Protein expression plasticity contributes to heat and drought tolerance of date palm.

Climate change is increasing the frequency and intensity of warming and drought periods around the globe, currently representing a threat to many plant species. Understanding the resistance and resilience of plants to climate change is, therefore, urgently needed. As date palm (Phoenix dactylifera) evolved adaptation mechanisms to a xeric environment and can tolerate large diurnal and seasonal temperature fluctuations, we studied the protein expression changes in leaves, volatile organic compound emissions, and photosynthesis in response to variable growth temperatures and soil water deprivation. Plants were grown under controlled environmental conditions of simulated Saudi Arabian summer and winter climates challenged with drought stress. We show that date palm is able to counteract the harsh conditions of the Arabian Peninsula by adjusting the abundances of proteins related to the photosynthetic machinery, abiotic stress and secondary metabolism. Under summer climate and water deprivation, these adjustments included efficient protein expression response mediated by heat shock proteins and the antioxidant system to counteract reactive oxygen species formation. Proteins related to secondary metabolism were downregulated, except for the P. dactylifera isoprene synthase (PdIspS), which was strongly upregulated in response to summer climate and drought. This study reports, for the first time, the identification and functional characterization of the gene encoding for PdIspS, allowing future analysis of isoprene functions in date palm under extreme environments. Overall, the current study shows that reprogramming of the leaf protein profiles confers the date palm heat- and drought tolerance. We conclude that the protein plasticity of date palm is an important mechanism of molecular adaptation to environmental fluctuations.

Optimization of photosynthesis and stomatal conductance in the date palm Phoenix dactylifera during acclimation to heat and drought.

We studied acclimation of leaf gas exchange to differing seasonal climate and soil water availability in slow-growing date palm (Phoenix dactylifera) seedlings. We used an extended Arrhenius equation to describe instantaneous temperature responses of leaf net photosynthesis (A) and stomatal conductance (G), and derived physiological parameters suitable for characterization of acclimation (Topt , Aopt and Tequ ). Optimum temperature of A (Topt ) ranged between 20-33°C in winter and 28-45°C in summer. Growth temperature (Tgrowth ) explained c. 50% of the variation in Topt , which additionally depended on leaf water status at the time of measurement. During water stress, light-saturated rates of A at Topt (i.e. Aopt ) were reduced to 30-80% of control levels, albeit not limited by CO2 supply per se. Equilibrium temperature (Tequ ), around which A/G and substomatal [CO2 ] are constant, remained tightly coupled with Topt . Our results suggest that acclimatory shifts in Topt and Aopt reflect a balance between maximization of photosynthesis and minimization of the risk of metabolic perturbations caused by imbalances in cellular [CO2 ]. This novel perspective on acclimation of leaf gas exchange is compatible with optimization theory, and might help to elucidate other acclimation and growth strategies in species adapted to differing climates.

Three physiological parameters capture variation in leaf respiration of Eucalyptus grandis, as elicited by short-term changes in ambient temperature, and differing nitrogen supply.

We used instantaneous temperature responses of CO2 -respiration to explore temperature acclimation dynamics for Eucalyptus grandis grown with differing nitrogen supply. A reduction in ambient temperature from 23 to 19 °C reduced light-saturated photosynthesis by 25% but increased respiratory capacity by 30%. Changes in respiratory capacity were not reversed after temperatures were subsequently increased to 27 °C. Temperature sensitivity of respiration measured at prevalent ambient temperature varied little between temperature treatments but was significantly reduced from ~105 kJ mol-1 when supply of N was weak, to ~70 kJ mol-1 when it was strong. Temperature sensitivity of respiration measured across a broader temperature range (20-40 °C) could be fully described by 2 exponent parameters of an Arrhenius-type model (i.e., activation energy of respiration at low reference temperature and a parameter describing the temperature dependence of activation energy). These 2 parameters were strongly correlated, statistically explaining 74% of observed variation. Residual variation was linked to treatment-induced changes in respiration at low reference temperature or respiratory capacity. Leaf contents of starch and soluble sugars suggest that respiratory capacity varies with source-sink imbalances in carbohydrate utilization, which in combination with shifts in carbon-flux mode, serve to maintain homeostasis of respiratory temperature sensitivity at prevalent growth temperature.

