Katharina Siebke

Spatiotemporal variation of metabolism in a plant circadian rhythm: The biological clock as an assembly of coupled individual oscillators

The complex dynamic properties of biological timing in organisms remain a central enigma in biology despite the increasingly precise genetic characterization of oscillating units and their components. Although attempts to obtain the time constants from oscillations of gene activity and biochemical units have led to substantial progress, we are still far from a full molecular understanding of endogenous rhythmicity and the physiological manifestations of biological clocks. Applications of nonlinear dynamics have revolutionized thinking in physics and in biomedical and life sciences research, and spatiotemporal considerations are now advancing our understanding of development and rhythmicity. Here we show that the well known circadian rhythm of a metabolic cycle in a higher plant, namely the crassulacean acid metabolism mode of photosynthesis, is expressed as dynamic patterns of independently initiated variations in photosynthetic efficiency (φPSII) over a single leaf. Noninvasive highly sensitive chlorophyll fluorescence imaging reveals randomly initiated patches of varying φPSII that are propagated within minutes to hours in wave fronts, forming dynamically expanding and contracting clusters and clearly dephased regions of φPSII. Thus, this biological clock is a spatiotemporal product of many weakly coupled individual oscillators, defined by the metabolic constraints of crassulacean acid metabolism. The oscillators operate independently in space and time as a consequence of the dynamics of metabolic pools and limitations of CO2 diffusion between tightly packed cells.

Expressing an RbcS Antisense Gene in Transgenic Flaveria bidentis Leads to an Increased Quantum Requirement for CO2 Fixed in Photosystems I and II.

It was previously shown with concurrent measurements of gas exchange and carbon isotope discrimination that the reduction of ribulose-1,5-bisphosphate carboxylase/oxygenase by an antisense gene construct in transgenic Flaveria bidentis (a C4 species) leads to reduced CO2 assimilation rates, increased bundle-sheath CO2 concentration, and leakiness (defined as the ratio of CO2 leakage to the rate of C4 acid decarboxylation; S. von Caemmerer, A. Millegate, G.D. Farquhar, R.T. Furbank [1997] Plant Physiol 113: 469–477). Increased leakiness in the transformants should result in an increased ATP requirement per mole of CO2 fixed and a change in the ATP-to-NADPH demand. To investigate this, we compared measurements of the quantum yield of photosystem I and II ([phi]PSI and [phi]PSII) with the quantum yield of CO2 fixation ([phi]CO2) in control and transgenic F. bidentis plants in various conditions. Both [phi]PSI/[phi]CO2 and [phi]PSII/[phi]CO2 increased with a decrease in ribulose-1,5-bisphosphate carboxylase/oxygenase content, confirming an increase in leakiness. In the wild type the ratio of [phi]PSI to [phi]PSII was constant at different irradiances but increased with irradiance in the transformants, suggesting that cyclic electron transport may be higher in the transformants. To evaluate the relative contribution of cyclic or linear electron transport to extra ATP generation, we developed a model that links leakiness, ATP/NADP requirements, and quantum yields. Despite some uncertainties in the light distribution between photosystem I and II, we conclude from the increase of [phi]PSII/[phi]CO2 in the transformants that cyclic electron transport is not solely responsible for ATP generation without NADPH production.

Hydraulically based stomatal oscillations and stomatal patchiness in Gossypium hirsutum

Slow stomatal oscillations (70-95 min), associated with feedback within the plant hydraulic systems, were studied in cotton (Gossypium hirsutum L.). Oscillations were only evident when the whole plant was exposed to light, and were not influenced by reductions in intercellular CO2 concentrations (Ci) in intact, attached leaves. Oscillations were synchronised among different leaves of the same plant, even when the leaf-to-air vapour pressure difference (VPD) was reduced in a cuvette enclosing one of the leaves. In the trough phase of stomatal oscillations the apparent Ci was higher than expected from the combination of the observed assimilation rate and the A(Ci) relationship measured in the absence of oscillations. Using chlorophyll fluorescence imaging we found evidence of stomatal heterogeneity in this phase. Finally, we found that stomatal oscillations appeared to be correlated with xylem embolism, with more vessels filled with gas at the peak than at the troughs of stomatal oscillations.

