Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxide-mediated electron transfer with the superoxide reductase neelaredoxin

Superoxide reductases are a class of non-haem iron enzymes which catalyse the monovalent reduction of the superoxide anion O2- into hydrogen peroxide and water. Treponema pallidum (Tp), the syphilis spirochete, expresses the gene for a superoxide reductase called neelaredoxin, having the iron protein rubredoxin as the putative electron donor necessary to complete the catalytic cycle. In this work, we present the first cloning, overexpression in Escherichia coli and purification of the Tp rubredoxin. Spectroscopic characterization of this 6 kDa protein allowed us to calculate the molar absorption coefficient of the 490 nm feature of ferric iron, epsilon=6.9+/-0.4 mM(-1) cm(-1). Moreover, the midpoint potential of Tp rubredoxin, determined using a glassy carbon electrode, was -76+/-5 mV. Reduced rubredoxin can be efficiently reoxidized upon addition of Na(2)IrCl(6)-oxidized neelaredoxin, in agreement with a direct electron transfer between the two proteins, with a stoichiometry of the electron transfer reaction of one molecule of oxidized rubredoxin per one molecule of neelaredoxin. In addition, in presence of a steady-state concentration of superoxide anion, the physiological substrate of neelaredoxin, reoxidation of rubredoxin was also observed in presence of catalytic amounts of superoxide reductase, and the rate of rubredoxin reoxidation was shown to be proportional to the concentration of neelaredoxin, in agreement with a bimolecular reaction, with a calculated k(app)=180 min(-1). Interestingly, similar experiments performed with a rubredoxin from the sulfate-reducing bacteria Desulfovibrio vulgaris resulted in a much lower value of k(app)=4.5 min(-1). Altogether, these results demonstrated the existence for a superoxide-mediated electron transfer between rubredoxin and neelaredoxin and confirmed the physiological character of this electron transfer reaction.     

Molecular Characterization of Desulfovibrio gigas Neelaredoxin, a Protein Involved in Oxygen Detoxification in Anaerobes

Desulfovibrio gigas neelaredoxin is an iron-containing protein of 15 kDa, having a single iron site with a His(4)Cys coordination. Neelaredoxins and homologous proteins are widespread in anaerobic prokaryotes and have superoxide-scavenging activity. To further understand its role in anaerobes, its genomic organization and expression in D. gigas were studied and its ability to complement Escherichia coli superoxide dismutase deletion mutant was assessed. In D. gigas, neelaredoxin is transcribed as a monocistronic mRNA of 500 bases as revealed by Northern analysis. Putative promoter elements resembling sigma(70) recognition sequences were identified. Neelaredoxin is abundantly and constitutively expressed, and its expression is not further induced during treatment with O(2) or H(2)O(2). The neelaredoxin gene was cloned by PCR and expressed in E. coli, and the protein was purified to homogeneity. The recombinant neelaredoxin has spectroscopic properties identical to those observed for the native one. Mutations of Cys-115, one of the iron ligands, show that this ligand is essential for the activity of neelaredoxin. In an attempt to elucidate the function of neelaredoxin within the cell, it was expressed in an E. coli mutant deficient in cytoplasmic superoxide dismutases (sodA sodB). Neelaredoxin suppresses the deleterious effects produced by superoxide, indicating that it is involved in oxygen detoxification in the anaerobe D. gigas.     

Inheritance of superoxide dismutase (<Emphasis Type="Italic">Sod-1</Emphasis>) in a perennial × annual ryegrass cross and its allelic distribution among cultivars