The carnivorous Venus flytrap uses prey-derived amino acid carbon to fuel respiration.

The present study was performed to elucidate the fate of carbon (C) and nitrogen (N) derived from protein of prey caught by carnivorous Dionaea muscipula. For this, traps were fed 13 C/15 N-glutamine (Gln). The release of 13 CO2 was continuously monitored by isotope ratio infrared spectrometry. After 46 h, the allocation of C and N label into different organs was determined and tissues were subjected to metabolome, proteome and transcriptome analyses. Nitrogen of Gln fed was already separated from its C skeleton in the decomposing fluid secreted by the traps. Most of the Gln-C and Gln-N recovered inside plants were localized in fed traps. Among nonfed organs, traps were a stronger sink for Gln-C compared to Gln-N, and roots were a stronger sink for Gln-N compared to Gln-C. A significant amount of the Gln-C was respired as indicated by 13 C-CO2 emission, enhanced levels of metabolites of respiratory Gln degradation and increased abundance of proteins of respiratory processes. Transcription analyses revealed constitutive expression of enzymes involved in Gln metabolism in traps. It appears that prey not only provides building blocks of cellular constituents of carnivorous Dionaea muscipula, but also is used for energy generation by respiratory amino acid degradation.

A novel mechanistic interpretation of instantaneous temperature responses of leaf net photosynthesis.

Steady-state rates of leaf CO2 assimilation (A) in response to incubation temperature (T) are often symmetrical around an optimum temperature. A/T curves of C3 plants can thus be fitted to a modified Arrhenius equation, where the activation energy of A close to a low reference temperature is strongly correlated with the dynamic change of activation energy to increasing incubation temperature. We tested how [CO2] < current atmospheric levels and saturating light, or [CO2] at 800 µmol mol(-1) and variable light affect parameters that describe A/T curves, and how these parameters are related to known properties of temperature-dependent thylakoid electron transport. Variation of light intensity and substomatal [CO2] had no influence on the symmetry of A/T curves, but significantly affected their breadth. Thermodynamic and kinetic (physiological) factors responsible for (i) the curvature in Arrhenius plots and (ii) the correlation between parameters of a modified Arrhenius equation are discussed. We argue that the shape of A/T curves cannot satisfactorily be explained via classical concepts assuming temperature-dependent shifts between rate-limiting processes. Instead the present results indicate that any given A/T curve appears to reflect a distinct flux mode, set by the balance between linear and cyclic electron transport, and emerging from the anabolic demand for ATP relative to that for NADPH.

Integration of trap- and root-derived nitrogen nutrition of carnivorous Dionaea muscipula.

Carnivorous Dionaea muscipula operates active snap traps for nutrient acquisition from prey; so what is the role of D. muscipula's reduced root system? We studied the capacity for nitrogen (N) acquisition via traps, and its effect on plant allometry; the capacity of roots to absorb NO3(-), NH4(+) and glutamine from the soil solution; and the fate and interaction of foliar- and root-acquired N. Feeding D. muscipula snap traps with insects had little effect on the root : shoot ratio, but promoted petiole relative to trap growth. Large amounts of NH4(+) and glutamine were absorbed upon root feeding. The high capacity for root N uptake was maintained upon feeding traps with glutamine. High root acquisition of NH4(+) was mediated by 2.5-fold higher expression of the NH4(+) transporter DmAMT1 in the roots compared with the traps. Electrophysiological studies confirmed a high constitutive capacity for NH4(+) uptake by roots. Glutamine feeding of traps inhibited the influx of (15)N from root-absorbed (15)N/(13)C-glutamine into these traps, but not that of (13)C. Apparently, fed traps turned into carbon sinks that even acquired organic carbon from roots. N acquisition at the whole-plant level is fundamentally different in D. muscipula compared with noncarnivorous species, where foliar N influx down-regulates N uptake by roots.

The Venus flytrap attracts insects by the release of volatile organic compounds.

