Sucrose phosphate synthase (SPS) is one of a number of sucrose-metabolizing enzymes that regulates the sucrose synthesis pathway. SPSs were purified from etiolated rice seedlings (ERS), green rice seedlings (GRS), rice grain suspension cells under osmotic stress (RGSO), and rice grain suspension cells under illumination (RGSI). A native molecular mass of ca. 420 and 520 kDa was found using native-PAGE. The SDS-PAGE analyses revealed SPSs to be homotetramers composed of subunits with a mass of 116-120 kDa. The maximum activity for SPSs was observed on the third day. As far as their biochemical characterization was concerned, the optimum pH of the enzyme reactions lay generally between 6-8, the optimum temperatures between 35-40°C. The ERS and RGSO SPS Km values for Fm 6-P and UDPG were 1.8 and 35mM, respectively. However, the GRS and RGSI SPS had similar Km values for Fm 6-P and UDPG of 1.5 and 28mM, respectively. GRS and RGSI SPS activities were allosterically regulated by Glc 6-P (activator) or Pi (inhibitor), but ERS and RGSO SPS had no effect. From their regulations and Km values two enzyme forms (SPS-I and SPS-II) could be discriminated in the rice. SPS-II was induced by illumination, but SPS-I by osmotic stress. All SPSs were activated by Mg(superscript 2+). The nucleotides AMP, ADP, ATP, UMP, UDP, GDP and UTP inhibited enzyme activity by about 25-50%. Thiol reagents became sensitized to the enzyme activity, but could be restored with DTT or β-ME. Glucose, galactose, glucosamine, maltose, and lactose activated the enzymes and were inhibited by δ-gluconolactone and mannose. SPSs were also inhibited by PCMBS, cibacron blue F3G-A, and DEP.
The aim of this study was to examine possible antioxidant and antiproliferative activities of 95% ethanol or water extract from water spinach (Ipomoea aquatica Forsk) organs. DPPH staining, total phenolic compounds, total flavonoid content, DPPH radical, reducing power method, FTC method, and inhibition of cancer cell prolifera- tion were employed. Ethanol extract of stem demonstrated a positive effect in DPPH staining when it was diluted to 6.25 mg dry matter/mL while all other fractions showed no effect at the same dilution. This fraction also had the highest content of the total phenolic compounds, as well as the highest reducing power and FTC activity. Ethanol extract of leaf had the highest amount of flavonoids. Using DPPH colorimetric method, it was found that ethanol extract of stem had the highest radical-scavenging activity, followed by ethanol extract of leaf. The antiproliferative activities of water spinach extracts were studied in vitro using human lymphoma NB4 cells, and the following results were found: water extract of stem had the highest antiproliferative activity with an EC 50 of 661.40 ± 3.36 µg dry matter/mL, followed by ethanol extract of stem and ethanol extract of leaf. The water extract of leaf had the lowest antiproliferative activity (EC 50 >1000 µg dry matter/mL) under the experimental conditions.
Basidiomes of Antrodia cinnamomea are used as a traditional medicine in Taiwan. Because fungal basidiomes are so rare and attempts to cultivate them have failed, the price they command in the marketplace is exorbitant. Here, one dikaryotic isolate from a pairing between two compatible monokaryons obtained from two different basidiomes was able to produce basidiomes on PDA and MEA media at temperatures of 20-28°C within 45 days of incubation. The basidia and basidiospores produced on the hymenial layer were microscopically observed.
Alpha-amylase inhibitor protein, which inhibits the activity of insect (Callosobruchus analis) alpha-amylase, was characterized from V. sublobata. The molecular weight of purified inhibitor protein was 14 kDa by SDS-PAGE. The inhibitor is non-glycosylated protein and its N-terminal sequence is similar (A P S P V...) to Phaseolus vulgaris alpha-AI-1. Its pI value is 6.0 and largely localised in cotyledons. The inhibitory activity decreased during germination from days one to five. In the developmental stages of seed formation from anthesis to 30 days the inhibitor content increased.