Identifying annual ryegrass contamination in perennial ryegrass seed lots has been of major interest in seed-testing laboratories and for seed regulatory agencies in the USA for many years. This study was conducted to characterize a superoxide dismutase locus (Sod-1) and determine its potential to distinguish cultivated ryegrass species. The inheritance of Sod-1 was evaluated in a three-generation annual 2 perennial ryegrass mapping population and segregation fitted an expected 1:2:1 ratio for a single locus with two alleles. The molecular form of the Sod-1 locus was determined by H₂O₂ and KCN inhibitor assays which indicated that the Sod-1, and a second independently segregating Sod-2, locus were both Cu/ZnSod enzymes. The common alleles at the Sod-1 locus were scored in 13 annual and 24 perennial ryegrass cultivars to determine the potential of using this locus for species separation. The Sod-1b allele was homozygous in 98% of perennial ryegrass individuals from 24 cultivars, but those not 100% homozygous for Sod-1b were seed lots with unknown contamination from annual ryegrass. These results indicate that the Sod-1b allele in the homozygous condition is a good indicator of perenniality. All eight annual ryegrass cultivars originating in Europe or Asia had a low frequency of Sod-1b homozygous individuals or none at all. The five cultivars originating in the Western Hemisphere, however, had genotype frequencies for homozygous Sod-1b of up to 56%. The potential of the Sod-1 locus to serve as a test to separate the two growth forms depends on the source of the annual-type contamination.     

Lactate dehydrogenase from the Antarctic eelpout, Lycodichthys dearborni

As part of our studies to examine the molecular basis of cold-adaptation, we have determined the kinetic properties, thermal stability and deduced amino acid sequence of the enzyme lactate dehydrogenase (LDH) from an Antarctic zoarcid fish, Lycodichthys dearborni. Unlike Antarctic notothenioid fish which are endemic to the Southern Ocean, zoarcid fish are cosmopolitan and have a substantially longer evolutionary history as a sub-order. The A₄-LDH isoform was isolated and purified from the white muscle of L. dearborni. The kinetic parameters K_m^PYR and k_cat were determined at temperatures from 0 to 25°C. K_m^PYR was substantially higher at low temperatures than those from Antarctic and temperate notothenioid fish, whereas k_cat at these temperatures was essentially the same as those of the other fish LDH in this study. The sequence of L. dearborni A₄-LDH was determined from cDNA derived from white muscle RNA and found to be similar to, but distinct from, the A₄-LDH sequences of Antarctic notothenioid fish. Molecular modelling based on the structure of the A₄-LDH from Pagothenia borchgrevinki suggested that three conservative amino acid changes within the core of the protein that are not directly part of the active site but which might nonetheless influence the active site, may be important in cold-adaptation in L. dearborni A₄-LDH, and that several other changes on the surface of the protein might also play a role in cold-adaptation.     

Limitation of stomatal conductance by hydraulic traits: sensing or preventing xylem cavitation?

We tested the hypothesis that hydraulic conductance per unit leaf surface area of plant shoots (K_SL) determines the maximum diurnal stomatal conductance (g_L) that can be reached by plants growing in the field. A second hypothesis was tested that some xylem cavitation cannot be avoided by transpiring plants and might act as a signal for regulating g_L. Eleven woody species were studied, differing from each other with respect to taxonomy, wood anatomy and leaf habit. Maximum diurnal g_L, transpiration rate (E_L), pre-dawn and minimum diurnal leaf water potential (O_pd and O_min, respectively) were measured in the field. The critical O level at which stem cavitation was triggered (O_cav) was measured on detached branches, using the acoustic method. A high-pressure flow meter was used to measure maximum K_SL of 1-year-old shoots. Both g_L and E_L were positively related to K_SL. The whole-plant hydraulic conductance per unit leaf area (K_WL) of all the species studied, calculated as the ratio of E_L to (O (=O_pd-O_min) was closely related to K_SL. In every case, O_min (ranging between -0.85 and -1.35 MPa in the different species) dropped to the O_cav range or was <O_cav (ranging between -0.71 and -1.23 MPa), thus suggesting that some cavitation-induced embolism could not be avoided. The possibility is discussed that some cavitation-induced reduction in K_SL is the signal for stomatal closure preventing runaway embolism. The lack of correlation of g_L to O_cav is discussed in terms of the inconsistency of O_cav as an indicator of the vulnerability of plants to cavitation. No differences in hydraulic traits were observed between evergreen and deciduous species.     

