The temperature sensor TWA1 is required for thermotolerance in Arabidopsis.

Plants exposed to incidences of excessive temperatures activate heat-stress responses to cope with the physiological challenge and stimulate long-term acclimation1,2. The mechanism that senses cellular temperature for inducing thermotolerance is still unclear3. Here we show that TWA1 is a temperature-sensing transcriptional co-regulator that is needed for basal and acquired thermotolerance in Arabidopsis thaliana. At elevated temperatures, TWA1 changes its conformation and allows physical interaction with JASMONATE-ASSOCIATED MYC-LIKE (JAM) transcription factors and TOPLESS (TPL) and TOPLESS-RELATED (TPR) proteins for repressor complex assembly. TWA1 is a predicted intrinsically disordered protein that has a key thermosensory role functioning through an amino-terminal highly variable region. At elevated temperatures, TWA1 accumulates in nuclear subdomains, and physical interactions with JAM2 and TPL appear to be restricted to these nuclear subdomains. The transcriptional upregulation of the heat shock transcription factor A2 (HSFA2) and heat shock proteins depended on TWA1, and TWA1 orthologues provided different temperature thresholds, consistent with the sensor function in early signalling of heat stress. The identification of the plant thermosensors offers a molecular tool for adjusting thermal acclimation responses of crops by breeding and biotechnology, and a sensitive temperature switch for thermogenetics.

Estimating Mesophyll Conductance from Measurements of C18OO Photosynthetic Discrimination and Carbonic Anhydrase Activity.

Carbonic anhydrase (CA) activity in leaves catalyzes the 18O exchange between CO2 and water during photosynthesis. This feature has been used to estimate the mesophyll conductance to CO2 (g m) from measurements of online C18OO photosynthetic discrimination (∆18O). Based on CA assays on leaf extracts, it has been argued that CO2 in mesophyll cells should be in isotopic equilibrium with water in most C3 species as well as many C4 dicot species. However, this seems incompatible with ∆18O data that would indicate a much lower degree of equilibration, especially in C4 plants under high light intensity. This apparent contradiction is resolved here using a new model of C3 and C4 photosynthetic discrimination that includes competition between CO2 hydration and carboxylation and the contribution of respiratory fluxes. This new modeling framework is used to revisit previously published data sets on C3 and C4 species, including CA-deficient plants. We conclude that (1) newly ∆18O-derived g m values are usually close but significantly higher (typically 20% and up to 50%) than those derived assuming full equilibration and (2) despite the uncertainty associated with the respiration rate in light, or the water isotope gradient between mesophyll and bundle sheath cells, robust estimates of ∆18O-derived g m can be achieved in both C3 and C4 plants.

Bryophyte gas-exchange dynamics along varying hydration status reveal a significant carbonyl sulphide (COS) sink in the dark and COS source in the light.

Carbonyl sulphide (COS) is a potential tracer of gross primary productivity (GPP), assuming a unidirectional COS flux into the vegetation that scales with GPP. However, carbonic anhydrase (CA), the enzyme that hydrolyses COS, is expected to be light independent, and thus plants without stomata should continue to take up COS in the dark. We measured net CO2 (AC ) and COS (AS ) uptake rates from two astomatous bryophytes at different relative water contents (RWCs), COS concentrations, temperatures and light intensities. We found large AS in the dark, indicating that CA activity continues without photosynthesis. More surprisingly, we found a nonzero COS compensation point in light and dark conditions, indicating a temperature-driven COS source with a Q10 (fractional change for a 10°C temperature increase) of 3.7. This resulted in greater AS in the dark than in the light at similar RWC. The processes underlying such COS emissions remain unknown. Our results suggest that ecosystems dominated by bryophytes might be strong atmospheric sinks of COS at night and weaker sinks or even sources of COS during daytime. Biotic COS production in bryophytes could result from symbiotic fungal and bacterial partners that could also be found on vascular plants.

Actin filament reorganisation controlled by the SCAR/WAVE complex mediates stomatal response to darkness.

Stomata respond to darkness by closing to prevent excessive water loss during the night. Although the reorganisation of actin filaments during stomatal closure is documented, the underlying mechanisms responsible for dark-induced cytoskeletal arrangement remain largely unknown. We used genetic, physiological and cell biological approaches to show that reorganisation of the actin cytoskeleton is required for dark-induced stomatal closure. The opal5 mutant does not close in response to darkness but exhibits wild-type (WT) behaviour when exposed to abscisic acid (ABA) or CaCl2 . The mutation was mapped to At5g18410, encoding the PIR/SRA1/KLK subunit of the ArabidopsisSCAR/WAVE complex. Stomata of an independent allele of the PIR gene (Atpir-1) showed reduced sensitivity to darkness and F1 progenies of the cross between opal5 and Atpir-1 displayed distorted leaf trichomes, suggesting that the two mutants are allelic. Darkness induced changes in the extent of actin filament bundling in WT. These were abolished in opal5. Disruption of filamentous actin using latrunculin B or cytochalasin D restored wild-type stomatal sensitivity to darkness in opal5. Our findings suggest that the stomatal response to darkness is mediated by reorganisation of guard cell actin filaments, a process that is finely tuned by the conserved SCAR/WAVE-Arp2/3 actin regulatory module.

