Realistic low-doses of two emerging contaminants change size distribution of an annual flowering plant population.

HHCB [1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta(g)-2-benzopyran] and 4-tert-octylphenol [4-(1,1,3,3-tetramethylbutyl)phenol] are widely used emerging contaminants that have the potential to cause adverse effects in the environment. The purpose of this study was to observe if and how environmentally realistic concentrations of these contaminants alter growth in plant populations. It was hypothesized that within an exposed Gypsophila elegans Bieb (annual baby's breath) population especially fast-growing seedlings are impaired even when the population mean is unaffected, and small doses can cause hormesis and, thus, an increase in shoot or root length. In a dose-response experiment, an experimental population of G. elegans was established (total 15.600 seeds, 50 seeds per replicate, 24 replicates per concentration, 5.2 seedlings/cm2) and exposed to 12 doses of HHCB or 4-tert-octylphenol. After five days, shoot and root length values were measured and population averages, as well as slow- and fast-growing subpopulations, were compared with unexposed controls. Growth responses were predominantly monophasic. HHCB seemed to selectively inhibit both root and shoot elongation among slow- and fast-growing individuals, while 4-tert-octylphenol selectively inhibited both root and shoot elongation of mainly fast-growing seedlings. The ED50 values (dose causing 50% inhibition) revealed that the slow-growing seedlings were more sensitive and fast-growing seedlings less sensitive than the average of all individuals. Although there was toxicant specific variation between the effects, selective toxicity was consistently found among both slow- and fast-growing plants starting already at concentrations of 0.0067 µM, that are usually considered to be harmless. This study indicates that these contaminants can change size distribution of a plant population at low concentrations in the nM/µM range.

Target-site and non-target-site-based resistance to tribenuron-methyl in multiply-resistant Myosoton aquaticum L.

Myosoton aquaticum L., a widespread and competitive winter weed of wheat in China, has evolved resistance to many classes of herbicides. In one M. aquaticum population (AH03), collected from Anhui Province, where tribenuron-methyl and florasulam had been used to control this weed resistance to both herbicides had evolved. Compared with the sensitive population, HN03(S), the resistant (R) population, AH03, was highly resistant to tribenuron-methyl, flucarbazone-Na and pyroxsulam, moderately resistant to pyrithiobac­sodium, and florasulam, and had low resistance to diflufenican. AH03 was still controlled by imazethapyr, 2,4-D butylate, fluroxypyr-meptyl, and isoproturon. Pretreatment with the P450 inhibitor malathion reduced the GR50 value of tribenuron-methyl by 43% in the R population, and by 25% in the S population. This indicates that P450-mediated enhanced metabolism is one likely mechanism for tribenuron-methyl resistance in M. aquaticum. Glutathione-S-transferase (GST) activity could be induced by tribenuron-methyl in both the R and S populations. However, both the basal and induced GST activity of the R population was lower than that of the S population. The in vitro ALS assay confirmed that the ALS from the R plants showed a high resistance (52.93-fold) to tribenuron-methyl. ALS gene sequencing revealed a Pro197Ala substitution in the R plants. Based on the ALS gene sequence analysis, molecular markers were also developed to identify the specific Pro197Ala mutation. This population of M. aquaticum has multiple resistance and target-site (ALS Pro197Ala) and non-target-site resistance mechanisms contribute to tribenuron-methyl resistance.

Non-target site-based resistance to tribenuron-methyl and essential involved genes in Myosoton aquaticum (L.).