Environmental controls on carbon dioxide flux from black spruce coarse woody debris

Carbon dioxide flux from coarse woody debris (CWD) is an important source of CO₂ in forests with moderate to large amounts of CWD. A process-based understanding of environmental controls on CWD CO₂ flux (R_CWD) is needed to accurately model carbon exchange between forests and the atmosphere. The objectives of this study were to: (1) use a laboratory incubation factorial experiment to quantify the effect of temperature (T_CWD), water content (W_C), decay status, and their interactions on R_CWD for black spruce [Picea mariana (Mill.) BSP] CWD; (2) measure and model spatial and temporal dynamics in T_CWD for a boreal black spruce fire chronosequence; and (3) validate the R_CWD model with field measurements, and quantify potential errors in estimating annual R_CWD from this model on various time steps. The R_CWD was positively correlated to T_CWD (R²=0.37, P<0.001) and W_C (R²=0.18, P<0.001), and an empirical R_CWD polynomial model that included T_CWD and W_C interactions explained 74% of the observed variation of R_CWD. The R_CWD estimates from the R_CWD model excellently matched the field measurements. Decay status of CWD significantly (P<0.001) affected R_CWD. The temperature coefficient (Q₁₀) averaged 2.5, but varied by 141% across the 5-42°C temperature range, illustrating the potential shortcomings of using a constant Q₁₀. The CWD temperature was positively correlated to air temperature (R²=0.79, P<0.001), with a hysteresis effect that was correlated to CWD decay status and stand leaf area index . Ignoring this temperature hysteresis introduced errors of -1% to +32% in annual R_CWD estimates. Increasing T_CWD modeling time step from hourly to daily or monthly introduced a 5-11% underestimate in annual R_CWD. The annual R_CWD values in this study were more than two-fold greater than those in a previous study, illustrating the need to incorporate spatial and temporal responses of R_CWD to temperature and water content into models for long-term R_CWD estimation in boreal forest ecosystems.     

The optimum substrate to biomass ratio to reduce net biomass yields and inert compounds in biological leachate treatment under pure-oxygen conditions

To investigate the effect of both initially present soluble inert COD (S_I) and soluble inert COD formed by microbial activities (S_PM) on the effluent soluble residual COD (S_R) and to determine biokinetic constants, the pure-oxygen was employed for the batch assays of biological leachate treatment. The results of this work showed that the effluent residual soluble COD was entirely composed of S_I and S_PM, therefore, could not be reduced below 7-10% of total influent soluble COD (S_T0.inf), corresponding to the organics removal efficiency of 93-90%. The value of S_I of leachate, which is associated with the types of wastewaters, was determined as approximately 7.84% of S_T0.inf, and the soluble inert COD by microbial activities was assessed by means of the coefficient f_PM of 0.0474. These results mean that significant amount of feed leachate COD may pass the biological system without any change. On the basis of the concept that microorganisms must satisfy their maintenance energy requirements prior to synthesizing new biomass, a set of batch assays with various ratios of S_T0.inf /X₀ were carried out to evaluate their effects on the excess biomass production. Decreasing the supply of substrate per unit biomass resulted in gradual decrease in the biomass yields, but, at the same time, it resulted in gradual increase in the bacteria mediated inert COD as a side effect. The optimum ratios of S_T0.inf /X₀ were concluded as 0.2-0.6 according to the careful consideration of both aspects on the reduction of net sludge yields and inert COD from microbial activities.     

