Comparative analysis of macrophage associated vectors for use in genetic vaccine

Background

Antigen presentation by non professional antigen presenting cells (APC) can lead to anergy. In genetic vaccines, targeting the macrophages and APC for efficient antigen presentation might lead to balanced immune response. One such approach is to incorporate APC specific promoter in the vector to be used.

Methods

Three promoters known to be active in macrophage were selected and cloned in mammalian expressing vector (pAcGFP1-N1) to reconstruct (pAcGFP-MS), (pAcGFP-EMR) and (pAcGFP-B5I) with macrosialin, EmrI and Beta-5 Integrin promoters respectively. As a positive control (pAcGFP-CMV) was used with CMV promoter and promoterless vector (pAcGFP-NIX) which served as a negative control. GFP gene was used as readout under the control of each of the promoter. The expression of GFP was analyzed on macrophage and non-macrophage cell lines using Flow cytometry and qRT-PCR with TaqMan probe chemistries.

Results

All the promoters in question were dominant to macrophage lineage cell lines as observed by fluorescence, Western blot and quantitative RT-PCR. The activity of macrosialin was significantly higher than other macrophage promoters. CMV promoter showed 1.83 times higher activity in macrophage cell lines. The expression of GFP driven by macrosialin promoter after 24 hours was 4.40 times higher in macrophage derived cell lines in comparison with non macrophage cell lines.

Conclusions

Based on this study, macrosialin promoter can be utilized for targeting macrophage dominant expression. In vivo study needs to be carried out for its utility as a vaccine candidate.     

Molecular dissection of the human antibody response to the structural repeat epitope of Plasmodium falciparum sporozoite from a protected donor

Background

The circumsporozoite surface protein is the primary target of human antibodies against Plasmodium falciparum sporozoites, these antibodies are predominantly directed to the major repetitive epitope (Asn-Pro-Asn-Ala)_n, (NPNA)_n. In individuals immunized by the bites of irradiated Anopheles mosquitoes carrying P. falciparum sporozoites in their salivary glands, the anti-repeat response dominates and is thought by many to play a role in protective immunity.

Methods

The antibody repertoire from a protected individual immunized by the bites of irradiated P. falciparum infected Anopheles stephensi was recapitulated in a phage display library. Following affinity based selection against (NPNA)₃ antibody fragments that recognized the PfCSP repeat epitope were rescued.

Results

Analysis of selected antibody fragments implied the response was restricted to a single antibody fragment consisting of V_H3 and V_κI families for heavy and light chain respectively with moderate affinity for the ligand.

Conclusion

The dissection of the protective antibody response against the repeat epitope revealed that the response was apparently restricted to a single V_H/V_L pairing (PfNPNA-1). The affinity for the ligand was in the μM range. If anti-repeat antibodies are involved in the protective immunity elicited by exposure to radiation attenuated P. falciparum sporozoites, then high circulating levels of antibodies against the repeat region may be more important than intrinsic high affinity for protection. The ability to attain and sustain high levels of anti-(NPNA)_n will be one of the key determinants of efficacy for a vaccine that relies upon anti-PfCSP repeat antibodies as the primary mechanism of protective immunity against P. falciparum.     

Varietal differences of quinoa’s tolerance to saline conditions

Aims

This study aimed to assess varietal differences of quinoa’s tolerance to salinity and to investigate physiological mechanisms conferring these differences.

Methods

Production of biomass in fourteen varieties grown under saline conditions was analysed in a pot experiment. For two contrasting varieties, the Danish variety Titicaca and the Bolivian variety Utusaya gas exchange, chlorophyll content index (CCI), fluorescence and ion relations were studied.

Results

Responses to salinity differed greatly among the varieties; least affected were two varieties from the Bolivian altiplano and a variety from Peru. Titicaca and Utusaya both had substantially increased K+ concentrations in the leaf sap. But, Utusaya was much more efficient in restricting xylem Na+ loading. Xylem Na+ and K+ loading were found to be uncoupled. Utusaya maintained a relatively high stomatal conductance resulting in an only 25% NaCl-induced reduction in net CO₂ assimilation compared to a 67% reduction in salt treated Titicaca plants. Maximum photochemical efficiency of PSII was not affected by salinity.