Genetically improved Autographa californica (Speyer) multiple nucleopolyhedrovirus (AcMNPV), the type species of the genus Nucleopolyhedrovirus, was constructed with an insect-selective toxin gene (LqhIT2) derived from the venom of the Israeli yellow scorpion, Leiurus quinquestriatus (Ehrenberg). LqhIT2 was expressed by two distinct and temporally regulated promoters. These two promoters were the early promoter p-PCm (a pu-enhanced minimal CMV promoter) and the very late p10 promoter, resulting in recombinant viruses named vAPcmIT2 and vAP10IT2, respectively. Western blot and bioassay analysis showed that the toxin gene under the control of the early p-PCm promoter was expressed 24 h earlier than under the very late p10 promoter control, and vAPcmIT2 killed faster than vAP10IT2. The EC50 (effective concentration, EC) values of wild type AcMNPV, vAP1OIT2, and vAPcmIT2 to the 3(superscript rd)-instar larvae of Trichoplusia ni (Hubner) were 1.00, 0.19, and 0.17 polyhedral inclusion bodies (PIBs)/mm^3, respectively. Compared to the very late promoter, the early promoter could shorten the effective time (ET50) by 14~17 h. Furthermore, for the 4(superscript th)-instar larvae, compared to the wild-type virus and vAP10G, a 19~23% and 30~33% reduction in ET50 were found using vAP10IT2 and vAPcmIT2, respectively. Field trials of these two viruses showed an effective paralysis of the infected larvae which then stopped feeding, resulting in a decrease in the leaf area eaten compared to those larvae infected with wild-type virus and another control virus vAP1OG, the GFP-expression recombinant AcMNPV. Scorpion toxin driven by the early p-PCm promoter shortened the time-to-paralysis of insect larvae, and thus provided an economical pest control advantage over that driven by the very late p10 promoter.
The aim of this study was to determine whether there were major differences in the seasonal incidence and abundance of pollen grains and pollen types in pollen fall between sites of different land use. The material was collected from 5 January until 20 December 2003 at three sites in central Croatia. The sampling sites were located in an average urban setting (Zagreb), a rural setting (Ivanic Grad), and a small town surrounded by a large woodland with partially thermophilic vegetation (Samobor). Using the volumetric method of pollen collection, pollen grains of 3537 taxa were identified, depending on the sampling site, eight of them producing the greatest amount of pollen (Alnus sp., Ambrosia sp., Betula sp., Carpinus sp., Poaceae, Quercus sp., Taxus/Juniperus, and Urticaceae). Differences among the sampling sites were recorded according to seasonal pollen concentration, total monthly pollen count, and total annual pollen count. The proportion of particular plant classes (tree, grass and weed) was quite comparable among the three sampling sites because of their relative geographic proximity (i.e. within the same climatic region). A difference was observed in the air pollen concentration. All three monitoring sites revealed the total annual airborne pollen concentration in inland Croatia to be dominated by highly allergenic pollen (Zagreb 54%, Samobor 58% and Ivanic Grad 82%) of the following taxa: Alnus sp., Ambrosia sp., Betula sp., Carpinus sp., Corylus sp., Poaceae, Urticaceae and Artemisia sp. Accordingly, there were no distinct phenologically induced differences in pollen species recorded at the three monitoring sites. However, substantial differences were observed in seasonal pollen grain count and in the percentage proportion of some allergenic pollen grains (Ambrosia sp., Betula sp., Carpinus sp., Poaceae), providing valuable information to individuals suffering from pollen allergy.
Blooms of ulvoid macroalgae (mainly Enteromorpha and Ulva) have covered 80% of the intertidal seagrass bed at Wanlitung, southern Taiwan, effectively shading the seagrasss species Thalassia hemprichii resulting in a decrease in photosynthetic performance and low inorganic carbon (C i ) uptake. We looked for evidence of C i limitation and investigated the C i utilization characteristics of ulvoid-free and ulvoid-covered T. hemprichii. The rapid light curve (RLC) function of the Diving-PAM (Diving-PAM, Walz, Germany) was used to measure in situ photosynthetic performance (based on the effective quantum yield of PSII (Y) values) of intact seagrasses that were placed in small incubating chambers. Significantly, a lower RETR max (maximum relative electron transport rate) and E k (light intensity at the onset of saturation) were noted in the ulvoid-covered compared to the ulvoid-free T. hemprichii, suggesting that the former has acclimatized to the low light environment becoming a shade type plant. The ulvoid-covered T. hemprichii showed some evidence of C i limitation since a significant increase in RETR max (up to 46%; P < 0.05) was noted after an increase in the concentration of NaHCO 3 from 2.2 (normal seawater) to 6.2 mM. In terms of C i utilization characteristics, T. hemprichii could directly absorb HCO 3 - as the major C i source but partially depended on the extracellular carbonic anhydrase (CA) to convert HCO 3 - to CO 2 prior to uptake in the ulvoid-free, high light- adapted populations. A wastewater stream with a high nutrient load coming from the urbanized area may have caused the frequent blooms of ulvoid macroalgae.
