Abscisic acid and CO2 signalling via calcium sensitivity priming in guard cells, new CDPK mutant phenotypes and a method for improved resolution of stomatal stimulus-response analyses

Background

Stomatal guard cells are the regulators of gas exchange between plants and the atmosphere. Ca²⁺-dependent and Ca²⁺-independent mechanisms function in these responses. Key stomatal regulation mechanisms, including plasma membrane and vacuolar ion channels have been identified and are regulated by the free cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt).

Scope

Here we show that CO₂-induced stomatal closing is strongly impaired under conditions that prevent intracellular Ca²⁺ elevations. Moreover, Ca²⁺ oscillation-induced stomatal closing is partially impaired in knock-out mutations in several guard cell-expressed Ca²⁺-dependent protein kinases (CDPKs) here, including the cpk4cpk11 double and cpk10 mutants; however, abscisic acid-regulated stomatal movements remain relatively intact in the cpk4cpk11 and cpk10 mutants. We further discuss diverse studies of Ca²⁺ signalling in guard cells, discuss apparent peculiarities, and pose novel open questions. The recently proposed Ca²⁺ sensitivity priming model could account for many of the findings in the field. Recent research shows that the stomatal closing stimuli abscisic acid and CO₂ enhance the sensitivity of stomatal closing mechanisms to intracellular Ca²⁺, which has been termed ‘calcium sensitivity priming’. The genome of the reference plant Arabidopsis thaliana encodes for over 250 Ca²⁺-sensing proteins, giving rise to the question, how can specificity in Ca²⁺ responses be achieved? Calcium sensitivity priming could provide a key mechanism contributing to specificity in eukaryotic Ca²⁺ signal transduction, a topic of central interest in cell signalling research. In this article we further propose an individual stomatal tracking method for improved analyses of stimulus-regulated stomatal movements in Arabidopsis guard cells that reduces noise and increases fidelity in stimulus-regulated stomatal aperture responses ( Box 1). This method is recommended for stomatal response research, in parallel to previously adopted blind analyses, due to the relatively small and diverse sizes of stomatal apertures in the reference plant Arabidopsis thaliana.

Box 1. Improved resolution of stimulus-induced stomatal movements in guard cells by tracking of individual stomatal apertures