Does Dionaea muscipula, the Venus flytrap, use a particular mechanism to attract animal prey? This question was raised by Charles Darwin 140 years ago, but it remains unanswered. This study tested the hypothesis that Dionaea releases volatile organic compounds (VOCs) to allure prey insects. For this purpose, olfactory choice bioassays were performed to elucidate if Dionaea attracts Drosophila melanogaster. The VOCs emitted by the plant were further analysed by GC-MS and proton transfer reaction-mass spectrometry (PTR-MS). The bioassays documented that Drosophila was strongly attracted by the carnivorous plant. Over 60 VOCs, including terpenes, benzenoids, and aliphatics, were emitted by Dionaea, predominantly in the light. This work further tested whether attraction of animal prey is affected by the nutritional status of the plant. For this purpose, Dionaea plants were fed with insect biomass to improve plant N status. However, although such feeding altered the VOC emission pattern by reducing terpene release, the attraction of Drosophila was not affected. From these results it is concluded that Dionaea attracts insects on the basis of food smell mimicry because the scent released has strong similarity to the bouquet of fruits and plant flowers. Such a volatile blend is emitted to attract insects searching for food to visit the deadly capture organ of the Venus flytrap.

Strategy of nitrogen acquisition and utilization by carnivorous Dionaea muscipula.

Plant carnivory represents an exceptional means to acquire N. Snap traps of Dionaea muscipula serve two functions, and provide both N and photosynthate. Using (13)C/(15)N-labelled insect powder, we performed feeding experiments with Dionaea plants that differed in physiological state and N status (spring vs. autumn plants). We measured the effects of (15)N uptake on light-saturated photosynthesis (A(max)), dark respiration (R(D)) and growth. Depending on N status, insect capture briefly altered the dynamics of R(D)/A(max), reflecting high energy demand during insect digestion and nutrient uptake, followed by enhanced photosynthesis and growth. Organic N acquired from insect prey was immediately redistributed, in order to support swift renewal of traps and thereby enhance probability of prey capture. Respiratory costs associated with permanent maintenance of the photosynthetic machinery were thereby minimized. Dionaea's strategy of N utilization is commensurate with the random capture of large prey, occasionally transferring a high load of organic nutrients to the plant. Our results suggest that physiological adaptations to unpredictable resource availability are essential for Dionaea's success with regards to a carnivorous life style.

Disentangling respiratory acclimation and adaptation to growth temperature by Eucalyptus.

• Respiratory acclimation to growth temperature differs between species, but underlying mechanisms are poorly understood. In the present study, we tested the hypothesis that respiratory acclimation of CO(2) release is a consequence of growth regulation such that growth rates of young foliage of Eucalyptus spp. are similar at contrasting growth temperatures. Further, we tested whether such a response is affected by adaptation of Eucalyptus to different thermal environments via growth at different altitudes in the Australian Alps. • We employed calorimetric methods to relate rates of CO(2) release (mainly from substrate oxidation) and rates of O(2) reduction to conservation of energy. Temperature responses of these processes provided insight into mechanisms that control energy conservation and expenditure, and helped define 'instantaneous enthalpic growth capacity' (CapG). • CapG increased with altitude, but was counteracted by other factors in species adapted to highland habitats. The acclimation response was partly driven by changes in respiratory capacity (CapR(CO2)), and partly by more pronounced dynamic responses of CO(2) release (δ(R(CO2))) to measurement temperature. We observed enhanced temperature sensitivity of O(2) reduction (E(o)(R(O2))) at higher altitudes. • Adaptation to growth temperature included differences in respiration and growth capacities, but there was little evidence that Eucalyptus species vary in metabolic flexibility.

Steps towards a mechanistic understanding of respiratory temperature responses.

Temperature crucially affects the speed of metabolic processes in poikilotherm organisms, including plants. The instantaneous temperature responses of O(2)-reduction and CO(2)-release can be approximated by Arrhenius kinetics, even though respiratory gas exchange of plants is the net effect of many constituent biochemical processes. Nonetheless, the classical Arrhenius equation must be modified to account for a dynamic response to measurement temperatures. We show that this dynamic response is readily explained by combining Arrhenius and Michaelis-Menten kinetics, as part of a fresh appraisal of metabolic interpretations of instantaneous temperature responses. In combination with recent experimental findings, we argue that control of mitochondrial electron flow is shared among cytochrome oxidase and alternative oxidase under in vivo conditions, and is continuously coordinated. In this way, upstream carbohydrate metabolism and downstream electron transport appear to be optimized according to the demand of ATP, TCA-cycle intermediates and anabolic reducing power under differing metabolic states. We provide a link to the 'Growth and Maintenance Paradigm' of respiration and argue that respiratory temperature responses can be used as a tool to probe metabolic states of plant tissue, such that we can learn more about the mechanisms that govern longer-term acclimatization responses of plant metabolism.