BACKGROUND: Water chickweed (Myosoton aquaticum (L.)) is a dicot broadleaf weed that is widespread in winter fields in China, and has evolved serious resistance to acetolactate synthase (ALS) inhibiting herbicides. RESULTS: We identified a M. aquaticum population exhibiting moderate (6.15-fold) resistance to tribenuron-methyl (TM). Target-site ALS gene sequencing revealed no known resistance mutations in these plants, and the in vitro ALS activity assays showed no differences in enzyme sensitivity between susceptible and resistant populations; however, resistance was reversed by pretreatment with the cytochrome P450 (CYP) monooxygenase inhibitor malathion. An RNA sequencing transcriptome analysis was performed to identify candidate genes involved in metabolic resistance, and the unigenes obtained by de novo transcriptome assembly were annotated across seven databases. In total, 34 differentially expressed genes selected by digital gene expression analysis were validated by quantitative real-time (qRT)-PCR. Ten consistently overexpressed contigs, including four for CYP, four for ATP-binding cassette (ABC) transporter, and two for peroxidase were further validated by qRT-PCR using additional plants from resistant and susceptible populations. Three CYP genes (with homology to CYP734A1, CYP76C1, and CYP86B1) and one ABC transporter gene (with homology to ABCC10) were highly expressed in all resistant plants. CONCLUSION: The mechanism of TM resistance in M. aquaticum is controlled by NTSR rather than TSR. Four genes, CYP734A1, CYP76C1, CYP86B1, and ABCC10 could play essential role in metabolic resistance to TM and justify further functional studies. To our knowledge, this is the first large-scale transcriptome analysis of genes associated with NTSR in M. aquaticum using the Illumina platform. Our data provide resource for M. aquaticum biology, and will facilitate the study of herbicide resistance mechanism at the molecular level in this species as well as in other weeds.

Enhanced Herbicide Metabolism and Metabolic Resistance Genes Identified in Tribenuron-Methyl Resistant Myosoton aquaticum L.

The evolved resistance of Myosoton aquaticum L. to acetolactate synthase (ALS) inhibitors is well established, but most research has focused on target-site resistance, while nontarget-site resistance remains neglected. Here, we investigated mechanisms of the latter. The pretreatment with the P450 inhibitor malathion significantly increased the sensitivity of resistant plants to tribenuron-methyl. The rapid P450-mediated tribenuron-methyl metabolism in resistant plants was confirmed by LC-MS/MS analysis. Besides, GST activity was higher among resistant than susceptible individuals. The next transcriptome analysis generated 544,102,236 clean reads from RNA sequencing libraries. De novo assembly yielded 102,529 unigenes with an average length of 866 bp, annotated across seven databases. Digital gene expression selected 25 differentially expressed genes, further validated with qRT-PCR. Three P450 genes, two GST genes, two glucosyltransferase genes, four ABC transporter genes, and four additional contigs were constitutively up-regulated in resistant individuals. Overall, our research confirmed that enhanced herbicide metabolism drives tribenuron-methyl resistance in M. aquaticum.

Effects of indole-3-butytric acid on lead and zinc accumulations in Pseudostellaria maximowicziana.

Plant hormones can improve the phytoremediation capabilities of heavy metal hyperaccumulator plants. In this study, different doses of indole-3-butytric acid (IBA) were sprayed on the leaves of the lead (Pb) and zinc (Zn) accumulator plant Pseudostellaria maximowicziana, which was planted in Pb-Zn contaminated soil, and the effects of IBA on Pb and Zn accumulation levels in P. maximowicziana were studied. Spraying 25- and 50-mg/L IBA doses increased the stem, leaf and shoot biomasses of P. maximowicziana compared with the control, while 75- and 100-mg/L IBA doses decreased them. The 50-mg/L IBA dose increased the P. maximowicziana contents of chlorophyll a, total chlorophyll and carotenoid of compared with the control, and other doses had no significant effects or decreased these values. Spraying IBA reduced the superoxide dismutase activity of P. maximowicziana compared with the control, but improved the peroxidase and catalase activities. The 50-, 75-, and 100-mg/L IBA doses increased the Pb and Zn contents in P. maximowicziana compared with the control and also increased the amounts of Pb and Zn extracted by P. maximowicziana. Thus, 50 mg/L of IBA could promote the growth and the Pb and Zn phytoremediation capabilities of P. maximowicziana.