Photosynthesis in an invasive grass and native forb at elevated CO2 during an El Niño year in the Mojave Desert

Annual and short-lived perennial plant performance during wet years is important for long-term persistence in the Mojave Desert. Additionally, the effects of elevated CO₂ on desert plants may be relatively greater during years of high resource availability compared to dry years. Therefore, during an El Niño year at the Nevada Desert FACE Facility (a whole-ecosystem CO₂ manipulation), we characterized photosynthetic investment (by assimilation rate-internal CO₂ concentration relationships) and evaluated the seasonal pattern of net photosynthesis (A_net) and stomatal conductance (g_s) for an invasive annual grass, Bromus madritensis ssp. rubens and a native herbaceous perennial, Eriogonum inflatum. Prior to and following flowering, Bromus showed consistent increases in both the maximum rate of carboxylation by Rubisco (V_Cmax) and the light-saturated rate of electron flow (J_max) at elevated CO₂. This resulted in greater A_net at elevated CO₂ throughout most of the life cycle and a decrease in the seasonal decline of maximum midday A_net upon flowering as compared to ambient CO₂. Eriogonum showed significant photosynthetic down-regulation to elevated CO₂ late in the season, but the overall pattern of maximum midday A_net was not altered with respect to phenology. For Eriogonum, this resulted in similar levels of A_net on a leaf area basis as the season progressed between CO₂ treatments, but greater photosynthetic activity over a typical diurnal period. While g_s did not consistently vary with CO₂ in Bromus, it did decrease in Eriogonum at elevated CO₂ throughout much of the season. Since the biomass of both plants increased significantly at elevated CO₂, these patterns of gas exchange highlight the differential mechanisms for increased plant growth. The species-specific interaction between CO₂ and phenology in different growth forms suggests that important plant strategies may be altered by elevated CO₂ in natural settings. These results indicate the importance of evaluating the effects of elevated CO₂ at all life cycle stages to better understand the effects of elevated CO₂ on whole-plant performance in natural ecosystems.     

Considerations of correlated fertility between genders on genetic diversity: the Pinus densiflora seed orchard as a model

The correlation between 99 clone female and male fertilities in a first generation seed orchard of Pinus densiflora was studied over 6 years. The effective number of the parent (N_p) and the variance effective population number [N_e^(v)] were used to assess the impact of total (O_T), female (N_f) and male (N_m) fertility variation. A theoretical framework was developed to account for female and male fertility correlations as well as the impact of possible pollen contamination. Total fertility variation was described by the sibling coefficient (O_T: the probability that two genes randomly chosen from the gamete gene pool originate from the same parent), which was further subdivided into N_f and N_m. These parameters were compared under various conditions including the total seed harvest, imposing on equal seed harvest among the orchard's clones and two contamination scenarios (M = 0 and 20%). Fertility variations among females, males and clones were observed within and among years. Sibling coefficients (O_T) were lower, but the effective number of parent (N_p) and variance effective population number (N_e^(v)) were higher in years with moderate female and good male strobilus production. N_p for female and male reproductive outputs varied from 49 to 82 and from 57 to 93, respectively. N_p was higher for males than females. When the crop of the 6 years was pooled, N_p for female, male and the clone were 73, 87 and 85, respectively. The impact of female-male fertility correlation for conditions with no-, positive- and negative-correlations were assessed and their impact on O_T, N_p and N_e^(v) was also evaluated. It was demonstrated that the practice of equal seed harvesting from every clone, or the mixing of seeds from several years, would substantially improve the genetic diversity and the genetic representation of the seed orchard population when a positive correlation between gender fertilities was observed. The relevance of these results to supplemental-mass-pollination was discussed under two cases where equal- and un-equal amounts of pollen from clones were included in the pollen mixes.     

Trends in wood density and structure are linked to prevention of xylem implosion by negative pressure

Wood density (D_t), an excellent predictor of mechanical properties, is typically viewed in relation to support against gravity, wind, snow, and other environmental forces. In contrast, we show the surprising extent to which variation in D_t and wood structure is linked to support against implosion by negative pressure in the xylem pipeline. The more drought-tolerant the plant, the more negative the xylem pressure can become without cavitation, and the greater the internal load on the xylem conduit walls. Accordingly, D_t was correlated with cavitation resistance. This trend was consistent with the maintenance of a safety factor from implosion by negative pressure: conduit wall span (b) and thickness (t) scaled so that (t/b)² was proportional to cavitation resistance as required to avoid wall collapse. Unexpectedly, trends in D_t may be as much or more related to support of the xylem pipeline as to support of the plant.     