Arabidopsis guard cells have become a prime model system for analysing signal transduction, since early research combining genetic and ion channel analyses in this system (Ichida et al., 1997; Pei et al., 1997, 1998; Roelfsema and Prins, 1997). Arabidopsis stomata are small relative to other stomatal model systems and stomatal apertures of various plant types including Arabidopsis are known to show variability in the size of individual stomatal complexes and also variability in the opening apertures of stomata of similar size in a given leaf (Gorton et al., 1988; Mott and Buckley, 2000; Mott and Peak, 2007). Thus stomatal aperture measurements are expected to show a clear degree of statistical variation. Use of blind experiments, in which the genotype and, when possible, the stimulus being applied to guard cells is unknown to the experimenter (Murata et al., 2001) has been employed by several laboratories, has become a standard in the field and has aided in addressing the above limitations of the range of stomatal aperture sizes found under any given condition.Research in our laboratory has shown that a major additional improvement in experiments can be made, by adding imaging of the same individual stomatal apertures over time (Allen et al., 2001; Mori et al., 2006; Vahisalu et al., 2008; Siegel et al., 2009), while performing blind experiments. In such ‘stomatal tracking’ experiments the lower side of a leaf is attached to a glass coverslip in an extracellular incubation medium (Webb et al., 2001; Young et al., 2006). The mesophyll and upper leaf epidermis are removed surgically for better optical resolution of stomatal apertures in the intact lower leaf epidermis (Young et al., 2006). For stimulus-induced stomatal closing analyses, a field of well-opened stomata is located and images are captured (e.g. using Scion Image software) for later analyses and data storage. The bottom (dry side) of coverslips can be marked with colour marker pens to label grids in the regions where apertures where imaged, for finding these same stomata subsequently if needed. Images of the same stomatal apertures are taken over time and can be stored for later analyses of individual stomatal apertures and for deposition of image files. While this approach has been used as a standard for imposed Ca²⁺ oscillation studies (Allen et al., 2001; Mori et al., 2006; Vahisalu et al., 2008; Fig. 4), we have found that this method also substantially improves stomatal movement response analyses to any given stimulus (Siegel et al., 2009; see Figs 1 and 4 and, Box Fig. 1). For example, while individual stomata are known to have diverse apertures (e.g. Box Fig. 1C), the relative responses of wide open stomata and smaller stomatal apertures to ABA or to CO₂ were comparable (Fig. 1 and Box Fig. 1; Siegel et al., 2009). Note that this method has previously been proposed and used in Vicia faba (Gorton et al., 1988), for which stomata exhibit relatively weak ABA and CO₂ responses, compared with, for example, Arabidopsis. We propose that this simple image-capturing approach, together with blind analyses, be used as a standard for stomatal response research in arabidopsis. Our research experience with this method shows that this approach will aid in greatly improving resolution and robustness and in defining the functions of individual Ca²⁺-independent and Ca²⁺-dependent components and mechanisms in stomatal response analyses. Open in a separate windowBox Fig. 1.ABA-induced stomatal closing of individually tracked stomatal apertures. (A) Average individually tracked stomatal apertures in the presence of 50 µm Ca²⁺ (open triangles) and in the presence of 200 nm free Ca²⁺ (open squares) in the bath solution from three experiments are shown and were normalized to the stomatal apertures at time = 0. (B, C) ABA-induced stomatal closing in the presence of 50 µm Ca²⁺ in five individually tracked stomatal apertures. In (A; open triangles) normalized stomatal apertures of the same stomata depicted in (B) and (C) are shown. Methods used in these experiments tracking individual stomatal apertures are described in Siegel et al. (2009). ABA-induced stomatal closing experiments are reproduced from Siegel et al. (2009) with permission of the publisher.     

Concepts on charge transfer through naturally vibrating DNA molecule

Delocalization of charges thorough DNA occurs due to the natural and continuous movements of molecule which stimulates the charge transfer through the molecule. A model is presented showing that the mechanism of electrical conduction occurs mainly by thermally-activated drift motion of holes under control of the localized carriers; where electrons are localized in the conduction band. These localized (stationary-trapped) electrons control the movements of the positive charges and do not play an effective role in the electrical conduction itself. It is found that the localized charge-carriers in the bands have characteristic relaxation times at 5×10(^-2)s, 1.94×10(^-4)s, 5×10(^-7)s, and 2×10(^-11)s respectively which are corresponding to four intrinsic thermal activation energies 0.56eV, 0.33eV, 0.24eV, and 0.05eV respectively. The ac-conductivity of some published data are well fitted with the presented model and the total charge density in DNA molecule is calculated to be n=1.88×10(^19)cm(^-3) at 300K which is corresponding to a linear electron density n=8.66×10(^3)cm(^-1) at 300K. The model shed light on the role of transfer and/or localization of charges through DNA which has multiple applications in medical, nano-technical, bio-sensing and different domains. So, repair DNA by adjusting the charge transport through the molecule is future challenges to new medical applications.     

Meta-analysis for surrogacy: accelerated failure time models and semicompeting risks modeling

There has been great recent interest in the medical and statistical literature in the assessment and validation of surrogate endpoints as proxies for clinical endpoints in medical studies. More recently, authors have focused on using metaanalytical methods for quantification of surrogacy. In this article, we extend existing procedures for analysis based on the accelerated failure time model to this setting. An advantage of this approach relative to proportional hazards model is that it allows for analysis in the semicompeting risks setting, where we model the region where the surrogate endpoint occurs before the true endpoint. Several estimation methods and attendant inferential procedures are presented. In addition, between- and within-trial methods for evaluating surrogacy are developed; a novel principal components procedure is developed for quantifying trial-level surrogacy. The methods are illustrated by application to data from several studies in colorectal cancer.     

