Chlorophyll thermofluorescence and thermoluminescence as complementary tools for the study of temperature stress in plants

The photosynthetic apparatus, especially the electron transport chain imbedded in the thylakoid membrane, is one of the main targets of cold and heat stress in plants. Prompt and delayed fluorescence emission originating from photosystem II have been used, most often separately, to monitor the changes induced in the photosynthetic membranes during progressive warming or cooling of a leaf sample. Thermofluorescence of F ₀ and F _M informs on the effects of heat on the chlorophyll antennae and the photochemical centers, thermoluminescence on the stabilization and movements of charges and Delayed Light Emission on the permeability of the thylakoid membranes to protons and ions. Considered together and operated simultaneously, these techniques constitute a powerful tool to characterize the effect of thermal stress on intact photosynthetic systems and to understand the mechanisms of constitutive or induced tolerance to temperature stresses.     

Escalating Plasmodium falciparum antifolate drug resistance mutations in Macha, rural Zambia

Background

Artemisinin and its derivatives have been used for falciparum malaria treatment in China since late 1970s. Monotherapy and uncontrolled use of artemisinin drugs were common practices for a long period of time. In vitro tests showed that the susceptibility of Plasmodium falciparum to artemisinins was declining in China. A concern was raised about the resistance to artemisinins of falciparum malaria in the country. It has been reported that in vitro artemisinin resistance was associated with the S769N mutation in the PfATPase6 gene. The main purpose of this study was to investigate whether that mutation has occurred in field isolates from China.

Methods

Plasmodium falciparum field isolates were collected in 2006–2007 from Hainan and Yunnan provinces, China. A nested PCR-sequencing assay was developed to analyse the genotype of the PfATPase6 S769N polymorphism in the P. falciparum field isolates.

Results

The genotyping results of six samples could not be obtained due to failure of PCR amplification, but no S769N mutation was detected in any of the 95 samples successfully analysed.

Conclusion

The results indicate that the S769N mutation in the PfATPase6 gene is not present in China, suggesting that artemisinin resistance has not yet developed, but the situation needs to be watched very attentively.     

Pre-clinical evaluation of an sLe x-glycosylated complement inhibitory protein in a non-human primate model of reperfused stroke

BACKGROUND: Soluble complement receptor-1 (sCR1), a potent complement inhibitor, confers neuroprotection in a murine stroke model. Additional neuroprotective benefit is achieved by sLe x-glycosylation of sCR1. In an effort to translate sCR1-sLe x to clinical trials, we evaluated this agent in a primate stroke model. METHODS: Adult male baboons randomly received either sCR1-sLe x or vehicle. Stroke volume was assessed on day 3, and neurological examinations were conducted daily. Complement activity (CH50) was measured at 30 minute, 2, 6, 12 hour, 3, and 10 days post-ischemia. RESULTS: The experiment was terminated prematurely following an interim analysis. In a preliminary cohort (n = 3 per arm), infarct volume was greater in the treated animals. No difference in neurological score was found between groups. CH50 levels were significantly reduced in the sCR1sLe x-treated groups. A hypotensive response was also observed in animals treated with sCR1-sLe x. Conclusions Further work is necessary to explain the hypotensive response observed in primates prior to further clinical development of sCR1-sLe x.     

Role of the oxidized secondary acceptor Q B of Photosystem II in the delayed ‘afterglow’ chlorophyll luminescence

Leaf discs of dark-adapted tobacco plants were excited by 2 flashes and kept in darkness at 20 °C for various time periods, then thermoluminescence emission was recorded without freezing the sample. The B band at 30 °C decreased with a half-time t_1/2~1 min and the AG band at 45 °C with a t_1/2~5 min. This corresponds to the decay kinetics of S_2/3 in PS II centres in the state S_2/3 Q_B^- (B band) or S_2/3 Q_B. Assuming that the 45 °C band is an ‘afterglow’ emission originating from those centres with an oxidized Q_B on which an electron is back-transferred from stroma reductants through a pathway induced by warming, the theoretical ratio of the B and AG band was compared to that measured experimentally. After 2 or 3 flashes producing mainly S₃, the intensity of AG band encompassed several fold that of the B band, because recombining S₃ recreated S₂ Q_B AG-emitting centres. In order to confirm that the AG band is governed by the heat-induced activation of a dark Q_B-reducing pathway rather than by PS II charge recombination, the AG emission was characterized in triazine-resistant Chenopodium album weed biotypes. In these mutants where the Q_B pocket is altered, the B band is strongly downshifted to 18 °C, compared to 32 °C in the wild type, whereas the AG band is only downshifted by 3 or 4 °C, demonstrating that S_2/3 Q_B^- is not the limiting step of the AG emission.     