Integrating two physiological approaches helps relate respiration to growth of Pinus radiata.

Correlation methods originating in the growth and maintenance paradigm (GMP) are traditionally used to calculate a 'growth coefficient' (g) or the 'growth potential' (1/g) of entire plants. The enthalpy balance approach is usually applied to plant organs and relies on determination of both CO(2) release and O(2) reduction to provide a measure of instantaneous rates of enthalpic growth (R(SG)DeltaH(B)). Aspects of both the approaches to explore physiological mechanisms that govern enthalpic growth (variation in rates of CO(2) release versus rates of O(2) reduction) were combined. Respiration and growth rates of apical buds of Pinus radiata were affected strongly by canopy position, and moderately by branching order. A linear relation between enthalpic growth and CO(2) respiration explained 69% of the observed variation. Despite faster rates of growth, enthalpic growth potential (1/g(H)) was comparatively low in the upper canopy. Low enthalpic growth potential entailed comparatively low enthalpy conversion efficiency (eta(H), ratio of R(SG)DeltaH(B) to R(CO(2)) DeltaH(CO(2)); proportional to CO(2):O(2) and to carbon conversion efficiency epsilon) at large R(SG)DeltaH(B). Maximizing enthalpic growth requires a large capacity for O(2) reduction. Relations between R(SG)DeltaH(B) and eta(H) could be described by hyperbolae using two parameters. One parameter, P(1), is equivalent to enthalpic growth potential (1/g(H)).

Three parameters comprehensively describe the temperature response of respiratory oxygen reduction.

Using an exponential model that relies on Arrhenius kinetics, we explored Type I, Type II and dynamic (e.g. declining Q(10) with increasing temperature) responses of respiration to temperature. Our Arrhenius model provides three parameters: R(REF) (the base of the exponential model, nmol g(-1) s(-1)), E(0) (the overall activation energy of oxygen reduction that dominates its temperature sensitivity, kJ mol(-1)) and delta (that describes dynamic responses of E(0) to measurement temperature, 10(3) K(2)). Two parameters, E(0) and delta, are tightly linked. Increases in overall activation energy at a reference temperature were inversely related to changes in delta. At an E(0) of ca. 45 kJ mol(-1), delta approached zero, and respiratory temperature response was strictly Arrhenius-like. Physiologically, these observations suggest that as contributions of AOX to combined oxygen reduction increase, E(0)(REF) decreases because of different temperature sensitivities for V(max), and delta increases because of different temperature sensitivities for K(1/2) of AOX and COX. The balance between COX and AOX activity helps regulate plant metabolism by adjusting the demand for ATP to that for reducing power and carbon skeleton intermediates. Our approach enables determination of respiratory capacity in vivo and opens a path to development of process-based models of plant respiration.

Temperature responses are a window to the physiology of dark respiration: differences between CO2 release and O2 reduction shed light on energy conservation.

We showed that temperature responses of dark respiration for foliage of Pinus radiata could be approximated by Arrhenius kinetics, whereby E(0) determines shape of the exponential response and denotes overall activation energy of respiratory metabolism. Reproducible and predictable deviation from strict Arrhenius kinetics depended on foliage age, and differed between R(CO2) and R(O2). Inhibition of oxygen reduction (R(O2)) by cyanide (inhibiting COX) or SHAM (inhibiting AOX) resulted in reproducible changes of the temperature sensitivity for R(O2), but did not affect R(CO2). Enthalpic growth--preservation of electrons in anabolic products--could be approximated with knowledge of four variables: activation energies (E(0)) for both R(CO2) and R(O2), and basal rates of respiration at a low reference temperature (R(REF)). Rates of enthalpic growth by P. radiata needles were large in spring due to differences between R(REF) of oxidative decarboxylation and that of oxygen reduction, while overall activation energies for the two processes were similar. Later during needle development, enthalpic growth was dependent on differences between E(0) for R(CO2) as compared with R(O2), and increased E(0)(R(O2)) indicated greater contributions of cytochrome oxidase to accompany the switch from carbohydrate sink to source. Temperature-dependent increments in stored energy can be calculated as the difference between R(CO2)DeltaH(CO2) and R(O2)DeltaH(O2).

