Accelerated vegetation succession but no hydrological change in a boreal fen during 20 years of recent climate change

Northern mires (fens and bogs) have significant climate feedbacks and contribute to biodiversity, providing habitats to specialized biota. Many studies have found drying and degradation of bogs in response to climate change, while northern fens have received less attention. Rich fens are particularly important to biodiversity, but subject to global climate change, fen ecosystems may change via direct response of vegetation or indirectly by hydrological changes. With repeated sampling over the past 20 years, we aim to reveal trends in hydrology and vegetation in a pristine boreal fen with gradient from rich to poor fen and bog vegetation. We resampled 203 semi‐permanent plots and compared water‐table depth (WTD), pH, concentrations of mineral elements, and dissolved organic carbon (DOC), plant species occurrences, community structure, and vegetation types between 1998 and 2018. In the study area, the annual mean temperature rose by 1.0°C and precipitation by 46 mm, in 20‐year periods prior to sampling occasions. We found that wet fen vegetation decreased, while bog and poor fen vegetation increased significantly. This reflected a trend of increasing abundance of common, generalist hummock species at the expense of fen specialist species. Changes were the most pronounced in high pH plots, where Sphagnum mosses had significantly increased in plot frequency, cover, and species richness. Changes of water chemistry were mainly insignificant in concentration levels and spatial patterns. Although indications toward drier conditions were found in vegetation, WTD had not consistently increased, instead, our results revealed complex dynamics of WTD as depending on vegetation changes. Overall, we found significant trend in vegetation, conforming to common succession pattern from rich to poor fen and bog vegetation. Our results suggest that responses intrinsic to vegetation, such as increased productivity or altered species interactions, may be more significant than indirect effects via local hydrology to the ecosystem response to climate warming.     

Over-expression of the Gerbera hybrida At-SOC1-like1 gene Gh-SOC1 leads to floral organ identity deterioration

Background and Aims

The family of MADS box genes is involved in a number of processes besides controlling floral development. In addition to supplying homeotic functions defined by the ABC model, they influence flowering time and transformation of vegetative meristem into inflorescence meristem, and have functions in roots and leaves. Three Gerbera hybrida At-SOC1-like genes (Gh-SOC1–Gh-SOC3) were identified among gerbera expressed sequence tags.

Methods

Evolutionary relationships between SOC1-like genes from gerbera and other plants were studied by phylogenetic analysis. The function of the gerbera gene Gh-SOC1 in gerbera floral development was studied using expression analysis, protein–protein interaction assays and reverse genetics. Transgenic gerbera lines over-expressing or downregulated for Gh-SOC1 were obtained using Agrobacterium transformation and investigated for their floral phenotype.

Key Results

Phylogenetic analysis revealed that the closest paralogues of At-SOC1 are Gh-SOC2 and Gh-SOC3. Gh-SOC1 is a more distantly related paralogue, grouping together with a number of other At-SOC1 paralogues from arabidopsis and other plant species. Gh-SOC1 is inflorescence abundant and no expression was seen in vegetative parts of the plant. Ectopic expression of Gh-SOC1 did not promote flowering, but disturbed the development of floral organs. The epidermal cells of ray flower petals appeared shorter and their shape was altered. The colour of ray flower petals differed from that of the wild-type petals by being darker red on the adaxial side and greenish on the abaxial surface. Several protein–protein interactions with other gerbera MADS domain proteins were identified.

Conclusions

The At-SOC1 paralogue in gerbera shows a floral abundant expression pattern. A late petal expression might indicate a role in the final stages of flower development. Over-expression of Gh-SOC1 led to partial loss of floral identity, but did not affect flowering time. Lines where Gh-SOC1 was downregulated did not show a phenotype. Several gerbera MADS domain proteins interacted with Gh-SOC1.     

Virus-induced gene silencing for Asteraceae-a reverse genetics approach for functional genomics in Gerbera hybrida

Virus-induced gene silencing (VIGS) is a natural defence mechanism in plants which leads to sequence-specific degradation of viral RNA. For identifying gene functions, Tobacco rattle virus (TRV)-based VIGS has been applied for silencing of endogenous genes in many plant species. Gerbera hybrida (Asteraceae) has emerged as a novel model for studies in flower development and secondary metabolism. For this highly heterozygous species, functional studies have been conducted through reverse genetic methods by producing stable transgenic lines, which, however, is labour-intensive and time-consuming. For the development of TRV-based VIGS system for gerbera, and for the first time for an Asteraceaeous species, we screened several gerbera cultivars and optimized the agroinfiltration methods for efficient silencing. Gene fragments for gerbera phytoene desaturase (GPDS) and Mg-chelatase subunits (GChl-H and GChl-I), expressed from a TRV vector, induced silencing phenotypes in leaves, scapes, and involucral bracts indicating their feasibility as markers for green tissues. In addition, robust silencing symptoms were achieved in gerbera floral tissues by silencing the anthocyanin pathway gene for chalcone synthase (GCHS1) and a gerbera B-type MADS-box gene globosa (GGLO1), confirming the phenotypes previously observed in stable transgenic lines. Unexpectedly, photobleaching induced by GPDS and GChl-H or GChl-I silencing, or by the herbicide norflurazon, resulted in silencing of the polyketide synthase gene G2PS1, which has no apparent connections to carotenoid or chlorophyll biosynthesis. We have shown feasibility of VIGS for functional studies in gerbera, but our results also show that selection of the marker gene for silencing must be critically evaluated.     

