and environment.

The catalytic efficiency of trehalose-6-phosphate synthase is effected by the N-loop at low temperatures.

The enzyme OtsA (trehalose-6-phosphate synthase) is ubiquitous in both prokaryotic and eukaryotic organisms, where it plays a critical role in stress resistance and glucose metabolism. Here, we cloned the otsA gene from Arthrobacter sp. Cjts, and expressed and then purified the recombinant proteins. Enzyme activity analysis indicated that the high catalytic efficiency of OtsA from Arthrobacter sp. Cjts resulted from the high affinity of the enzyme for uridine 5'-diphosphoglucose (UDP-Glc) at low temperatures. We also confirmed that the N-loop sequence of OtsA has a large effect on its affinity for UDP-Glc. Sequence analysis indicated that the flexibility of the N-loop may be directly related to the catalytic efficiency of OtsA at low temperatures.

Spatial patterns and interspecific relationships of two dominant cushion plants at three elevations on the Kunlun Mountain, China.

One of the most important ecological processes is the formation of interspecific relationships in relation to spatial patterns among alpine cushion plants in extreme environmental habitats. However, such relationships remain poorly understood. Here, we examined the spatial patterns of alpine cushion plants along an altitudinal gradient of environmental severity and the interspecific relationship between two cushion species (Thylacospermum caespitosum and Androsace tangulashanensis) on the eastern Kunlun Mountain of China. Our results showed that the two species were highly aggregated within a distance of 2.5-5 m at the mid (S2) altitude, whereas they were randomly distributed at the low (S1) and high (S3) altitudes. A positive spatial interaction between the two species was observed over shorter distances at the mid (S2) altitude, and the spatial patterns were related to the size of individuals of the two species. Moreover, the impact of A. tangulashanensis on T. caespitosum (RIIT. caespitosum) was negative in all the study plots, and a positive impact of T. caespitosum on A. tangulashanensis (RIIA. tangulashanensis) was only observed at the mid (S2) altitude. Together, these results demonstrated that the spatial patterns of these two cushions varied with environmental severity, since the outcome of the interactions were different, to some extent, at the three altitudes. Plant size is the main factor affecting the spatial correlation and interspecific relationship between two cushions. Therefore, its potential influence should be considered when discussing interspecific relationships among cushions and their community construction at small scales in alpine ecosystems.

Defense strategy of old and modern spring wheat varieties during soil drying.

Different defense mechanisms of three spring wheat (Triticum aestivum L.) varieties were studied by withholding watering in well-watered pots to gradually increase water deficit of plants grown in containers. The strategies of plant adaptation were divided into three phases according to the severity of drought: first, a positive defense phase that started from commencement of non-hydraulic root-sourced signals (nHRS) and ended at onset of hydraulic root-sourced signals (HRS)--the plant responded to imminent drought by decreasing stomatal aperture to lessen water loss and no membrane injury occurred. The second defense phase occurred between the onset of HRS and temporary wilting (TW), characterized by enhancement of reactive oxygen species (ROS), marked enzyme activity and increased MDA content. Mild lipid membrane peroxidation came mainly from a dynamic imbalance between free radical production and enzymatic defense reaction, which indicated that injury by ROS had not been completely repaired by increasing enzymatic activity. The third defense phase was from TW to permanent wilting (PW), the synthesis of SOD and CAT during TW could not deal with the collapse of antioxidant enzymes, and SOD and CAT activities began to decrease, which caused the excessive ROS production and thus serious membrane lipid peroxidation. The defense strategies to drought are similar among the varieties, but modern varieties LC8275 and GY602 bred after 1975 had relatively higher defense levels at all three defense phases, which suggest that modern varieties are more resistant than old ones, and artificial selection would lead to a different direction in evolution from natural selection.

Targeting silencing androgen receptor gene by shRNA with low-intensity focused ultrasonic irradiation inhibits growth of prostate cancer xenografts in nude mice.

The androgen receptor (AR) plays a pivotal role in prostate cancer, making it a potential therapeutic target. Short-hairpin RNA (shRNA) inhibits gene expression and offers a novel strategy to eradicate disease. Ultrasound-mediated gene transfection is a promising gene delivery method. This study sought to determine whether targeting silencing androgen receptor gene by shRNA with low-intensity focused ultrasonic irradiation could be used as effective therapy for prostate cancers in vivo. A plasmid-based short-hairpin RNA combined with low-intensity focused ultrasonic irradiation approach was used to specifically knock down the expression of AR in prostate cancer 22RV1 cells in vivo. The growth of 22RV1 tumors that had been subcutaneously xenografted was evaluated and expression level of AR was determined by immunohistochemical staining. The proliferative index (PI) and the apoptotic index (AI) were respectively derived from the percentage of positive cells by Ki-67 immunohistochemical staining and TUNEL assay. The plasmid-based AR shRNA administrated intravenously significantly inhibited the tumor growth and AR expression. These inhibitory effects of AR shRNA were augmented when the region of tumor received low-intensity focused ultrasound irradiation. Immunohistochemical staining and TUNEL assay confirmed AR shRNA with low-intensity focused ultrasonic irradiation exhibited growth-inhibitory, antiproliferative, and apoptotic effects on prostate cancer xenografts. The authors showed for the first time that the knockdown of AR expression by plasmid-based AR shRNA with low-intensity focused ultrasonic irradiation significantly suppressed the tumor growth of prostate cancer in vivo.