Proteomic analysis of naturally occurring boron tolerant plant Gypsophila sphaerocephala L. in response to high boron concentration.

Gypsophila sphaerocephala is a naturally Boron (B) tolerant species that can grow around the B mining areas in Turkey, where the B concentration in soil reaches a lethal dose for plants (up to ∼8900mgkg-1 (∼140mM). While its interesting survival capacity in extremely B containing soils, any molecular research has been conducted to understand its high tolerance mechanism yet. In the present study, we have performed a proteomic analysis of this plant to understand its high tolerance towards B-stress. Seedlings of G. sphaerocephala were collected from B mining area and were adapted to greenhouse conditions. An excessive level of Boric acid (3mM)was applied to the plantlets for 24h. Total proteins were precipitated by using TCA/Acetone method. 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis) analysis of the proteins was carried out. Out of 121 protein spots, 14 were differentially expressed between the control and B-exposed G. sphaerocephala roots. The peptide profile of each protein was determined by MALDI-TOF mass spectrometer after in-gel trypsin digestion. The identified proteins are involved in different mechanisms in the cell such as in antioxidant mechanism, energy metabolism, protein degradation, lipid biosynthesis and signaling pathways, indicating that G. sphaerocephala has multiple cooperating mechanisms to protect itself from high B levels. Overall, this study sheds light on to the possible regulatory switches (gene/s) controlling the B-tolerance proteins and their possible roles in plant's defense mechanism.

Moderate irrigation intervals facilitate establishment of two desert shrubs in the Taklimakan Desert Highway Shelterbelt in China.

BACKGROUND: Water influences various physiological and ecological processes of plants in different ecosystems, especially in desert ecosystems. The purpose of this study is to investigate the response of physiological and morphological acclimation of two shrubs Haloxylon ammodendron and Calligonum mongolicunl to variations in irrigation intervals. METHODOLOGY/PRINCIPAL FINDINGS: The irrigation frequency was set as 1-, 2-, 4-, 8- and 12-week intervals respectively from March to October during 2012-2014 to investigate the response of physiological and morphological acclimation of two desert shrubs Haloxylon ammodendron and Calligonum mongolicunl to variations in the irrigation system. The irrigation interval significantly affected the individual-scale carbon acquisition and biomass allocation pattern of both species. Under good water conditions (1- and 2-week intervals), carbon assimilation was significantly higher than other treatments; while, under water shortage conditions (8- and 12-week intervals), there was much defoliation; and under moderate irrigation intervals (4 weeks), the assimilative organs grew gently with almost no defoliation occurring. CONCLUSION/SIGNIFICANCE: Both studied species maintained similar ecophysiologically adaptive strategies, while C. mongolicunl was more sensitive to drought stress because of its shallow root system and preferential belowground allocation of resources. A moderate irrigation interval of 4 weeks was a suitable pattern for both plants since it not only saved water but also met the water demands of the plants.

Cryopreservation of in vitro Shoot Tips of Ulluco (Ullucus tuberosus Cal.) Using D Cryo-Plate Method.