Shoot tips from 4- to 6-week-old in vitro grown plantlets of six different cultivars of papaya were cryopreserved by vitrification. Shoot tips were treated in a 2 ml cryo-tube filled with a solution of 2 M glycerol and 0.4 M sucrose at 25degreesC for 20 min, then dehydrated with 1 ml pre-cooled vitrification solution at 4degreesC for 60 min. Treated samples were kept in liquid nitrogen for at least one week. The recovery percentages of six cultivars' shoot tips after vitrification were between 48% and 93%. Cryopreserved shoot tips after being warmed resumed growth within 7 days and developed shoots directly without intermediate callus formation. Prolonged storage in the liquid nitrogen up to 2 years had no affect on regeneration. This study has successfully developed a simple and effective protocol for the cryopreservation of papaya shoot tips.
The properties and tissue compositions of reaction wood in the leaning trunks or branches of trees have been extensively investigated, but studies on the influence of the related reaction on the secondary phloem are few and incomplete, in this work, formation of the vascular and cork cambium and the secondary phloem in the upper and lower sides of branches of Zelkova serrata Makino were studied. Vascular cambium is formed first in the second internode of shoot. In the third internode, cork cambium began to initiate subepidermally, meanwhile two to four layers of grouped gelatinous fibers were first found in the cortex outside of all the primary phloem. Gelatinous fibers were also found in both the secondary xylem and secondary phloem. The leaning branches exhibited pronounced radial secondary growth promotion to the upper side, and the reaction wood formed eccentrically. The secondary phloem formed layers of conducting sieve elements alternately with gelatinous fibers. Comparing the secondary phloem in the upper side (reaction phloem) of the branches with that in the lower side (opposite phloem), there is no obvious difference in thickness. Nevertheless, it was found that in the cross sections, the gelatinous fibers formed earlier, and there were more continuous cell senates and a much larger area ratio in the upper side. Besides, the sieve tubes in the upper side of secondary phloem were longer and wider and possessed a very horizontally-orientated sieve plate between two sieve elements. These features may imply that, in branches of Zelkova serrata, the secondary phloem of the upper side (reaction phloem) may have a higher translocation efficiency.
A new species, Ephedra dawuensis Y. Yang, is described and illustrated. This species is similar to E. equisetina Bunge in having thin branchlets 0.8-1 mm in diam. and brown scale-like leaves, and to E. saxatilis Royle ex Florin in having oblong-ellipsoid seeds and a short micropylar tube ca. 0.5 mm long. It differs from E equisetina in having female cones bearing 2 or 3 pairs of bracts (vs. 4 or 3 pairs of bracts), purplish black, oblong-ellipsoid seeds (vs. ovoid seeds), and a shorter micropylar tube (vs. 1.5-2 mm long), and from E. saxatilis in having thinner branchlets and brown scale-like leaves (vs. yellowish, narrow triangular, leaves). This new species is assigned to E. sect. Ephedra in that the bracts of female cones become fleshy at maturity. Intermediate character states of this new species provide evidence for the hypothesized affinity between E. equisetina and E. saxatilis.
The pollen morphology of five taxa of Cuscuta, C. australis, C. campestris, C. chinensis, C. japonica var. formosana and C. japonica var. japonica (Convolvulaceae) in Taiwan was investigated by using LM, SEM and TEM. The pollen of Cuscuta is three to six colpate, zonocolpate or pantocolpate colpus with granules, tectum with scabrate processes, ektexine reticulate or finely reticulate, and endexine thinner than ektexine. Two distinct pollen types of Cuscuta spp. in Taiwan are recognized. Type 1 is small, and has colpus with granules, and ektexine finely reticulate, including C. australis, C. campestris and C. chinensis. Type 2 is medium in size, and has colpus with granules, scabrate processes on surface of granule and ektexine reticulate, including C. japonica var. formosana and C. japonica var. japonica. Pollen types correspond with Yuncker's subdivision of the genus Cuscuta, type 1 for subgenus Grammica and type 2 for subgenus Monogyna. Meanwhile, pollen characteristics are also helpful in identifying species.