K L a Evaluation during the course of fermentation using data reconciliation techniques

The oxygen mass transfer coefficient often serves to compare the efficiency of bioreactors and their mixing devices as well as being an important scale-up factor. In submerged fermentation, four methods are available to estimate the overall oxygen mass transfer coefficient (K_La): the dynamic method, the stationary method based on a previous determination of the oxygen uptake rate (Q_O2X), the gaseous oxygen balance and the carbon dioxide balance. Each method provides a distinct estimation of the value of K_La. Data reconciliation was used to obtain a more probable value of K_La during the production of Saccharomyces cerevisiae, performed in 22.5-l fed-batch bioreactor. The estimate of K_La is obtained by minimising an objective function that includes measurement terms and oxygen conservation models, each being weighted according to their level of confidence. Weighting factors of measurement terms were taken as their respective inverse variance whereas weighting factors of oxygen conservation models were obtained using Monte Carlo simulations. Results show that more coherent and precise estimations of K_La are obtained.     

Exogenous passive heating during torpor arousal in free-ranging rock elephant shrews, <Emphasis Type="Italic">Elephantulus myurus</Emphasis>

In the laboratory rock elephant shrews (Elephantulus myurus; mean body mass 56.6 g) displayed the lowest torpor T_{b min} yet recorded (ca. 5°C) in a placental daily heterotherm. It was unknown whether these low T_bs were characteristic of daily heterothermy in free-ranging animals. It was also unclear how cost effective these low T_bs were since considerable energy is required to arouse from low T_bs on a daily basis. We continuously measured body temperature once every hour for 85 days in 13 free-ranging E. myurus from May to August 2001 (winter) in Weenen Game Reserve, KwaZulu-Natal, South Africa. We recorded a total of 412 torpor bouts. Free-ranging E. myurus had a high propensity for torpor with females displaying higher torpor frequency than males. The lowest T_b recorded was 7.5°C at T_a=2.7°C and the minimum torpor T_b was strongly correlated with ambient temperature. Torpor arousal was tightly coupled with ambient temperature cycles. Low torpor T_{b min} at low T_as was therefore cost-effective because the animals offset the high cost of arousal through exogenous passive heating. Laboratory studies under constant ambient temperatures may therefore underestimate the energetic benefits of torpor in free-ranging small mammals that inhabit regions where seasonality is moderate.     

The mechanism of superoxide scavenging by Archaeoglobus fulgidus neelaredoxin

Neelaredoxin is a mononuclear iron protein widespread among prokaryotic anaerobes and facultative aerobes, including human pathogens. It has superoxide scavenging activity, but the exact mechanism by which this process occurs has been controversial. In this report, we present the study of the reaction of superoxide with the reduced form of neelaredoxin from the hyperthermophilic archaeon Archaeoglobus fulgidus by pulse radiolysis. This protein reduces superoxide very efficiently (k = 1.5 x 10(9) m(-1)s(-1)), and the dismutation activity is rate-limited, in steady-state conditions, by the much slower superoxide oxidation step. These data show unambiguously that the superfamily of neelaredoxin-like proteins (including desulfoferrodoxin) presents a novel type of reactivity toward superoxide, a result of particular relevance for the understanding of both oxygen stress response mechanisms and, in particular, how pathogens may respond to the oxidative burst produced by the defense cells in eukaryotes. The actual in vivo functioning of these enzymes will depend strongly on the cell redox status. Further insight on the catalytic mechanism was obtained by the detection of a transient intermediate ferric species upon oxidation of neelaredoxin by superoxide, detectable by visible spectroscopy with an absorption maximum at 610 nm, blue-shifted approximately 50 nm from the absorption of the resting ferric state. The role of the iron sixth ligand, glutamate-12, in the reactivity of neelaredoxin toward superoxide was assessed by studying two site-directed mutants: E12Q and E12V.     