Nitrogen allocation and the fate of absorbed light in 21-year-old Pinus radiata.

We investigated effects of nitrogen (N) fertilizer and canopy position on the allocation of N to Rubisco and chlorophyll as well as the distribution of absorbed light among thermal energy dissipation, photochemistry, net CO2 assimilation and alternative electron sinks such as the Mehler reaction and photorespiration. The relative reduction state of the primary quinone receptor of photosystem II (QA) was used as a surrogate for photosystem II (PSII) vulnerability to photoinactivation. Measurements were made on needles from the lower, mid and upper canopy of 21-year-old Pinus radiata D. Don trees grown with (N+) and without (N0) added N fertilizer. Rubisco was 45 to 60% higher in needles of N+ trees than in needles of N0 trees at all canopy positions. Chlorophyll was approximately 80% higher in lower- and mid-canopy needles of N+ trees than of N0 trees, but only approximately 20% higher in upper-canopy needles. Physiological differences between N+ and N0 trees were found only in the lower- and mid- canopy positions. Needles of N+ trees dissipated up to 30% less light energy as heat than needles of N0 trees and had correspondingly more reduced QA. Net CO2 assimilation and the proportions of electrons used by alternative electron sinks such as the Mehler reaction and photorespiration were unaffected by N treatment regardless of canopy position. We conclude that the application of N fertilizer mainly affected the biochemistry and light-use physiology in lower- and mid-canopy needles by increasing the amount of chlorophyll and hence the amount of light harvested. This, however, did not improve photochemistry or safe dissipation, but increased PSII vulnerability to photoinactivation, an effect with likely significant consequences during sunflecks or sudden gap formation.

Elevated pCO2 affects N-metabolism of young poplar plants (Populus tremula × P. alba) differently at deficient and sufficient N-supply.

• The effects of N-availability and elevated atmospheric CO 2 partial pressure ( pCO2 ) on growth, allometry and N-metabolism of poplar plants are reported here. • Poplar plants were grown hydroponically at deficient and sufficient N-supply under ambient and elevated pCO2 . The N-fluxes within the plants were estimated by comparing the fate of newly acquired 15 N-NO3 - in plants either severely N-limited or with sufficient N-supply. • At deficient N-supply, plants accumulated less biomass and exhibited an increased root : shoot ratio compared with sufficient N-supply; a larger fraction of newly acquired 15 N was allocated to the youngest leaves immediately after exchange of the nutrient solution. Increasing the external N-supply from deficient to sufficient shifted the site of nitrate reduction from roots to leaves. • Elevated pCO2 increased total biomass and the root : shoot ratio at deficient N-supply, but had no effect at sufficient N-supply. Elevated pCO2 decreased rates of N-uptake in both treatments. Increased root : shoot ratio at deficient N-supply coincided with enhanced nitrate reduction in the root and elevated pCO2 also enhanced the allocation of newly acquired 15 N to the youngest leaves. Root nitrate reduction as a possible factor controlling the root : shoot ratio and N-allocation is discussed.

The Dionaea muscipula ammonium channel DmAMT1 provides NH4⁺ uptake associated with Venus flytrap's prey digestion.