BYPASS1-LIKE, A DUF793 Family Protein, Participates in Freezing Tolerance via the CBF Pathway in Arabidopsis.

The C-REPEAT BINDING FACTOR signaling pathway is strictly modulated by numerous factors and is essential in the cold response of plants. Here, we show that the DUF793 family gene BYPASS1-LIKE modulates freezing tolerance through the CBFs in Arabidopsis. The expression of B1L was rapidly induced under cold treatment. Comparing to wild type, B1L knockout mutants were more sensitive to freezing treatment, whereas B1L-overexpressing lines were more tolerant. The expression of CBFs and CBF target genes was significantly decreased in b1l mutant. Using yeast two-hybrid screening system, 14-3-3λ was identified as one of proteins interacting with B1L. The interaction was confirmed with bimolecular fluorescence complementation assay and co-immunoprecipitation assay. Biochemical assays revealed that b1l mutation promoted the degradation of CBF3 compared to wild type, whereas 14-3-3κλ mutant and b1l 14-3-3κλ mutant suppressed the degradation of CBF3. Consistently, 14-3-3κλ and b1l 14-3-3κλ mutants showed enhanced freezing tolerance compared to wild type. These results indicate that B1L enhances the freezing tolerance of plants, at least partly through stabilizing CBF. Our findings improve our understanding of the regulation of CBF in response to cold stress.

The cooperative relation between non-hydraulic root signals and osmotic adjustment under water stress improves grain formation for spring wheat varieties.

Non-hydraulic root signals (nHRS) and osmotic adjustment (OA) are two important adaptive responses of plants to water stress. There is little understanding of their relationships during water stress. The threshold range of soil water potential to occurrence of nHRS, the capacity for OA, grain yield and water use efficiency (WUE) were examined in three spring wheat (Triticum aestivum L.) varieties (two bred after 1975 and one bred before 1900) under water stress conditions. The threshold range of nHRS was significantly correlated with the maintenance rate of grain yield (MRGY) (r = 0.99, P < 0.05) under moderate drought (-0.49 to -0.55 MPa) but not under severe drought (-0.70 to -0.76 MPa). There were similar correlations between OA and the MRGY. However, regulation of nHRS precedes OA during gradual water stress. The threshold range of nHRS and OA was positively correlated (r = 0.93, P < 0.05), suggesting a mechanism for adapting to drought. WUE was higher for modern than for old varieties and was correlated with the root efficiency (full biomass weight including root per root weight, r = 0.78, P < 0.05) and the root water uptake efficiency (water consumption per root weight, r = 0.72, P < 0.05). However, there was a significant negative correlation between WUE and root weight (r = -0.84, P < 0.01). The cooperative relationship between the threshold range of nHRS and OA under water stress was beneficial for improving grain formation for spring wheat varieties.

Thylacospermum caespitosum population structure and cushion species community diversity along an altitudinal gradient.

As alpine plants, cushion species are particularly susceptible to environment changes. Thus, understanding population structure and community diversity variation of cushion plants along elevational gradients is crucial for estimating their response to predicted climate changes. In this study, Thylacospermum caespitosum populations from three elevations (low, medium, and high) in three climate zones of China (the Kunlun, Qilian, and Tianshan Mountains) were selected to evaluate the effect of elevation on the structure of T. caespitosum populations and species diversity of cushion communities. Results showed that elevation substantially influenced T. caespitosum populations (size structure, density, and death rate), as well as richness (α-diversity) and microhabitat species pool (species pool) of cushion communities. In the low elevations, T. caespitosum populations were in decline due to a lower ratio of small plants and higher mortality compared with populations at medium and high elevations. The α-diversity and species pool in cushion communities were significantly increased with decreased elevation, but the importance value of T. caespitosum decreased accordingly. Moreover, there was a significant positive correlation between elevation and relative importance value (the importance of one species in the community) of T. caespitosum (r = 0.883; P < 0.01). Elevation was significantly negatively correlated with the mortality rate of T. caespitosum (r = - 0.855; P < 0.01), α-diversity (r = - 0.933; P < 0.001), and species pool (r = - 0.885; P < 0.01). The declining characters of T. caespitosum population structure were obvious in low elevation populations. This decline may directly or indirectly relate to environmental change. Effects of elevation can provide an early indication of range contractions and population declines of cushion species with future climate warming. We call for more mechanistic studies of climate change impacts on cushion populations, particularly in alpine systems near the snow line.