Dwarf mistletoe affects whole-tree water relations of Douglas fir and western larch primarily through changes in leaf to sapwood ratios

Dwarf mistletoes induce abnormal growth patterns and extreme changes in the biomass allocation of their hosts as well as directly parasitizing them for resources. Because biomass allocation can affect the resource use and efficiency of conifers, we studied the influences of dwarf mistletoe infection on above-ground biomass allocation of Douglas fir and western larch, and the consequences of such changes on whole-tree water use and water relations. Sap flow, tree water potentials, leaf:sapwood area ratios (A_L:A_S), leaf carbon isotope ratios, and nitrogen content were measured on Douglas fir and western larch trees with various degrees of mistletoe infection during the summer of 1996 in western Montana. Heavy dwarf mistletoe infection on Douglas fir and western larch was related to significant increases in A_L:A_S. Correspondingly, water transport dynamics were altered in infected trees, but responses were different for the two species. Higher A_L:A_S ratios in heavily infected Douglas firs were offset by increases in sapwood area-based sap flux densities (Q_SW) such that leaf area-based sap flux densities (Q_L) and predawn leaf water potentials at the end of the summer did not change significantly with mistletoe infection. Small (but statistically insignificant) decreases of Q_L for heavily infected Douglas firs were enough to offset increases in leaf area such that whole-tree water use was similar for uninfected and heavily infected trees. Increased A_L:A_S ratios of heavily infected western larch were not offset by increases of Q_SW. Consequently, Q_L was reduced, which corresponded with significant decreases of water potential at the end of the summer. Furthermore, mistletoe-infection-related changes in A_L:A_S as a function of tree size resulted in greater whole-tree water use for large infected larches than for large uninfected trees. Such changes may result in further depletion of limited soil water resources in mature infected stands late in the growing season. Foliage from infected trees of both species had lower water use efficiencies than non-infected trees. Our results demonstrate substantial changes of whole-tree processes related to mistletoe infection, and stress the importance of integrating whole-tree physiological and structural processes to fully understand the mechanisms by which pathogens suppress forest productivity.     

Regional and local impact on species diversity – from pattern to processes

The impact of regional factors (such as speciation or dispersal) on the species richness in local communities (S_L) has received increasing attention. A prominent method to infer the impact of regional factors is the comparison of species richness in local assemblages (S_L) with the total number of species in the region (S_R). Linear relations between S_R and S_L have been interpreted as an indication of strong regional influence and weak influence of interactions within local communities. We propose that two aspects bias the outcome of such comparisons: (1) the spatial scale of local and regional sampling, and (2) the body size of the organisms. The impact of the local area reflects the scales of ecological interactions, whereas the ratio between local and regional area reflects the inherent moment of autocorrelation. A proposed impact of body size on the relation is based on the high dispersal and high abundance of small organisms. We predict strongest linearity between S_R and S_L for large organisms, for large local areas (less important ecological interactions) and for sampling designs where the local habitat area covers a high proportion of the regional area (more important autocorrelation). We conducted a meta-analysis on 63 relations obtained from the literature. As predicted, the linearity of the relationship between S_L and S_R increased with the proportion of local to regional sampling area. In contrast, neither the body size of the organisms nor the local area itself was significantly related to the relation between S_L and S_R. This indicated that ecological interactions played a minor role in the shape of local to regional richness plots, which instead was mainly influenced by the sampling design. We found that the studies published so far were highly biased towards larger organisms and towards high similarity between the local and regional area. The proposed prevalence of linear relationships may thus be an artefact and plots of S_L to S_R are not a suitable tool with which to infer the strength of local interactions.     