Probing the FQR and NDH activities involved in cyclic electron transport around Photosystem I by the 'afterglow' luminescence

Far-red illumination of plant leaves for a few seconds induces a delayed luminescence rise, or afterglow, that can be measured with the thermoluminescence technique as a sharp band peaking at around 40-45 degrees C. The afterglow band is attributable to a heat-induced electron flow from the stroma to the plastoquinone pool and the PSII centers. Using various Arabidopsis and tobacco mutants, we show here that the electron fluxes reflected by the afterglow luminescence follow the pathways of cyclic electron transport around PSI. In tobacco, the afterglow signal relied mainly on the ferredoxin-quinone oxidoreductase (FQR) activity while the predominant pathway responsible for the afterglow in Arabidopsis involved the NAD(P)H dehydrogenase (NDH) complex. The peak temperature T(m) of the afterglow band varied markedly with the light conditions prevailing before the TL measurements, from around 30 degrees C to 45 degrees C in Arabidopsis. These photoinduced changes in Tm followed the same kinetics and responded to the same light stimuli as the state 1-state 2 transitions. PSII-exciting light (leading to state 2) induced a downward shift while preillumination with far-red light (inducing state 1) caused an upward shift. However, the light-induced downshift was strongly inhibited in NDH-deficient Arabidopsis mutants and the upward shift was cancelled in plants durably acclimated to high light, which can perform normal state transitions. Taken together, our results suggest that the peak temperature of the afterglow band is indicative of regulatory processes affecting electron donation to the PQ pool which could involve phosphorylation of NDH. The afterglow thermoluminescence band provides a new and simple tool to investigate the cyclic electron transfer pathways and to study their regulation in vivo.     

Cytochrome c550 in the cyanobacterium Thermosynechococcus elongatus: study of redox mutants

Cytochrome c(550) is one of the extrinsic Photosystem II subunits in cyanobacteria and red algae. To study the possible role of the heme of the cytochrome c(550) we constructed two mutants of Thermosynechococcus elongatus in which the residue His-92, the sixth ligand of the heme, was replaced by a Met or a Cys in order to modify the redox properties of the heme. The H92M and H92C mutations changed the midpoint redox potential of the heme in the isolated cytochrome by +125 mV and -30 mV, respectively, compared with the wild type. The binding-induced increase of the redox potential observed in the wild type and the H92C mutant was absent in the H92M mutant. Both modified cytochromes were more easily detachable from the Photosystem II compared with the wild type. The Photosystem II activity in cells was not modified by the mutations suggesting that the redox potential of the cytochrome c(550) is not important for Photosystem II activity under normal growth conditions. A mutant lacking the cytochrome c(550) was also constructed. It showed a lowered affinity for Cl(-) and Ca(2+) as reported earlier for the cytochrome c(550)-less Synechocystis 6803 mutant, but it showed a shorter lived S(2)Q(B)(-) state, rather than a stabilized S(2) state and rapid deactivation of the enzyme in the dark, which were characteristic of the Synechocystis mutant. It is suggested that the latter effects may be caused by loss (or weaker binding) of the other extrinsic proteins rather than a direct effect of the absence of the cytochrome c(550).     

CstF-64 and 3′-UTR cis-element determine Star-PAP specificity for target mRNA selection by excluding PAPα

Almost all eukaryotic mRNAs have a poly (A) tail at the 3′-end. Canonical PAPs (PAPα/γ) polyadenylate nuclear pre-mRNAs. The recent identification of the non-canonical Star-PAP revealed specificity of nuclear PAPs for pre-mRNAs, yet the mechanism how Star-PAP selects mRNA targets is still elusive. Moreover, how Star-PAP target mRNAs having canonical AAUAAA signal are not regulated by PAPα is unclear. We investigate specificity mechanisms of Star-PAP that selects pre-mRNA targets for polyadenylation. Star-PAP assembles distinct 3′-end processing complex and controls pre-mRNAs independent of PAPα. We identified a Star-PAP recognition nucleotide motif and showed that suboptimal DSE on Star-PAP target pre-mRNA 3′-UTRs inhibit CstF-64 binding, thus preventing PAPα recruitment onto it. Altering 3′-UTR cis-elements on a Star-PAP target pre-mRNA can switch the regulatory PAP from Star-PAP to PAPα. Our results suggest a mechanism of poly (A) site selection that has potential implication on the regulation of alternative polyadenylation.     

Effects of prenatal diclofenac sodium exposure on newborn testis: a histomorphometric study

Diclofenac sodium (DS) is used primarily to treat fever and to alleviate pain and inflammation. We investigated the effects of DS exposure during gestation on the testes of rat pups to investigate the safety of its use during the prenatal period. Pregnant rats were separated into control, saline, low dose, medium dose and high dose groups. DS was given between weeks 15 and 21 of gestation. Total numbers of spermatogonia and Sertoli cells were counted in the testes of 7-day-old male rats using the physical disector method. By the end of the study, the total number of Sertoli cells was decreased significantly in a dose dependent manner in the medium and high dose groups compared to controls. No significant differences were found in the total number of spermatogonia in the control, saline and low dose DS groups. Medium and high dose DS administration reduced the total number of spermatogonia compared to other groups. We suggest that prenatal administration of DS can cause deleterious effects on the testis development, especially in high doses.     

Intraspecific chemical variability of the leaf essential oil of Juniperus phoenicea subsp. turbinata from Corsica

The composition of 50 samples of essential oil of individual plants of Juniperus phoenicea subsp. turbinata from Corsica was investigated by GC, GC-MS and 13C NMR. alpha-Pinene, beta-phellandrene, alpha-terpinyl acetate, Delta-3-carene, myrcene and alpha-phellandrene were found to be the main constituents. The results were submitted to cluster analysis and discriminant analysis which allowed two groups of essential oils to be distinguished with respect to the content of alpha-pinene, beta-phellandrene and alpha-terpinyl acetate.