Conclusion

In addition to maintaining high gas exchange, tolerant varieties better control xylem Na+ loading. To what extent this control is related to radial root Na+ uptake or to the activity of Na+/H+-exchangers at the xylem parenchyma boundary remains to be studied.     

Hot receptors in the brain

Background

The complex neuronal circuitry of the dorsal horn of the spinal cord is as yet poorly understood. However, defining the circuits underlying the transmission of information from primary afferents to higher levels is critical to our understanding of sensory processing. In this study, we have examined phosphodiesterase 1C (Pde1c) BAC transgenic mice in which a green fluorescent protein (GFP) reporter gene reflects Pde1c expression in sensory neuron subpopulations in the dorsal root ganglia and spinal cord.

Results

Using double labeling immunofluorescence, we demonstrate GFP expression in specific subpopulations of primary sensory neurons and a distinct neuronal expression pattern within the spinal cord dorsal horn. In the dorsal root ganglia, their distribution is restricted to those subpopulations of primary sensory neurons that give rise to unmyelinated C fibers (neurofilament 200 negative). A small proportion of both non-peptidergic (IB4-binding) and peptidergic (CGRP immunoreactive) subclasses expressed GFP. However, GFP expression was more common in the non-peptidergic than the peptidergic subclass. GFP was also expressed in a subpopulation of the primary sensory neurons immunoreactive for the vanilloid receptor TRPV1 and the ATP-gated ion channel P2X₃. In the spinal cord dorsal horn, GFP positive neurons were largely restricted to lamina I and to a lesser extent lamina II, but surprisingly did not coexpress markers for key neuronal populations present in the superficial dorsal horn.

Conclusion

The expression of GFP in subclasses of nociceptors and also in dorsal horn regions densely innervated by nociceptors suggests that Pde1c marks a unique subpopulation of nociceptive sensory neurons.     

Elevated CO2 temporally enhances phosphorus immobilization in the rhizosphere of wheat and chickpea

Aims

The efficient management of phosphorus (P) in cropping systems remains a challenge due to climate change. We tested how plant species access P pools in soils of varying P status (Olsen-P 3.2–17.6 mg?kg^?1), under elevated atmosphere CO₂ (eCO₂).

Methods

Chickpea (Cicer arietinum L.) and wheat (Triticum aestivum L.) plants were grown in rhizo-boxes containing Vertosol or Calcarosol soil, with two contrasting P fertilizer histories for each soil, and exposed to ambient (380 ppm) or eCO₂ (700 ppm) for 6 weeks.

Results

The NaHCO₃-extractable inorganic P (Pi) in the rhizosphere was depleted by both wheat and chickpea in all soils, but was not significantly affected by CO₂ treatment. However, NaHCO₃-extractable organic P (Po) accumulated, especially under eCO₂ in soils with high P status. The NaOH-extractable Po under eCO₂ accumulated only in the Vertosol with high P status. Crop species did not exhibit different eCO₂-triggered capabilities to access any P pool in either soil, though wheat depleted NaHCO₃-Pi and NaOH-Pi in the rhizosphere more than chickpea. Elevated CO₂ increased microbial biomass C in the rhizosphere by an average of 21 %. Moreover, the size in Po fractions correlated with microbial C but not with rhizosphere pH or phosphatase activity.

Conclusion

Elevated CO₂ increased microbial biomass in the rhizosphere which in turn temporally immobilized P. This P immobilization was greater in soils with high than low P availability.     

Soil nitrogen balance resulting from N fixation and rhizodeposition by the symbiotic association Anthyllis vulneraria/Mesorhizobium metallidurans grown in highly polluted Zn,Pb and Cd mine tailings

Background and aims

The association of the legume Anthyllis vulneraria and the grass Festuca arvernensis, was found to be very efficient for the phytostabilisation of highly multi-metal contaminated mine tailings. Our objective was to quantify the contribution of Anthyllis inoculated with its symbiotic bacteria Mesorhizobium metallidurans to the soil N pool and to test whether a starter nitrogen fertilization may improve symbiotic nitrogen fixation and the growth of Festuca.