Elevated CO2 increases the leaf temperature of two glasshouse-grown C4 grasses

This study investigates the effect of elevated CO2 partial pressure (pCO2)-induced stomatal closure on leaf temperature and gas exchange of C4 grasses. Two native Australian C4 grasses, Astrebla lappacea (Lindl.) Domin and Bothriochloa bladhii Kuntze, were grown at three different pCO2 (35, 70 and 120 Pa) in three matched, temperature-controlled glasshouse compartments. The difference between leaf and air temperature (ΔT) was monitored diurnally with thermocouples. ΔT increased with both step-increases of ambient pCO2. Average noon leaf temperature increased by 0.4 and 0.3°C for A. lappacea with the 35-70 and 70-120 Pa steps of pCO2 elevation, respectively. For B. bladhii, the increases were 0.5°C for both pCO2 steps. ΔT was strongly dependent on irradiance, pCO2 and air humidity. Leaf gas exchange was measured at constant temperature and high irradiance at the three growth pCO2. Under these conditions, CO2 assimilation saturated at 70 Pa, while stomatal conductance decreased by the same extent (0.58-fold) with both step-increases in pCO2, suggesting that whole-plant water use efficiency of C4 grasses would increase beyond a doubling of ambient pCO2. The ratio of intercellular to ambient pCO2 was not affected by short- or long-term doubling or near-tripling of pCO2, in either C4 species when measured under standard conditions.

Leaf cooling curves: measuring leaf temperature in sunlight

Despite the obvious benefits of using thermography under field conditions, most infrared studies at the leaf level are generally conducted in the laboratory. One reason for this bias is that accuracy can potentially be compromised in sunlight because reflected radiation from the leaf might affect the calculation of the temperature measurement. We have developed a method for measuring leaf temperature in sunlight by using thermal imagery to generate cooling curves from which the time constant for cooling, τ, can be calculated. The original temperature of the sunlit leaf may be determined by extrapolating backwards in time. In the absence of specular reflection, there is close agreement between the extrapolated sunlit temperature and the sunlit temperature recorded by the camera. However, when reflected radiation is high, the difference between the initial (incorrect) temperature determined from the sunlit image and the temperature extrapolated from the cooling curve can be > 2°C. Notably, our results demonstrate a close agreement between the extrapolated sunlit temperature and the temperature of the leaf approximately 1 s after being shaded, suggesting that this shaded image provides a good estimate of the original sunlit temperature. Thus, our technique provides two means for measuring leaf surface temperature in sunlight.

Infrared video thermography: a technique for assessing cold adaptation in insects

Insects can survive subzero temperatures by two main strategies: freeze tolerance and freeze avoidance. An array of techniques have been used to investigate the physiological limits of insects to low temperatures, such as differential scanning calorimetry, temperature-controlled cooling apparatus, thermocouples, and computer-controlled chart recording equipment. However, these techniques require animals to be stationary, precluding behavioral data. We used infrared video thermography to investigate cold adaptation in an alpine insect, expanding such investigations to include behavioral response as an indicator of physiological stress. This technique is noninvasive and provides a large amount of physiological information, such as supercooling points, lower lethal temperatures, and hemolymph melting points. Insect supercooling points in response to a constant cooling rate were variable; however, temperatures at the initiation of behavioral stress response were less variable. Assessments of supercooling points and lower lethal temperatures obtained in this way are more biologically meaningful because allowing unhindered movement of insects more closely resembles natural environments.

Metabolic control of photosynthetic electron transport in crassulacean acid metabolism-induced Mesembryanthemum crystallinum

This paper originates from a presentation at the IIIrd International Congress on Crassulacean Acid Metabolism, Cape Tribulation, Queensland, Australia, August 2001. We investigated photosynthetic electron transport in leaves of the facultative crassulacean acid metabolism (CAM) plant Mesembryanthemum crystallinum L. After CAM induction, electron transport exhibited variable redox kinetics during the diurnal CAM cycle. In CAM Phase IV, most of PSI (P700) and chlorophyll a fluorescence relaxed with a halftime of 20 ms after a saturating light pulse. This time-constant may reflect the overall linear electron flux from PSII to PSI in saturating light. Comparable relaxation kinetics were also determined for C3 plants. At the end of CAM Phase I and during Phase II, slow components (> 50% of signal amplitude) appeared in both P700 reduction and fluorescence relaxation. They displayed halftimes > 250 ms and > 1 s, suggesting a strong restriction of the linear electron flux from H2O to NADP. The appearance of the slow redox components was accompanied by a decrease in the Fv/Fm ratio of chlorophyll a fluorescence, suggesting a reversible detachment of light-harvesting complex (LHC) II from PSII. The slow redox fractions and the depression of Fv/Fm disappeared again in parallel to malate decarboxylation during CAM Phase III. We discuss a reversible downregulation of linear electron flux during CAM Phases II and III, due to a reversible deprivation of cytochrome-b6f complexes (cyt-bfs) and PSI from the linear system. In parallel, a redistribution of some LHCIIs could also occur. This could be an adaptive response to a reduced metabolic demand for NADPH due to a limited carbon flux through the Calvin cycle, resulting from low Rubisco activation. Furthermore, the cyt-bfs and PSIs deprived of linear electron transport could support cyclic electron flux to cover an increased ATP demand during gluconeogenesis in CAM Phase III.