Modelling mixing parameters in concentric-tube airlift bioreactors

The mixing behaviour of the liquid phase in concentric-tube airlift bioreactors of different scale (RIMP: V_L=0.070 m³; RIS-1: V_L=2.50 m³; RIS-2: V_L=5.20 m³) in terms of mixing time was investigated. This mixing parameter was determined from the output curves to an initial Dirac pulse, using the classical tracer response technique, and analyzed in relation to process and geometrical parameters, such as: gas superficial velocity, x_SGR; top clearance, h_S; bottom clearance, h_B, and ratio of the resistances at downcomer entrance, A_d/A_R. A correlation between the mixing time and the specified operating and geometrical parameters was developed, which was particularized for two flow regimes: bubbly and transition (x_SGRА.08 m/s) and churn turbulent flow (x_SGR> 0.08 m/s) respectively. The correlation was applied in bioreactors of different scale with a maximum error of ᆲ%.     

Gas exchange and chlorophyll fluorescence responses of <Emphasis Type="Italic">Pinus radiata</Emphasis> D. Don seedlings during and after several storage regimes and their effects on post-planting survival

Post-storage gas exchange parameters like CO₂ assimilation, stomatal conductance, transpiration, water use efficiency and intercellular CO₂ concentrations, together with several chlorophyll a fluorescence parameters: F_o, F_v, F_v/F_m, F_m/F_o and F_v/F_o were examined in radiata pine (Pinus radiata D. Don) seedlings that were stored for 1, 8 or 15 days at 4° or 10°C with or without soil around the roots. Results were analysed in relation to post-storage water potential and electrolyte leakage in order to forecast their vitality (root growth potential) following cold storage, and post-planting survival potential under optimal conditions. During storage at 4° and 10°C, photosynthesis was reduced, being more pronounced in bare-root seedlings than in seedlings with soil around the roots. The depletion of CO₂ assimilation seemed not to be solely a stomatal effect as effects on chloroplasts contributed to this photosynthetic inhibition. Thus, the fall in the ratios F_v/F_m, F_v/F_o and F_m/F_o indicated photochemical apparatus damage during storage. Photosynthetic rate was positively correlated with the root growth index and new root length showing that new root growth is dependent primarily on current photosynthesis. Pre-planting exposure of bare-root radiata pine seedlings to temperatures of 10°C for more than 24 h during transportation or storage is not recommended.     

Transmission of herbicide resistance from a monoecious to a dioecious weedy Amaranthus species

The genus Amaranthus includes several important monoecious and dioecious weed species, and several populations of these species have developed resistance to herbicides. These species are closely related and two or more species often coexist in agricultural settings. Collectively, these attributes raise the concern that herbicide resistance might transfer from one weedy Amaranthus species to another. We performed research to determine if a dominant allele encoding a herbicide-insensitive form of acetolactate synthase (ALS) could be transferred from a monoecious species, A. hybridus, to a dioecious species, A. rudis. Numerous F₁ hybrids were obtained from controlled crosses in a greenhouse between A. rudis and herbicide-resistant A. hybridus, and most (85%) of these hybrids were herbicide-resistant. Molecular analysis of the ALS gene was used to verify that herbicide-resistant hybrids contained both an A. rudis and an A. hybridus ALS allele. Although hybrids had greatly reduced fertility, 42 BC₁ plants were obtained by backcrossing 33 hybrids with male A. rudis. Fertility was greatly restored in BC₁ progeny, and numerous BC₂ progeny were obtained from a second backcross to A. rudis. The herbicide-resistance allele from A. hybridus was transmitted to 50% of the BC₁ progeny. The resistance allele was subsequently transmitted to and conferred herbicide resistance in 39 of 110 plants analyzed from four BC₂ families. Parental species, hybrids, and BC₂ progeny were compared for 2C nuclear DNA contents. The mean hybrid 2C nuclear DNA content, 1.27 pg, was equal to the average between A. rudis and A. hybridus, which had 2C DNA contents of 1.42 and 1.12 pg, respectively. The mean 2C DNA content of BC₂ plants, 1.40 pg, was significantly (! < 0.01) less than that of the recurring A. rudis parent and indicated that BC₂ plants were not polyploid. This report demonstrates that herbicide resistance can be acquired by A. rudis through a hybridization event with A. hybridus.