　　BACKGROUND: Maintenance of in vitro collections of ulluco (Ullucus tuberosus Cal.) is cumbersome and costly in an ex-situ genebank. An alternative method for long term preservation which is safe and cost-effective is required. OBJECTIVE: To apply a novel cryopreservation procedure using the cryo-plate system to improve the long-term conservation of ulluco. MATERIALS AND METHODS: Initially V and D cryo-plate methods were tested, subsequently the D cryo-plate method was selected for ulluco cryopreservation. The D cryo-plate procedures were optimized for post-LN regrowth procedures including cold-hardening, sucrose addition in alginate gel, and duration of LS treatment. Optimized procedures were tested with 11 ulluco lines. RESULTS: Shoot tips were isolated from cold-hardened shoots for 3-4 weeks at 5 degree C were excised to 1.0-1.5 mm long and 0.5 mm wide and precultured for 16h at 25 degree C on MS with 0.3 M sucrose. The shoot tips were attached on the cryo-plates by alginate gel with 0.4M sucrose. The cryo-plates with attached shoot tips were treated with 2.0 M glycerol and 1.0 M sucrose solution for 90 min at 25 degree C and dehydrated on filter paper in a Petri dish by air current flow at 25 degree C for 45 min before direct immersion in LN. This optimized procedure was applied to shoot tips of 11 ulluco lines, resulting regrowth ranging from 73 % to 97 %, with an average of 90 % post-LN regrowth. CONCLUSION: D cryo-plate is a practical and simple procedure for cryo-storage of in vitro grown ulluco shoot tips in an ex situ genebank.

Uniconazole (S-3307) strengthens the growth and cadmium accumulation of accumulator plant Malachium aquaticum.

The effects of uniconazole (S-3307) application on the growth and cadmium (Cd) accumulation of accumulator plant Malachium aquaticum (L.) Fries. were studied through a pot experiment. The application of S-3307 increased the biomass and photosynthetic pigment content of M. aquaticum in Cd-contaminated soil, and also improved the superoxide dismutase (SOD) and peroxidase (POD) activities in M. aquaticum. Application of S-3307 increased Cd content in shoots and decreased Cd content in roots of M. aquaticum, but the translocation factor (TF) of M. aquaticum increased with the increase of S-3307 concentration. For phytoextraction, the application of S-3307 increased Cd extractions by roots, shoots and whole plants of M. aquaticum, and the maxima were obtained at 75 mg L-1 S-3307, which increased by 22.07%, 37.79% and 29.07%, respectively, compared with their respective controls. Therefore, S-3307 can be used for enhancing the Cd extraction ability of M. aquaticum, and 75 mg L-1 S-3307 was the optimal dose.

A novel Pro197Glu substitution in acetolactate synthase (ALS) confers broad-spectrum resistance across ALS inhibitors.

Water chickweed (Myosoton aquaticum L.), a competitive broadleaf weed, is widespread in wheat fields in China. Tribenuron and pyroxsulam failed to control water chickweed in the same field in Qiaotian Village in 2011 and 2012, respectively. An initial tribenuron resistance confirmation test identified a resistant population (AH02). ALS gene sequencing revealed a previously unreported substitution of Glu for Pro at amino acid position 197 in resistant individuals. A purified subpopulation (WRR04) that was individually homozygous for the Pro197Glu substitution was generated and characterized in terms of its response to different classes of ALS inhibitors. A whole-plant experiment showed that the WRR04 population exhibited broad-spectrum resistance to tribenuron (SU, 318-fold), pyrithiobac sodium (PTB, > 197-fold), pyroxsulam (TP, 81-fold), florasulam (TP, > 36-fold) and imazethapyr (IMI, 11-fold). An in vitro ALS assay confirmed that the ALS from WRR04 showed high resistance to all the tested ALS inhibitors. These results established that the Pro197Glu substitution endows broad-spectrum resistance across ALS inhibitors in water chickweed. In addition, molecular markers were developed to rapidly identify the Pro197Glu mutation.

Polar auxin transport is essential for gall formation by Pantoea agglomerans on Gypsophila.