Phylogenetic analysis of lichen-forming fungi Rhizoplaca zopf from China based on ITS data and morphology.

A molecular phylogenetic analysis of Rhizoplaca melanophthalma, Rhizoplaca chrysoleuca, Rhizoplaca peltata and Rhizoplaca haydenii is presented based on the nuclear ribosomal internal transcribed spacer (ITS) regions and morphology. Rhizoplaca species were collected at 3400-3900 m in Tianshan Mountains, Xinjiang province, China. Rhizoplaca haydenii is reported for the first time in China. Maximum parsimony (MP) analysis of ITS sequences obtained from Tianshan Mountains samples and GenBank reveals that the evolution relationship of Rhizoplaca melanophthalma and Rhizoplaca chrysoleuca is closer to each other than to Rhizoplaca peltata, and Rhizoplaca haydenii showed closer relatedness to Rhizoplaca melanophthalma. When the four species groups from Tianshan Mountains were analyzed alone through the neighbour-joining (NJ) and minimum evolution method, we obtained the same result. The morphology analysis of Rhizoplaca Zopf which reveals the pruinose discs and apothecial discs of species did not show convincing evidences to prove phylogenetic relationship among Rhizoplaca species In our study, the result further proved that Rhizoplaca should be rejected as a genus separate from Lecanora.

Effects of the extract of Ammopiptanthus mongolicus cheng f. (JA1) on induction of apoptosis of HepG2 in vitro and its molecular mechanisms.

OBJECTIVE: To study the effects and the mechanisms of extract from a leguminous plant (Ammopiptanthus mongolicus cheng f.) (JA1) in northwest China on inducing apoptosis and inhibiting proliferation of HepG2 hepatocarcinoma cell in vitro. METHODS: The HepG2 cell line was used as target cells. The effect of JA1 on HepG2 cell growth was detected by microculture tetrazolium assay (MTT), flow cytometry assay, DNA agarose gel electrophoresis and transmission electronic microscopy. The expressive effect of the wt-p53 in HepG2 cells was analyzed with p53 protein test-reagent. RESULTS: JAI not only had significant anti-proliferative effects depending upon time and dosage, but also induced apoptosis of HepG2 cells. Apoptotic typical morphological changes were observed in JA1-treated HepG2 cells under transmission electronic microscope, "Sub-G1" phase peak occurred in flow cytometry and DNA "ladder" was found in DNA agarose gel electrophoresis. The expression of the wt-p53 increased in vitro, and JA1-treated HepG2 and the positive cell percentage of the wt-p53 protein also increased. CONCLUSIONS: JA1 could obviously induce apoptosis and inhibit proliferation of HepG2 cells in vitro, and these effects are closely related with the increase of wt-p53 expression. JA1 can be used as a good source of medicinal plant for the treatment of hepatocarcinoma.

[Alleviative effects of nitric oxide on the biological damage of spirulina platensis induced by enhanced ultraviolet-B].

Continuing depletion of the stratospheric ozone layer by atmospheric pollutants, in particular chlorofluorocarbons (CFCs), has resulted in an increasing incidence of solar UV-B (280-320 nm) at the Earth's surface. Enhanced UV-B radiation has been considered as important global environmental problem and results in important effects to mankind and the entire global ecosystem. Nitric oxide (NO) is not only a toxic molecule, one of reactive nitrogen species (RNS), but also an important redox-active signaling molecule. NO is really a double-edged sword, it can be either beneficial and activate defense responses in plants and animals or toxic, together with ROS. Besides those, NO can also act as a signal molecule and play very important roles in life of organisms. To study the effects of NO on the biological specific property of enhanced UV-B stressed Spirulina platensis, the chlorophyll-a, protein contents and biomass were investigated under enhanced UV-B radiation and its combination with different chemical treatment. The changes of chlorophyll-a, protein contents and biomass confirmed that 0.5 mmol/L sodium nitroprusside (SNP), a donor of nitric oxide (NO), could markedly alleviate the biological damage of cyanobacteria-Spirulina platensis 794 caused by enhanced ultraviolet-B. Further results proved that NO significantly increase the content of protein and proline. Meanwhile, the accumulation of reduced glutathione (GSH) in S. platensis cells were raised under normal growth condition. But exogenous NO could decrease the increasing of reduced glutathione (GSH) in enhanced UV-B stressed S. platensis cells. These results suggest that NO has protective effect and can strongly alleviate biological damage caused by UV-B stress in S. platensis 794 cells. For the first time, reported the effect of NO on the regulating ability of biological damage of S. platensis induced by enhanced UV-B. Therefore, further investigations will be necessary to inquire into the interaction and inter-correlation of signal molecules and the mechanism in cyanobacterium under enhanced UV-B stress.