Methods

Plants of Festuca and of Anthyllis inoculated with M. metallidurans were grown separately during eight months in pots filled with mine contaminated soil. Estimation of the N fluxes was realized using ¹⁵?N isotopic methods.

Results

Starter N fertilization (28 kg N ha^?1) improved symbiotic N₂ fixation and the growth of both species. Belowground N balance (N rhizodeposition – soil N uptake) of the non-fertilized Anthyllis at maturity was negative (?30.6 kg N ha^?1). However, the amount of N derived from fixation, including above- and belowground parts, was 78.6 kg N ha^?1, demonstrating the ability of this symbiotic association to improve soil N content after senescence.

Conclusions

i) soil N enrichment by the N₂-fixing symbiotic association occurs after plant senescence, when decaying leaves and shoots are incorporated into the soil; ii) application of a starter fertilization is an efficient solution to improve phytostabilisation of highly contaminated sites.     

Growth factor withdrawal in combination with sodium butyrate addition extends culture longevity and enhances antibody production in CHO cells

The effect of growth factor (GF) and sodium butyrate (NaBu) on Chinese hamster ovary (CHO) cell growth, cell viability and antibody production was investigated using shaking flasks in GF-containing and GF-deficient medium containing 0, 1 and 3 mM NaBu. The withdrawal of GF and the addition of NaBu suppressed cell growth, but they significantly increased specific antibody productivity, q_Ab. Interestingly, the withdrawal of GF in combination with the addition of NaBu markedly retarded cell death, leading to extended culture longevity. For instance, at 3 mM NaBu, cell viability fell below 80% after day 4 in GF-containing medium, but it remained over 80% until day 18 in GF-deficient medium. Due to the enhanced q_Ab and the extended culture longevity, approximately 2-fold increase in total antibody production was achieved in pseudo-perfusion culture with 1 mM NaBu in GF-deficient medium, compared to the culture in GF-containing medium. The effect of GF and NaBu on the change in the expression and activity of cellular proteins, c-Myc, Bcl-2 and pyruvate dehydrogenase (PDH), was also investigated. Both the withdrawal of GF and the addition of NaBu decreased the expression of c-Myc. The expression of Bcl-2 was enhanced by the addition of NaBu in a dose-dependent manner while it was not affected by the withdrawal of GF. In addition, both the withdrawal of GF and the addition of NaBu reduced metabolic rates, q_Glc, q_Lac and Y_Lac/Glc, and increased PDH activity while not affecting PDH expression, suggesting that they may reduce the glycolytic rates, but enhance the conversion rates of pyruvate to TCA intermediates. Taken together, the withdrawal of GF in combination with the addition of NaBu can be considered as a relevant strategy for alleviating NaBu-induced cell apoptosis and enhancing antibody production since it can be easily implemented as well as enhance q_Ab and extend culture longevity.     

Net root growth and nutrient acquisition in response to predicted climate change in two contrasting heathland species

Background and aims

Accurate predictions of nutrient acquisition by plant roots and mycorrhizas are critical in modelling plant responses to climate change.

Methods

We conducted a field experiment with the aim to investigate root nutrient uptake in a future climate and studied root production by ingrowth cores, mycorrhizal colonization, and fine root N and P uptake by root assay of Deschampsia flexuosa and Calluna vulgaris.

Results

Net root growth increased under elevated CO₂, warming and drought, with additive effects among the factors. Arbuscular mycorrhizal colonization increased in response to elevated CO₂, while ericoid mycorrhizal colonization was unchanged. The uptake of N and P was not increased proportionally with root growth after 5 years of treatment.

Conclusions

While aboveground biomass was unchanged, the root growth was increased under elevated CO₂. The results suggest that plant production may be limited by N (but not P) when exposed to elevated CO₂. The species-specific response to the treatments suggests different sensitivity to global change factors, which could result in changed plant competitive interactions and belowground nutrient pool sizes in response to future climate change.     

Nitrate-use traits of understory plants as potential regulators of vegetation distribution on a slope in a Japanese cedar plantation

Background and aims

Plant physiological traits and their relation to soil N availability was investigated as regulators of the distribution of understory shrub species along a slope in a Japanese cedar (Cryptomeria japonica) plantation in central Japan.