Reduction of Bacillus cereus spores in sikhye, a traditional Korean rice beverage, by modified tyndallization processes with and without carbon dioxide injection

Aims: The objective of this study was to inactivate Bacillus cereus spores in sikhye using a modified tyndallization process involving injection with carbon dioxide (CO₂). Methods and Results: Heat tolerance of B. cereus spores in tryptic soy broth and sikhye was evaluated. The D_95°C values of the B. cereus spores were 2·8–4·9 min, dependent of type of heating medium or inoculum level. The lethality of conventional heat treatment and modified tyndallization with or without CO₂ injection against B. cereus spores in sikhye was determined. The order of effectiveness was modified tyndallization with CO₂ > modified tyndallization without CO₂ > conventional heat treatment. Modified tyndallization with CO₂ reduced the number of B. cereus spores in sikhye by 5·8 log CFU ml^?1. The increased CO₂ concentration and decreased pH of sikhye resulting from CO₂ injection rapidly reverted to near‐normal values after heat treatment. Conclusions: Modified tyndallization with CO₂ was more effective than conventional heat treatment or modified tyndallization without CO₂ in reducing B. cereus spores in sikhye. Significance and Impact of the Study: Results of this study will be useful when developing strategies to control B. cereus spores in sikhye and may have application to other beverages.     

TFT6 and TFT7, two different members of tomato 14-3-3 gene family, play distinct roles in plant adaption to low phosphorus stress

14‐3‐3 proteins are a large family of proteins but exact roles of their members in plant response to abiotic stresses are not clear, especially under nutrient deficiency. We investigated the expressions of all the tomato 14‐3‐3 gene family members (TFT1–TFT12) under low phosphorus stress (LP) and found that TFT6 belongs to the later responsive gene while TFT7 belongs to the early responsive gene. When the two genes were separately introduced into Arabidopsis and overexpressed, their plant growth under LP was much enhanced compared with wild‐type plant. TFT6 overexpressing plants showed reduced starch synthase activity, reduced starch content but enhanced sucrose loading into phloem in the shoot under LP. TFT7 overexpressing plants had much enhanced H⁺ flux along their root tip and activity of plasma membrane H⁺‐ATPase in the roots under LP. Our results suggest that TFT6 and TFT7 play different roles in plant adaption to LP. TFT6 acts mainly in leaves and is involved in the systemic response to LP by regulating leaf carbon allocation and increasing phloem sucrose transport to promote root growth, while TFT7 directly functions in root by activating root plasma membrane H⁺‐ATPase to release more protons under LP.     

Cadmium exerts its toxic effects on photosynthesis via a cascade mechanism in the cyanobacterium,Synechocystis PCC 6803

Despite intense research, the mechanism of Cd²⁺ toxicity on photosynthesis is still elusive because of the multiplicity of the inhibitory effects and different barriers in plants. The quick Cd²⁺ uptake in Synechocystis PCC 6803 permits the direct interaction of cadmium with the photosynthetic machinery and allows the distinction between primary and secondary effects. We show that the CO₂‐dependent electron transport is rapidly inhibited upon exposing the cells to 40 µm Cd²⁺ (50% inhibition in ～15 min). However, during this time we observe only symptoms of photosystem I acceptor side limitation and a build of an excitation pressure on the reaction centres, as indicated by light‐induced P700 redox transients, O₂ polarography and changes in chlorophyll a fluorescence parameters. Inhibitory effects on photosystem II electron transport and the degradation of the reaction centre protein D1 can only be observed after several hours, and only in the light, as revealed by chlorophyll a fluorescence transients, thermoluminescence and immunoblotting. Despite the marked differences in the manifestations of these short‐ and long‐term effects, they exhibit virtually the same Cd²⁺ concentration dependence. These data strongly suggest a cascade mechanism of the toxic effect, with a primary effect in the dark reactions.     