In this study, the clearing method, LM, SEM, and TEM were used to examine the morphology and ultra-structures of laminar hydathodes of Ficus formosana Maxim. f. Shimadai Hayata. Morphologically, the laminar-hydathode of F. formosana is complex epithemal type, consisting of water pores. tracheid-ends, epithem cells, and a bounding sheath layer. Water pores are made up of two guard cells, and their pores open permanently. Ultrastructural and cytological data demonstrated that the epithem cell has a dense cytoplasm, numerous mitochondria, an extended ER system, and many small vesicles derived from Golgi bodies. It has proliferate peroxisomes, and their numbers increase with the maturation and aging of the epithem. In addition, abundant plasmodesmata were observed on the contacted cell wall between epithem cells. Variable structures of plasmalemmasome were also observed on the plasma membrane of the epithem. Plasmalemmasome is the result of endocytosis caused by the plasmolysis-deplasmolysis cycle, which is induced by repeating transpiration and guttation day and night. We suggest that the hydathode is an ideal system for studying endocytosis in plants.
All Phalaenopsis species have the same chromosome number (2n = 2x = 38), but the sizes of their genomes vary markedly. In this study, we investigated genome organization and relationships of Phalaenopsis species by genomic in situ hybridization (GISH) of seven interspecific hybrids derived from crosses between species with similar or dissimilar sizes of genomes. In the hybrid R aphrodite x R sanderiana, in which both parents possess small genomes, the two parental chromosome sets could not be distinguished by the strength and distribution of hybridization signals. Similar results were obtained from the hybrid P. mannii x P. violacea, in which both parents have large genomes. These results suggest that the two parents of these hybrids have similar genomes. In hybrids in which one parent possesses a large and the other parent a small genome, such as P. amboinensis x P. stuartiana, the two parental chromosome sets could readily be distinguished by GISH with or without the application of blocking DNA. Examination of hybridization signals on chromosomes revealed that species with large genomes have much more repetitive sequences, both in type and amount; however, species with small genomes do have their own specific sequences. In general, genome relationships obtained from GISH are in agreement with those from traditional genome analysis of other investigators. This study demonstrates that GISH is a useful tool for investigating genome organization and relationships of plant species, especially when analysis of meiotic behavior is technically difficult.
AtTRP1 is an Arabidopsis protein that binds duplex telomeric DNA in vitro. Here we showed that knockout of AtTRP1 did not change significantly the telomere length in plant. This implies that either AtTRP1 does not participate in the regulation of telomere length or the Arabidopsis genome contains other genes functionally redundant to AtTRP1. Sequence analysis of Arabidopsis genome together with molecular cloning enabled us to identify two additional genes AtTRP3 and AtTRP4 and the corresponding cDNA clones encoding AtTRP1-like proteins. The C-terminal regions of both AtTRP3 and AtTRP4 proteins bind specifically duplex telomeric DNA in vitro. The amino acid sequence of AtTRP4 is identical to that of another Arabidopsis protein TRFL1 except with an internal deletion of six amino acids, suggesting that AtTRP4 and TRFL1 may be derived from the same gene by alternative splicing. This speculation was further confirmed by DNA sequence analysis of RT-PCR products specific for AtTRP4 and TRFL1 transcripts. Our data together with reports from other researchers revealed that Arabidopsis contains at least seven different duplex telomeric DNA-binding proteins encoded by a six-member gene family, named AtTRP. We proposed that some members of the AtTRP family may be functionally redundant in the regulation of telomere length in Arabidopsis.
The cDNA of tomato chloroplastic Cu/Zn-superoxide dismutase was used to construct transgenic tobacco (Nicotiana plumbaginifolia). It was found that the gene expression of Cu/Zn-superoxide dismutase can constitutively be reduced in the transgenic tobacco plants because of double-stranded (dsRNA) expressed in the form of intron-spliced hairpin structures and antisense suppression. Furthermore, the endogenous chloroplastic Cu/Zn-superoxide dismutase gene in tobacco was established as a target for silencing due to the operation of hpRNA and antisense RNA constructs. However, under salt and PEG-induced stress, the cytosolic Cu/Zn-superoxide dismutase activity in transformed plants obviously increased. At the same time, the PEG pretreatment was able to promote tolerance of the transgenic plants to the salt stress. These results indicated that the use of hpRNA and anti-sense was able to successfully knockout the transcript encoding a chloroplast superoxidase dismutase. The null transformed plants grown under stress produced or retained other superoxide dismutase to compensate for the loss of the chloroplast. Consequently, hpRNA constructs would be helpful in discovering and validating the endogenous chloroplastic Cu/Zn-superoxide dismutase gene, and to prove the cytosolic alternative pathway of plant antioxidation associated with the function of Cu/Zn-superoxide dismutase gene under an unfavorable environment. Meanwhile, the experiment will provide an important technique for the antisensing strategies operating in tobacco.