Methods

At the study site, previous studies demonstrated that both net and gross soil nitrification rates are high on the lower slope and there are dramatic declines in different sections of the slope gradient. We examined the distributions of understory plant species and their nitrate (NO ₃ ^? -N) use traits, and compared the results with the soil traits.

Results

Our results show that boundaries between different dominant understory species correspond to boundaries between different soil types. Leucosceptrum stellipilum occurs on soil with high net and gross nitrification rates. Hydrangea hirta is dominant on soil with high net and low gross nitrification rates. Pieris japonica occurs on soil with very low net and gross nitrification rates. Dominant understory species have species-specific physiological traits in their use of NO ₃ ^? -N. Pieris japonica lacks the capacity to use NO ₃ ^? -N as a N source, but other species do use NO ₃ ^? -N. Lindera triloba, whose distribution is unrelated to soil NO ₃ ^? -N availability, changes the extent to which it uses NO ₃ ^? -N in response to soil NO ₃ ^? -N availability.

Conclusions

Our results indicate that differences in the physiological capabilities and adaptabilities of plant species in using NO ₃ ^? -N as a N source regulate their distribution ranges. The identity of the major form of available soil N is therefore an environmental factor that influences plant distributions.     

D-amino acids in the cell pool of bacteria

About 30 different bacterial species were tested for the possible presence of freed-amino acids in their cell pool. Gram-positive bacteria particularly the species of the genusBacillus have a fairly large pool of freely extractabled-amino acids. Varied quantities of freed-amino acids were detected inBacillus subtilis B₃,Bacillus subtilis Marburg,Bacillus licheniformis, Bacillus brevis, Bacillus stearothermophilus, Lactobacillus fermenti, Lactobacillus delbrueckii, Staphylococcus aureus andClostridium acetobutylicum. The individual components ofd-amino acids were identified in 5Bacillus species referred to above,d-alanine is the major component; the otherd-amino acids identified are aspartic acid, glutamic acid, histidine, leucines, proline, serine and tyrosine. Thed-amino acid pool size inBacillus subtilis B₃ varies with different culture conditions. The pool size is maximum when growth temperature is 30°C and it fluctuates with change in pH of the medium. The maximum quantity ofd-amino acids could be recovered when the culture was at mid log phase. O₂ supply to the medium has little effect ond-amino acid pool size. The starvation of cells leads to depletion of thed-amino acid pool which is exhausted almost completely within 4 hours by incubation in nutrient-free medium.     

Contributions of different mosquito species to the transmission of lymphatic filariasis in central Nigeria: implications for monitoring infection by PCR in mosquito pools

Background

Monitoring and evaluation are essential to the successful implementation of mass drug administration programmes for LF elimination. Monitoring transmission when it is low requires both large numbers of mosquito vectors and sensitive methods for detecting Wuchereria bancrofti infections in them. PCR-based methods are preferred over classical dissections but the best protocol so far achieved detection of one L₃ Wuchereria bancrofti larva in a pool of 35–50 Anopheles mosquitoes. It also lacks consistency and remains still a costly tool. Hence we decided to improve upon this to achieve detection in a pool of 100 or more by enhancing the quality of the template DNA. Prior to this we also evaluated three vector sampling methods in the context of numbers for monitoring.

Methods

Human landing, pyrethrium spray and light traps catches were conducted concurrently at sites in an LF endemic district in Ghana and the numbers obtained compared. Two DNA extraction methods; Bender buffer and phenol/chloroform purification, and DNAeasy Tissue kit (Quaigen Inc) were used on pools of 25, 50, 75 100 and 150 mosquitoes each seeded with one L₃ or its quivalent amount of DNA. Then another set of extracted DNA by the two methods was subjected to Dynal bead purification method (using capture oligonucleotide primers). These were used as template DNA in PCR to amplify W. bancrofti sequences. The best PCR result was then evaluated in the field at five sites by comparing its results (infections per 1000 mosquitoes) with that of dissection of roughly equal samples sizes.