Trace amounts of furan-2-carboxylic acids determine the quality of solid agar plates for bacterial culture

Background

Many investigators have recognised that a significant proportion of environmental bacteria exist in a viable but non-culturable state on agar plates, and some researchers have also noticed that some of such bacteria clearly recover their growth on matrices other than agar. However, the reason why agar is unsuitable for the growth of some bacteria has not been addressed.

Methodology/Principal Findings

According to the guide of a bioassay for swarming inhibition, we identified 5-hydroxymethylfuran-2-carboxylic acid (5-HMFA) and furan-2-carboxylic acid (FA) as factors that inhibit bacterial swarming and likely inhibit extracellular polysaccharide production on agar. The furan-2-carboxylic acids 5-HMFA and FA effectively inhibited the swarming and swimming of several environmental bacteria at concentrations of 1.8 and 2.3 µg L⁻¹ (13 and 21 nmol L⁻¹), respectively, which are equivalent to the concentrations of these compounds in 0.3% agar. On Luria-Bertani (LB) plates containing 1.0% agar that had been previously washed with MeOH, a mixture of 5-HMFA and FA in amounts equivalent to their original concentrations in the unwashed agar repressed the swarming of Escherichia coli K12 strain W3110, a representative swarming bacterium.

Conclusions/Significance

Agar that contains trace amounts of 5-HMFA and FA inhibits the proliferation of some slow-growing or difficult-to-culture bacteria on the plates, but it is useful for single colony isolation due to the ease of identification of swarmable bacteria as the non-swarmed colonies.     

Cavitation induced by a surfactant leads to a transient release of water stress and subsequent 'run away' embolism in Scots pine (Pinus sylvestris) seedlings

Cavitation decreases the hydraulic conductance of the xylem and has, therefore, detrimental effects on plant water balance. However, cavitation is also hypothesized to relieve water stress temporarily by releasing water from embolizing conduits to the transpiration stream. Stomatal closure in response to decreasing water potentials in order to avoid excessive cavitation has been well documented in numerous previous studies. However, it has remained unclear whether the stomata sense cavitation events themselves or whether they act in response to a decrease in leaf water potential to a level at which cavitation is initiated. The effects of massive cavitation on leaf water potential, transpiration, and stomatal behaviour were studied by feeding a surfactant into the transpiration stream of Scots pine (Pinus sylvestris) seedlings. The stomatal response to cavitation in connection with the capacitive effect was also studied. A major transient increase in leaf water potential was found due to cavitation in the seedlings. As cavitation was induced by lowering the surface tension, the two mechanisms could be uncoupled, as the usual relation between xylem water potential and the onset of cavitation did not hold. Our results indicate that the seedlings responded more to leaf water potential and less to cavitation itself, as stomatal closure was insufficient to prevent the seedlings from being driven to 'run-away' cavitation in a manner of hours.     

Surprisingly fast disappearance of beta-lactam selection pressure in cultivation as detected with novel biosensing approaches

Tetracycline and beta-lactam resistances among others are used as selection markers in the production of recombinant proteins. The beta-lactam resistance is based on degradation, i.e. the selection pressure gradually disappears from the culture, whereas tetracycline resistance is based on active efflux. We have studied the kinetics of the stability of antibiotic selection pressure in culture using a simple model system (pBR322 in Escherichia coli). Concentrations of ampicillin, carbenicillin and tetracycline were measured with novel sensor cells developed in our lab. These cells are specifically induced to produce light in the presence of the drugs and here their performance was shown to be excellent in monitoring antibiotic concentrations in cell culture. The sensor cells are cheap to produce and use and a high number of samples can be analysed simultaneously. To our surprise, ampicillin and carbenicillin were completely degraded after 2.5-3.0 h of culture, although it has been widely claimed that especially carbenicillin is a good selective agent, whereas tetracycline was stable in culture. beta-lactamase activity in culture was found to correlate with the kinetics of ampicillin degradation.     