The virulence of the bacterium Pantoea agglomerans pv. gypsophilae (Pag) on Gypsophila paniculata depends on a type III secretion system (T3SS) and its effectors. The hypothesis that plant-derived indole-3-acetic acid (IAA) plays a major role in gall formation was examined by disrupting basipetal polar auxin transport with the specific inhibitors 2,3,5-triiodobenzoic acid (TIBA) and N-1-naphthylphthalamic acid (NPA). On inoculation with Pag, galls developed in gypsophila stems above but not below lanolin rings containing TIBA or NPA, whereas, in controls, galls developed above and below the rings. In contrast, TIBA and NPA could not inhibit tumour formation in tomato caused by Agrobacterium tumefaciens. The colonization of gypsophila stems by Pag was reduced below, but not above, the lanolin-TIBA ring. Following Pag inoculation and TIBA treatment, the expression of hrpL (a T3SS regulator) and pagR (a quorum-sensing transcriptional regulator) decreased four-fold and that of pthG (a T3SS effector) two-fold after 24 h. Expression of PIN2 (a putative auxin efflux carrier) increased 35-fold, 24 h after Pag inoculation. However, inoculation with a mutant in the T3SS effector pthG reduced the expression of PIN2 by two-fold compared with wild-type infection. The results suggest that pthG might govern the elevation of PIN2 expression during infection, and that polar auxin transport-derived IAA is essential for gall initiation.

Effect of acid solutions on plants studied by the optical beam deflection method.

The optical beam deflection method was applied to study the effects of acid solution on both a terrestial and aquatic plants Egeria and Cerastium, which are common aquatic plant and terrestial weed respectively. A probe beam from a He-Ne laser was passed through a vicinity of a leaf of the plants, which were put in culture dishes filled with acid solutions. Deflection signals of the probe beam were monitored and compared for acid solutions with different pH values. The results of Egria showed that the deflection signals changed dramatically when pH values of acid solutions were 2.0 and 3.0, while little at pH of 4.0 and 5.0. For Cerastium when pH were below 3.0, deflection signals changed greatly with time at the begining. After a certain period of time, deflection signals changed little with time. When pH value was above 4.0, deflection signals of Cerastium were still changing with time even after 20 hours. The results suggested that the damage threshold of pH was between 3.0 and 4.0 for both the land and aquatic plants.

A comparative transcriptomic analysis of the extremely boron tolerant plant Puccinellia distans with the moderately boron tolerant Gypsophila arrostil.

UNLABELLED: The Turkish ecotype of Puccinellia distans displays exceptional boron (B) tolerance, >1,250 mg B L⁻¹, compared to <50 mg B L⁻¹ for Gypsophila arrostil. In the present study, we compare the molecular basis for the difference in B tolerance between the two species by constructing high B-responsive suppression subtractive hybridization libraries to identify the upregulated genes. A total of 219 and 113 unique non-redundant expressed sequence tags (ESTs) were identified and functionally classified in P. distans and G. arrostil, respectively. In addition, 63 ESTs were down-regulated in P. distans in response to high B. The majority of the high B upregulated genes belong to four categories: metabolism, protein synthesis, cellular organization, and stress/defense. We hypothesize that the superior B tolerance exhibited by P. distans may be due to its ability to restrict the accumulation of B in plant tissues through the upregulated expression of efflux transporters comparable to the Bot1 transporter of barley. In addition, our results are consistent with the view that other molecular mechanisms involved in stress/defense, such as detoxification, anti-oxidative, and signaling pathways, are needed to tolerate B-toxicity stress. KEY MESSAGE: The molecular basis of boron tolerance of two plant species (Puccinellia distans and Gypsophila arrostil) that differ greatly in their boron tolerance was studied in this manuscript.

Bioactive clerodane diterpenes from roots of Carex distachya.

Two new clerodane diterpenes were isolated from roots of Carex distachya Desf., a perennial plant widely distributed in the coastal area of the Mediterranean basin. Chemical characterization of the metabolites was carried out mainly by 1D and 2D NMR spectroscopy. The isolated compounds influenced either positively or negatively the plant growth (root and shoot elongation) of three coexisting herbaceous species.

Mechanisms of boron tolerance and accumulation in plants: a physiological comparison of the extremely boron-tolerant plant species, Puccinellia distans, with the moderately boron-tolerant Gypsophila arrostil.