Results

The largest numbers of mosquitoes were obtained with the human landing catches at all the sites sampled. Although PCR detection of one L₃ in pools of 25, 50 and 75 mosquitoes was consistent irrespective of the extraction method, that of one L₃ in 100 was only achieved with the kit-extracted DNA/Dynal bead purification method. Infections were found at only two sites by both dissection and pool-screening being 14.3 and 19 versus 13.4 and 20.1 per 1000 Anopheles mosquitoes respectively, which were not statistically significant

Discussion and conclusion

HLC still remains the best option for sampling for the large numbers of mosquitoes required for monitoring transmission during MDA programmes, when vector population densities are high and classical indices of transmission are required. One – in – 100 detection is an improvement on previous PCR pool-screening methods, which in our opinion was a result of the introduction of the extra step of parasite DNA capture using Dynal/beads. As pool sizes increase the insects DNA will swamp parasite DNA making the latter less available for an efficient PCR, therefore we propose either additional steps of parasite DNA capture or real-time PCR to improve further the pool screening method. The study also attests also to the applicability of Katholi et al's algorithm developed for determining onchocerciasis prevalence in LF studies.     

Efficient genetic transformation of okra (Abelmoschus esculentus (L.) Moench) and generation of insect-resistant transgenic plants expressing the cry1Ac gene

Key message

Agrobacterium -mediated transformation system for okra using embryos was devised and the transgenic Bt plants showed resistance to the target pest, okra shoot, and fruit borer ( Earias vittella ).

Abstract

Okra is an important vegetable crop and progress in genetic improvement via genetic transformation has been impeded by its recalcitrant nature. In this paper, we describe a procedure using embryo explants for Agrobacterium-mediated transformation and tissue culture-based plant regeneration for efficient genetic transformation of okra. Twenty-one transgenic okra lines expressing the Bacillus thuringiensis gene cry1Ac were generated from five transformation experiments. Molecular analysis (PCR and Southern) confirmed the presence of the transgene and double-antibody sandwich ELISA analysis revealed Cry1Ac protein expression in the transgenic plants. All 21 transgenic plants were phenotypically normal and fertile. T₁ generation plants from these lines were used in segregation analysis of the transgene. Ten transgenic lines were selected randomly for Southern hybridization and the results confirmed the presence of transgene integration into the genome. Normal Mendelian inheritance (3:1) of cry1Ac gene was observed in 12 lines out of the 21 T₀ lines. We selected 11 transgenic lines segregating in a 3:1 ratio for the presence of one transgene for insect bioassays using larvae of fruit and shoot borer (Earias vittella). Fruit from seven transgenic lines caused 100 % larval mortality. We demonstrate an efficient transformation system for okra which will accelerate the development of transgenic okra with novel agronomically useful traits.     

Engineering of N. benthamiana L. plants for production of N-acetylgalactosamine-glycosylated proteins - towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylation

Background

Mucin type O-glycosylation is one of the most common types of post-translational modifications that impacts stability and biological functions of many mammalian proteins. A large family of UDP-GalNAc polypeptide:N-acetyl-α-galactosaminyltransferases (GalNAc-Ts) catalyzes the first step of mucin type O-glycosylation by transferring GalNAc to serine and/or threonine residues of acceptor polypeptides. Plants do not have the enzyme machinery to perform this process, thus restricting their use as bioreactors for production of recombinant therapeutic proteins.

Results

The present study demonstrates that an isoform of the human GalNAc-Ts family, GalNAc-T2, retains its localization and functionality upon expression in N. benthamiana L. plants. The recombinant enzyme resides in the Golgi as evidenced by the fluorescence distribution pattern of the GalNAc-T2:GFP fusion and alteration of the fluorescence signature upon treatment with Brefeldin A. A GalNAc-T2-specific acceptor peptide, the 113-136 aa fragment of chorionic gonadotropin β-subunit, is glycosylated in vitro by the plant-produced enzyme at the "native" GalNAc attachment sites, Ser-121 and Ser-127. Ectopic expression of GalNAc-T2 is sufficient to "arm" tobacco cells with the ability to perform GalNAc-glycosylation, as evidenced by the attachment of GalNAc to Thr-119 of the endogenous enzyme endochitinase. However, glycosylation of highly expressed recombinant glycoproteins, like magnICON-expressed E. coli enterotoxin B subunit: H. sapiens mucin 1 tandem repeat-derived peptide fusion protein (LTBMUC1), is limited by the low endogenous UDP-GalNAc substrate pool and the insufficient translocation of UDP-GalNAc to the Golgi lumen. Further genetic engineering of the GalNAc-T2 plants by co-expressing Y. enterocolitica UDP-GlcNAc 4-epimerase gene and C. elegans UDP-GlcNAc/UDP-GalNAc transporter gene overcomes these limitations as indicated by the expression of the model LTBMUC1 protein exclusively as a glycoform.