Plants have evolved complex regulatory mechanisms in adapting to various environmental stresses. One of the consequences of many stresses is an increase in the cellular concentration of reactive oxygen species (ROS), which are subsequently converted to hydrogen peroxide (H2O2). An oxidative burst caused by biotic or abiotic stress leads to a disturbance in the cellular redox balance and is highly toxic to cells. Recently, H2O2, in addition to being a toxicant, has been regarded as a signaling molecule and a regulator of the expression of some genes in cells. These include genes encoding antioxidants, cell rescue/defense proteins, and signaling proteins such as kinase, phosphatase, and transcription factors. Here, we review the function of H2O2 as a signal molecule in the transduction of stress signals to the alteration of expression profiles of target genes, and we summarize the evidence that H2O2 acts as a stress signal in plants.
Glyptostrohus pensilis (Staunton ex D. Don) K. Koch is a critically endangered species, only distributed in South China and Vietnam. Genetic variation within and between populations was investigated using inter-simple sequence repeat (ISSR). One hundred and seventy individuals from fourteen populations representing five regions in southern and southeastern China were sampled. The results show that genetic diversity of G. pensilis is rather low: P=10.2%, Ae=1.070 and Hpop=0.058 at population level; P=24.7%, Ae=1.140 and Hsp=0.122 at species level. The results further indicate significant genetic differentiation between regions (16.10%, analysis of molecular variance [AMOVA], p＜0.005), between populations within particular regions (29.76%, P＜0.0002), and between individuals within populations (54.14%, P＜0.0002). High levels of genetic differentiation between populations were also detected based on Nei's genetic diversity analysis (51.36%), and Shannon's diversity index (52.54%). Possible reasons for the high genetic differentiation and its low level of diversity within populations are discussed. Possible in situ and ex situ conservation strategies for G. pensilis are suggested.
Experiments were conducted to find the difference among green gram (Vigna radiata L. Wilczek) genotypes for salinity tolerance during germination and during vegetative and reproductive growth stages under NaCl treatment. Data revealed significant genotypic differences in germination percentage and post-germination survival of seedlings, symptoms of salt injury, changes in the levels of Na+, Cl- and K+ and total chlorophyll at the vegetative stage, and in pod development, seed yield, and yield components at the reproductive stage. Salinity tolerance in green gram was related to greater final germination and post-germination seedling survival, low scorching, chlorosis and necrosis of aerial parts, reduced Na+ and Cl-, slightly enhanced K+, and greater chlorophyll content. Appearance of increased symptoms on the aerial parts was positively related to increased Na+ and Cl- and negatively to increased K+. At maturity, pod weight, seed:hull weight ratio, 100-seed weight, seed yield and harvest index were also greater in M-6601. Maintenance of a steady seed:hull weight ratio in M-6601 indicated that a higher seed yield in this genotype is principally due to a greater partitioning of photoassimilates to seed rather than hull during pod development under saline conditions. Based on these criteria, M-6601 and 241/11 were declared salt tolerant and sensitive, respectively. Essentially then, ion-toxicity is the dominant factor modulating the salt tolerance of green gram during growth periods,
Flow cytometry (FCM), simple sequence repeats (SSR) and cleaved amplified polymorphic sequence (CAPS) were used to characterize the ploidy level and the nuclear and cytoplasmic compositions of two intergeneric somatic hybrids of Changshou kumquat (Fortunella obovata), leaf parent, and Dancy tangerine (Citrus reticulata), suspension parent. FCM showed that both somatic hybrids were tetraploids. One out of two SSR primer pairs was able to detect polymorphisms between the fusion parents, and the somatic hybrids showed additive banding profiles, showing them to be allotetraploid hybrids with inherited nuclear DNA from both parents. CAPS showed that 4 and I polymorphic loci were obtained for parental chloroplast and mitochondrial genomes, respectively. The somatic hybrid plants had the same banding patterns as the suspension parent for all of the polymorphic chloroplast markers, and their mitochondrial banding patterns approximated those of the suspension parent with loss of some mtDNA fragments. The present research plays a pivotal role in understanding the relationship between nuclear and cytoplasmic compositions and the likely field performance of somatic hybrids in the future.