The physiological characteristics of the extremely boron (B)-tolerant plant species, Puccinellia distans, were compared with those of the moderately tolerant Gypsophila arrostil, two species collected from a B-mining area of Eskisehir, Turkey. Boron was supplied to plants hydroponically at B concentrations ranging from 0.5 to 50 mg B/L for G. arrostil, and from 0.5 to 2000 mg B/L for P. distans. The results show that P. distans has a strikingly greater tolerance to B than G. arrostil. While G. arrostil was unable to survive B supply concentrations greater than 50 mg B/L, P. distans grew at B supply concentrations exceeding 1250 mg B/L. Our research supports the conclusion that from 0.5 to 50 mg B/L, P. distans is better able to restrict the accumulation of B in the whole plant, and the transport of B from root to shoot, than G. arrostil. We propose that P. distans uses several strategies to achieve B tolerance including the ability to restrict the accumulation of B relative to its accumulation of biomass, the ability to restrict the transport of B from root to shoot, and, to a lesser extent, the ability to tolerate high concentrations of B in its shoot and root tissues.

Identification of differentially expressed transcripts from leaves of the boron tolerant plant Gypsophila perfoliata L.

Very recently some of the species of Gypsophila genus collected from the boron rich soils in Turkey were shown to be remarkably tolerant to high levels of boron. A limited amount of boron is necessary for the normal development of plants; however, a high level of boron in soil is generally toxic. Nevertheless, the adaptability of plant species allows them to withstand the presence of extreme amounts of metal ion by various strategies. This study is conducted on highly boron tolerant Gypsophila perfoliata L. collected from a location in the boron mining area. The plant samples were transferred into plant nutritional medium in the presence high; approximately 500 (35 mg/kg), 1,000, and 30 microM (considered normal) boron concentrations. We compared the transcriptome of the plant sample treated with the excess levels of boron to that of the samples grown under normal concentration using differential display PCR (DDRT-PCR) method. Thirty bands showing differential expression levels (presence or absence of bands or varying intensities) in either of approximately 500 or 30 microM B concentrations at varying time points were excised, cloned, and sequenced. Among which, 18 of them were confirmed via quantitative reverse transcription real time PCR (qRT-PCR). We are reporting the first preliminary molecular level study of boron tolerance on this organism by attempting to identify putative genes related in the tolerance mechanism. The gene fragments are consistent with the literature data obtained from a proteomics study and a metabolomics study performed in barley under varying boron concentrations.

Low toxicant concentrations decrease the frequency of fast-growing seedlings at high densities of annual baby's breath (Gypsophila elegans).

Very low toxicant concentrations reduce the frequency of most vital seedlings in dense stands in vitro.

Effect of salinity and temperature on the germination of Spergularia marina seeds and ameliorating effect of ascorbic and salicylic acids.

Spergularia marina (Caryophyllaceae) is a halophytic species and widely distributed among the sea shores of Turkey. Its seeds maybe unwinged or winged. Laboratory experiments were conducted to determine the effects of salinity (0, 50, 100 and 500 mM NaCl) and temperature (10, 15, 25, 30, 35 degrees C) on seed germination. S. marina showed 73.3% germination in non-saline controls at 25 degrees C. No germination occurred at 30 degrees C and 35 degrees C. Seed germination of Spergularia marina was significantly affected by salinity levels, temperature and their interactions. Maximum final germination percentage occured under the lowest salinity condition (50 mM) and distilled water at 25 degrees C. Seed germination was completely inhibited by 500 mM NaCl, although seed germination rate was not affected by salinity. Recovery germination was greatest in 500 mM and at 25 degrees C. The rate of germination was significantly affected by temperature (p < 0.01). The germination percentage of the seeds pretreated with 40 mM and 60 mM L-ASA in 50 mM and 100 mM NaCl was improved compared with that of untreated L-ASA. Addition of 0.5 mM salicylic acid (SA) improved the germination in 50 mM NaCl.