Conclusion

Plant bioreactors can be engineered that are capable of producing Tn antigen-containing recombinant therapeutics.     

Responses of CO2 efflux from an alpine meadow soil on the Qinghai Tibetan Plateau to multi-form and low-level N addition

Aims

To assess the effects of atmospheric N deposition on the C budget of an alpine meadow ecosystem on the Qinghai–Tibetan Plateau, it is necessary to explore the responses of soil-atmosphere carbon dioxide (CO₂) exchange to N addition.

Methods

Based on a multi-form, low-level N addition experiment, soil CO₂ effluxes were monitored weekly using the static chamber and gas chromatograph technique. Soil variables and aboveground biomass were measured monthly to examine the key driving factors of soil CO₂ efflux.

Results

The results showed that low-level N input tended to decrease soil moisture, whereas medium-level N input maintained soil moisture. Three-year N additions slightly increased soil inorganic N pools, especially the soil NH ₄ ⁺ -N pool. N applications significantly increased aboveground biomass and soil CO₂ efflux; moreover, this effect was more significant from NH ₄ ⁺ -N than from NO ₃ ^? -N fertilizer. In addition, the soil CO₂ efflux was mainly driven by soil temperature, followed by aboveground biomass and NH ₄ ⁺ -N pool.

Conclusions

These results suggest that chronic atmospheric N deposition will stimulate soil CO₂ efflux in the alpine meadow on the Qinghai–Tibetan Plateau by increasing available N content and promoting plant growth.     

Enhanced cell disruption strategy in the release of recombinant hepatitis B surface antigen from Pichia pastoris using response surface methodology

Background

Solely in Europoe, Salmonella Typhimurium causes more than 100,000 infections per year. Improved detection of livestock colonised with S. Typhimurium is necessary to prevent foodborne diseases. Currently, commercially available ELISA assays are based on a mixture of O-antigens (LPS) or total cell lysate of Salmonella and are hampered by cross-reaction. The identification of novel immunogenic proteins would be useful to develop ELISA based diagnostic assays with a higher specificity.

Results

A phage display library of the entire Salmonella Typhimurium genome was constructed and 47 immunogenic oligopeptides were identified using a pool of convalescent sera from pigs infected with Salmonella Typhimurium. The corresponding complete genes of seven of the identified oligopeptids were cloned. Five of them were produced in E. coli. The immunogenic character of these antigens was validated with sera from pigs infeced with S. Tyhimurium and control sera from non-infected animals. Finally, human antibody fragments (scFv) against these five antigens were selected using antibody phage display and characterised.

Conclusion

In this work, we identified novel immunogenic proteins of Salmonella Typhimurium and generated antibody fragments against these antigens completely based on phage display. Five immunogenic proteins were validated using a panel of positive and negative sera for prospective applications in diagnostics of Salmonela Typhimurium.     

Degradation kinetics of biochar from pyrolysis and hydrothermal carbonization in temperate soils

Background and Aims

Estimates of biochar residence times in soils range over three orders of magnitude. We present the first direct comparison between the biodegradation of a char from hydrothermal carbonization (htcBC) and pyrolysis (pyrBC) with high temporal resolution.

Methods

Mineralization of the biochars and their shared Miscanthus feedstock in three soils was determined directly by the ¹³CO₂ efflux using a novel method incorporating wavelength scanned cavity ring-down spectroscopy. Biochar half-life (t_1/2) was estimated with three empirical models.