Non-destructive measurement of chlorophyll b : a ratios and identification of photosynthetic pathways in grasses by reflectance spectroscopy

Equations for non-destructive determination of chlorophyll b : a ratios in grasses were developed from reflectance spectra of intact leaves of barley (Hordeum vulgare L.) and two barley mutants: clorina f2, which lacks chlorophyll b and clorina f104, which has a low chlorophyll b content. These plants enabled separation of effects of chlorophyll composition on reflectance spectra due to differential light absorption by chlorophylls a and b and to measure the effects of chlorophyll b on the contribution of fluorescence emitted by chlorophyll a to the reflectance spectra. Indices developed from these data were then tested on growth chamber-grown leaves from six C3 and 17 C4 grass species (7 NAD-ME and 10 NADP-ME subtypes). We used the chlorophyll b : a ratio because the data were less skewed than the chlorophyll a : b ratio. The best index for determination of the chlorophyll b : a ratio utilised wavelengths affected by chlorophyll absorbance: [R626 - 0.5 (R603 + R647)]/[R552- R626]. The chlorophyll b : a ratio was significantly lower in the C4 than C3 grasses, but was not sufficient in itself to separate these two functional groups. However, because of differences in fluorescence characteristics, C3 and C4 species could be distinguished by an index based on wavelengths affected by chlorophyll fluorescence: [R696 to 709/R545 to 567].

Cold hardiness of Apteropanorpa tasmanica Carpenter (Mecoptera: Apteropanorpidae)

There are very few investigations of cold hardiness in native Australian insects, and no such studies on insects from Tasmania. The Apteropanorpidae is a family of wingless Mecoptera endemic to Tasmania, comprising four described species that can be active in winter. In this study, we used infrared video thermography to investigate the physiological and behavioural responses of Apteropanorpa tasmanica to fast (0.3 degrees Cmin(-1)) and slow (0.03 degrees Cmin(-1)) rates of temperature reduction down to -10 degrees C. No adults survived cooling to -10 degrees C at either cooling rate. Mean supercooling points (SCPs) from fast cooling were -7.0 and -4.6 degrees C in 2002 and 2003, respectively. Ice nucleation always began in the abdomen, however, the position of nucleation within the abdomen varied between individuals. There was no relationship between SCP and body length, and no significant difference in SCPs between males and females. Stress-induced fast walking began when insects reached approximately -1.5 degrees C. Cooling rate did not affect the SCP or the temperature at which the behavioural stress response began. Adults survived for only short periods of time in the supercooled state; however they survived in the laboratory for up to 60 days at 4 degrees C, indicating their longevity at more favourable temperatures. Members of the Apteropanorpidae are adapted to the relatively warm, maritime climate currently influencing Tasmania.

Stomatal Regulation in Transgenic Tobacco Plants with Reduced Levels of b 6 f Complex

The control of stomatal aperture is multi-factorial. The signal perception and transduction chains within the guard cells are still unknown. In transgenic tobacco plants (Nicotiana tobaccum) with a reduced amount of b6f complexes the capacity of the linear electron transport rate is decreased. Because of it’s role in the control of stomatal opening, it should be expected, that the transgenic plants have a lower stomatal opening. Different signals were given to induce short term opening and responses were examined. Although the aim of the experiments was primarily to show a different response of wildtype and B6F plants to opening signals, which involve the linear electron transport chain, we also want to draw your attention to the differences in the kinetic response of stomata depending on the quality of the triggering signal regardless of the plants.