BACKGROUND: Ammonium transporter (AMT/MEP/Rh) superfamily members mediate ammonium uptake and retrieval. This pivotal transport system is conserved among all living organisms. For plants, nitrogen represents a macronutrient available in the soil as ammonium, nitrate, and organic nitrogen compounds. Plants living on extremely nutrient-poor soils have developed a number of adaptation mechanisms, including a carnivorous lifestyle. This study addresses the molecular nature, function, and regulation of prey-derived ammonium uptake in the Venus flytrap, Dionaea muscipula, one of the fastest active carnivores. RESULTS: The Dionaea muscipula ammonium transporter DmAMT1 was localized in gland complexes where its expression was upregulated upon secretion. These clusters of cells decorating the inner trap surface are engaged in (1) secretion of an acidic digestive enzyme cocktail and (2) uptake of prey-derived nutrients. Voltage clamp of Xenopus oocytes expressing DmAMT1 and membrane potential recordings with DmAMT1-expressing Dionaea glands were used to monitor and compare electrophysiological properties of DmAMT1 in vitro and in planta. DmAMT1 exhibited the hallmark biophysical properties of a NH4(+)-selective channel. At depolarized membrane potentials (Vm = 0), the Km (3.2 ± 0.3 mM) indicated a low affinity of DmAMT1 for ammonium that increased systematically with negative going voltages. Upon hyperpolarization to, e.g., -200 mV, a Km of 0.14 ± 0.015 mM documents the voltage-dependent shift of DmAMT1 into a NH4(+) transport system of high affinity. CONCLUSIONS: We suggest that regulation of glandular DmAMT1 and membrane potential readjustments of the endocrine cells provide for effective adaptation to varying, prey-derived ammonium sources.

Sulfur uptake in the ectomycorrhizal fungus Laccaria bicolor S238N.

The importance of the ectomycorrhiza symbiosis for plant acquisition of phosphorus and nitrogen is well established whereas its contribution to sulfur nutrition is only marginally understood. In a first step to investigate the role of ectomycorrhiza in plant sulfur nutrition, we characterized sulfate and glutathione uptake in Laccaria bicolor. By studying the regulation of sulfate uptake in this ectomycorrhizal fungus, we found that in contrast to bacteria, yeast, and plants, sulfate uptake in L. bicolor was not feedback-inhibited by glutathione. On the other hand, sulfate uptake was increased by sulfur starvation as in other organisms. The activity of 3'-phosphoadenosine 5'-phosphosulfate reductase, the key enzyme of the assimilatory sulfate reduction pathway in fungi, was increased by sulfur starvation and decreased after treatment with glutathione revealing an uncoupling of sulfate uptake and reduction in the presence of reduced sulfur compounds. These results support the hypothesis that L. bicolor increases sulfate supply to the plant by extended sulfate uptake and the plant provides the ectomycorrhizal fungus with reduced sulfur.

Elevated pCO(2 )favours nitrate reduction in the roots of wild-type tobacco (Nicotiana tabacum cv. Gat.) and significantly alters N-metabolism in transformants lacking functional nitrate reductase in the roots.

The impact of elevated pCO(2 )on N-metabolism of hydroponically grown wild-type and transformed tobacco plants lacking root nitrate reduction was studied in order to elucidate the effects on (i) nitrate uptake, (ii) long-distance transport of N, (iii) nitrate reduction with emphasis on root-NR, and (iv) the allocation of N between the root and shoot. The findings were related to alterations of growth rates. At elevated pCO(2 )the wild type exhibited higher growth rates, which were accompanied by an increase of NO(3)(-)-uptake per plant, due to a higher root:shoot ratio. Furthermore, elevated pCO(2 )enhanced nitrate reduction in the roots of the wild type, resulting in enhanced xylem-loading of organic N (amino-N) to supply the shoot with sufficient nitrogen, and decreased phloem-transport of organic N in a basipetal direction. Transformed tobacco plants lacking root nitrate reduction were smaller than the wild type and exhibited lower growth rates. Nitrate uptake per plant was decreased in transformed plants as a consequence of an impeded root growth and, thus, a significantly decreased root:shoot ratio. Surprisingly, transformed plants showed an altered allocation of amino-N between the root and the shoot, with an increase of amino-N in the root and a substantial decrease of amino-N in the shoot. In transformed plants, xylem-loading of nitrate was increased and the roots were supplied with organic N via phloem transport. Elevated pCO(2 )increased shoot-NR, but only slightly affected the growth rates of transformed plants, whereas carbohydrates accumulated at elevated pCO(2 )as indicated by a significant increase of the C/N ratio in the leaves of transformed plants. Unexpectedly, the C/N balance and the functional equilibrium between root and shoot growth was disturbed dramatically by the loss of nitrate reduction in the root.