Results

(1) The htcBC was readily biodegradable, whereas pyrBC was more recalcitrant. (2) Cumulative degradation of both biochars increased with soil organic carbon and nitrogen content. (3) The corrected Akaike information criterion (AIC_C) showed an overall preference for the double exponential model (DEM) reflecting a labile and a recalcitrant C-pool, over the first-order degradation model (FODM) and a logarithmic model. (4) The DEM resulted in t_1/2 ranging from 19.7–44.5, 0.7–2.1 and 0.8–1.3 years for pyrBC, htcBC and feedstock, respectively.

Conclusion

The degradation was rather similar between feedstock and htcBC but one order of magnitude slower for pyrBC. The AIC_C preferred FODM in two cases, where the DEM parameters indicated no distinction between a labile and recalcitrant carbon pool.     

Deriving fluorometer-specific values of relative PSI fluorescence intensity from quenching of F 0 fluorescence in leaves of Arabidopsis thaliana and Zea mays

The effect of stepwise increments of red light intensities on pulse-amplitude modulated (PAM) chlorophyll (Chl) fluorescence from leaves of A. thaliana and Z. mays was investigated. Minimum and maximum fluorescence were measured before illumination (F ₀ and F _M, respectively) and at the end of each light step ( $ F^{\prime}_{0} $ and $ F^{\prime}_{\text{M}} $ , respectively). Calculated $ F^{\prime}_{0} $ values derived from F ₀, F _M and $ F^{\prime}_{\text{M}} $ fluorescence according to Oxborough and Baker (1997) were lower than the corresponding measured $ F^{\prime}_{0} $ values. Based on the concept that calculated $ F^{\prime}_{0} $ values are under-estimated because the underlying theory ignores PSI fluorescence, a method was devised to gain relative PSI fluorescence intensities from differences between calculated and measured $ F^{\prime}_{0} $ . This method yields fluorometer-specific PSI data as its input data (F ₀, F _M, $ F^{\prime}_{0} $ and $ F^{\prime}_{\text{M}} $ ) depend solely on the spectral properties of the fluorometer used. Under the present conditions, the PSI contribution to F ₀ fluorescence was 0.24 in A. thaliana and it was independent on the light acclimation status; the corresponding value was 0.50 in Z. mays. Correction for PSI fluorescence affected Z. mays most: the linear relationship between PSI and PSII photochemical yields was clearly shifted toward the one-to-one proportionality line and maximum electron transport was increased by 50 %. Further, correction for PSI fluorescence increased the PSII reaction center-specific parameter, 1/F ₀ ? 1/F _M, up to 50 % in A. thaliana and up to 400 % in Z. mays.     

Influence of CdCl2 and CdSO4 supplementation on Cd distribution and ligand environment in leaves of the Cd hyperaccumulator Noccaea (Thlaspi) praecox

Background and aims

The aim was to investigate whether different Cd salts in the nutrient solution of the Cd/Zn hyperaccumulator Noccaea (Thlaspi) praecox alter leaf Cd distribution and Cd ligand environment, and plant fitness.

Methods

Plants were grown for 8 weeks with 100/300 μM CdCl₂ or CdSO₄. Leaf biomass, and total chlorophyll, anthocyanin, Cd, Cl, S and P concentrations were monitored. Cd localisation and ligand environment in leaves were analysed using quantitative synchrotron-based micro-X-ray fluorescence imaging, and Cd K-edge X-ray absorption fine structure and Cd L₃-edge micro-X-ray absorption near-edge structure measurements.

Results

Cd uptake and plant fitness were comparable for CdCl₂ and CdSO₄ treatments, and depended on applied Cd concentration. In all treatments, Cd preferentially accumulated with high concentrations of Cl in vacuoles of large vacuolarised epidermal cells, bound mainly to oxygen-based (O)-ligands. In the mesophyll of CdCl₂? treated plants, Cd was preferentially sequestered in vacuoles, while for CdSO₄, Cd accumulated preferentially in the apoplast. In the symplast, O-ligands increased with increasing Cd concentrations; in the apoplast, sulphur-based (S)-ligands prevailed.

Conclusions

Cd partitioning between leaf mesophyll apoplast and symplast and the Cd ligand environment in N. praecox depend on the Cd salt type and concentration added to the nutrient solution.