The critical role of carboxy-terminal amino acids in ligand-dependent and -independent transactivation of the constitutive androstane receptor

The mouse constitutive androstane receptor (CAR) is a unique member of the nuclear receptor superfamily, for which an inverse agonist, the testosterone metabolite 5alpha-androstan-3alpha-ol (androstanol), and an agonist, the xenobiotic 1,4-bis[2-(3, 5-dichloropyridyloxy)] benzene, are known. In this study the role of the transactivation domain 2 (AF-2) of CAR was investigated, which is formed by the seven most carboxy-terminal amino acids of the receptor. The AF-2 domain was shown to be critical for the constitutive activity by mediating a ligand-independent interaction of CAR with coactivator (CoA) proteins. In addition this domain increased and decreased contact with CoAs in the presence of agonist and inverse agonist, respectively. In analogy to classical endocrine nuclear receptors, in CAR the charge clamp between K187 (in helix 3) and E355 (within the AF-2 domain) was expected to be critical for its interaction with CoAs. However, the hydrophobic amino acids L352, L353, and I356 on the surface of the AF-2 domain were found to be more important for this protein-protein interaction. Moreover, these amino acids and C357 were shown to be involved in the response of CAR to androstanol. Interestingly, the cysteine at position 357 appears to block classical endocrine responsiveness of CAR to agonists, since mutagenesis of this amino acid both reduced CoA interaction in the absence of ligand and drastically increased inducibility by 1,4-bis[2-(3, 5-dichloropyridyloxy)] benzene. We showed that this blockade is not due to an intramolecular disulfide bridge, but is probably caused by an interaction between C357 and Y336.     

Suppression of a key gene involved in chlorophyll biosynthesis by means of virus-inducing gene silencing

The ChlH gene coding the H subunit of magnesium chelatase, an enzyme involved in chlorophyll biosynthesis, was silenced in Nicotiana benthamiana plants by infection with tobacco mosaic virus vectors (pTMV-30b) containing 67, 214 or 549 nt long ChlH inserts. Silencing of the nuclear ChlH gene induced a chimeric phenotype with green and yellow/white leaves associated with alterations of chloroplast shape and ultrastructure. The symptoms became first evident around veins of young leaves, and only later in the mesophyll tissues. The efficiency of gene silencing was not dependent on the insert orientation, but was strongly correlated with the size of the ChlH insert, providing a flexible method to modulate the level of gene suppression. Silencing efficiency seemed to be strongly dependent on endogenous ChlH mRNA level of the target tissue. Silencing of the ChlH gene with the longest fragment of 549 nt also lowered the accumulation of ChlD and chlorophyll synthetase mRNAs, i.e. other genes involved in chlorophyll biosynthesis.     

Refilling of a hydraulically isolated embolized xylem vessel: model calculations

When they are hydraulically isolated, embolized xylem vessels can be refilled, while adjacent vessels remain under tension. This implies that the pressure of water in the refilling vessel must be equal to the bubble gas pressure, which sets physical constraints for recovery. A model of water exudation into the cylindrical vessel and of bubble dissolution based on the assumption of hydraulic isolation is developed. Refilling is made possible by the turgor of the living cells adjacent to the refilling vessel, and by a reflection coefficient below 1 for the exchange of solutes across the interface between the vessel and the adjacent cells. No active transport of solutes is assumed. Living cells are also capable of importing water from the water-conducting vessels. The most limiting factors were found to be the osmotic potential of living cells and the ratio of the volume of the adjacent living cells to that of the embolized vessel. With values for these of 1.5 MPa and 1, respectively, refilling times were in the order of hours for a broad range of possible values of water conductivity coefficients and effective diffusion distances for dissolved air, when the xylem water tension was below 0.6 MPa and constant. Inclusion of the daily pattern for xylem tension improved the simulations. The simulated gas pressure within the refilling vessel was in accordance with recent experimental results. The study shows that the refilling process is physically possible under hydraulic isolation, while water in surrounding vessels is under negative pressure. However, the osmotic potentials in the refilling vessel tend to be large (in the order of 1 MPa). Only if the xylem water tension is, at most, twice atmospheric pressure, the reflection coefficient remains close to 1 (0.95) and the ratio of the volume of the adjacent living cells to that of the embolized vessel is about 2, does the osmotic potential stay below 0.4 MPa.     

Process control and drug analysis with an on-line capillary electrophoresis system

Inorganic and organic ions in pulp and paper process waters and drug mixtures were studied. Real-time measurements and on-line simulated analyses were made. A novel on-line capillary electrophoresis instrument equipped with a fixed wavelength UV detector having a 254-nm filter was used for the analyses. Three dynamic sample and electrolyte micromodules provided on-line sampling, sample introduction and solution feedings. The system was used to monitor process samples containing thiosulphate, chloride, sulphate, oxalate, acetate and carbonate. It approved good performance in separation of ethacrynic acid, furosemide, probenecid, bumetanide and hydrochlorothiazide as well as that of normethanephrine, methanephrine and dopamine standard mixtures. The LOD and LOQ values of the ions and drugs ranged from 0.1 to 1 mg/l for LOD and from 1 to 10 mg/l for LOQ. Sensitivity of the drugs was higher due to the single wavelength available in the system. It required that the drugs were to be identified by indirect UV detection. Repeatability of the analyses was good (RSD% below 5).