The influence of light quality on C4 photosynthesis under steady-state conditions in Zea mays and Miscanthus×giganteus: changes in rates of photosynthesis but not the efficiency of the CO2 concentrating mechanism

Differences in light quality penetration within a leaf and absorption by the photosystems alter rates of CO₂ assimilation in C₃ plants. It is also expected that light quality will have a profound impact on C₄ photosynthesis due to disrupted coordination of the C₄ and C₃ cycles. To test this hypothesis, we measured leaf gas exchange, ¹³CO₂ discrimination (Δ¹³C), photosynthetic metabolite pools and Rubisco activation state in Zea mays and Miscanthus × giganteus under steady‐state red, green, blue and white light. Photosynthetic rates, quantum yield of CO₂ assimilation, and maximum phosphoenolpyruvate carboxylase activity were significantly lower under blue light than white, red and green light in both species. However, similar leakiness under all light treatments suggests the C₄ and C₃ cycles were coordinated to maintain the photosynthetic efficiency. Measurements of photosynthetic metabolite pools also suggest coordination of C₄ and C₃ cycles across light treatments. The energy limitation under blue light affected both C₄ and C₃ cycles, as we observed a reduction in C₄ pumping of CO₂ into bundle‐sheath cells and a limitation in the conversion of C₃ metabolite phosphoglycerate to triose phosphate. Overall, light quality affects rates of CO₂ assimilation, but not the efficiency of CO₂ concentrating mechanism.     

Stable isotope evidence for sex- and status-based variations in diet and life history at medieval Trino Vercellese, Italy

The medieval period in Europe was a time of unprecedented social complexity that affected human diet. The diets of certain subgroups-for example, children, women, and the poor-are chronically underrepresented in historical sources from the medieval period. To better understand diet and the distribution of foods during the medieval period, we investigated stable carbon and nitrogen isotope ratios of 30 individuals from Trino Vercellese, Northern Italy (8th-13th c.). Specifically, we examined diet differences between subgroups (males and females, and high- and low-status individuals), and diet change throughout the life course among these groups by comparing dentine and bone collagen. Our results show a diet based on terrestrial resources with input from C(4) plants, which could include proso and/or foxtail millet. Diets of low-status males differ from those of females (both status groups) and of high-status males. These differences develop in adulthood. Childhood diets are similar among the subgroups, but sex- and status-based differences appear in adulthood. We discuss the possibility of cultural buffering and dietary selectivity of females and high-status individuals.     

Multi-isotopic analysis reveals individual mobility and diet at the Early Iron Age monumental tumulus of Magdalenenberg, Germany

For the Early Iron Age western Hallstatt culture, which includes the site of Magdalenenberg in southwest Germany, it has been proposed that people were mobile and maintained far reaching social and trading networks throughout Europe. We tested this hypothesis by analyzing multiple isotopes (strontium, oxygen, sulfur, carbon, and nitrogen) of the preserved skeletons from the Magdalenenberg elite cemetery to determine diets and to look for evidence of mobility. The analysis of carbon, nitrogen, and sulfur isotope ratios in collagen of humans (n = 50) and associated domestic fauna (n = 10) indicates a terrestrial-based diet. There was a heterogeneous range of isotope values in both strontium (0.70725 to 0.71923, n = 76) and oxygen (13.4‰ to 18.5‰, n = 78) measured in tooth enamel. Although many of the individuals had values consistent with being from Hallstatt culture sites within southwest Germany, some individuals likely originated from further afield. Possible areas include the Alps of Switzerland and Austria or even locations in Italy. Our study strongly supports the assumption of far reaching social and economic networks in the western Hallstatt culture.