A Specific Transcriptome Signature for Guard Cells from the C4 Plant Gynandropsis gynandra.

C4 photosynthesis represents an excellent example of convergent evolution that results in the optimization of both carbon and water usage by plants. In C4 plants, a carbon-concentrating mechanism divided between bundle sheath and mesophyll cells increases photosynthetic efficiency. Compared with C3 leaves, the carbon-concentrating mechanism of C4 plants allows photosynthetic operation at lower stomatal conductance, and as a consequence, transpiration is reduced. Here, we characterize transcriptomes from guard cells in C3 Tareneya hassleriana and C4 Gynandropsis gynandra belonging to the Cleomaceae. While approximately 60% of Gene Ontology terms previously associated with guard cells from the C3 model Arabidopsis (Arabidopsis thaliana) are conserved, there is much less overlap between patterns of individual gene expression. Most ion and CO2 signaling modules appear unchanged at the transcript level in guard cells from C3 and C4 species, but major variations in transcripts associated with carbon-related pathways known to influence stomatal behavior were detected. Genes associated with C4 photosynthesis were more highly expressed in guard cells of C4 compared with C3 leaves. Furthermore, we detected two major patterns of cell-specific C4 gene expression within the C4 leaf. In the first, genes previously associated with preferential expression in the bundle sheath showed continually decreasing expression from bundle sheath to mesophyll to guard cells. In the second, expression was maximal in the mesophyll compared with both guard cells and bundle sheath. These data imply that at least two gene regulatory networks act to coordinate gene expression across the bundle sheath, mesophyll, and guard cells in the C4 leaf.

Control of hydrogen photoproduction by the proton gradient generated by cyclic electron flow in Chlamydomonas reinhardtii.

Hydrogen photoproduction by eukaryotic microalgae results from a connection between the photosynthetic electron transport chain and a plastidial hydrogenase. Algal H2 production is a transitory phenomenon under most natural conditions, often viewed as a safety valve protecting the photosynthetic electron transport chain from overreduction. From the colony screening of an insertion mutant library of the unicellular green alga Chlamydomonas reinhardtii based on the analysis of dark-light chlorophyll fluorescence transients, we isolated a mutant impaired in cyclic electron flow around photosystem I (CEF) due to a defect in the Proton Gradient Regulation Like1 (PGRL1) protein. Under aerobiosis, nonphotochemical quenching of fluorescence (NPQ) is strongly decreased in pgrl1. Under anaerobiosis, H2 photoproduction is strongly enhanced in the pgrl1 mutant, both during short-term and long-term measurements (in conditions of sulfur deprivation). Based on the light dependence of NPQ and hydrogen production, as well as on the enhanced hydrogen production observed in the wild-type strain in the presence of the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, we conclude that the proton gradient generated by CEF provokes a strong inhibition of electron supply to the hydrogenase in the wild-type strain, which is released in the pgrl1 mutant. Regulation of the trans-thylakoidal proton gradient by monitoring pgrl1 expression opens new perspectives toward reprogramming the cellular metabolism of microalgae for enhanced H2 production.

The dual effect of abscisic acid on stomata.

The classical view that the drought-related hormone ABA simply acts locally at the guard cell level to induce stomatal closure is questioned by differences between isolated epidermis and intact leaves in stomatal response to several stimuli. We tested the hypothesis that ABA mediates, in addition to a local effect, a remote effect in planta by changing hydraulic regulation in the leaf upstream of the stomata. By gravimetry, porometry to water vapour and argon, and psychrometry, we investigated the effect of exogenous ABA on transpiration, stomatal conductance and leaf hydraulic conductance of mutants described as ABA-insensitive at the guard cell level. We show that foliar transpiration of several ABA-insensitive mutants decreases in response to ABA. We demonstrate that ABA decreases stomatal conductance and down-regulates leaf hydraulic conductance in both the wildtype Col-0 and the ABA-insensitive mutant ost2-2. We propose that ABA promotes stomatal closure in a dual way via its already known biochemical effect on guard cells and a novel, indirect hydraulic effect through a decrease in water permeability within leaf vascular tissues. Variability in sensitivity of leaf hydraulic conductance to ABA among species could provide a physiological basis to the isohydric or anisohydric behaviour.

Perturbation of indole-3-butyric acid homeostasis by the UDP-glucosyltransferase UGT74E2 modulates Arabidopsis architecture and water stress tolerance.

Reactive oxygen species and redox signaling undergo synergistic and antagonistic interactions with phytohormones to regulate protective responses of plants against biotic and abiotic stresses. However, molecular insight into the nature of this crosstalk remains scarce. We demonstrate that the hydrogen peroxide-responsive UDP-glucosyltransferase UGT74E2 of Arabidopsis thaliana is involved in the modulation of plant architecture and water stress response through its activity toward the auxin indole-3-butyric acid (IBA). Biochemical characterization of recombinant UGT74E2 demonstrated that it strongly favors IBA as a substrate. Assessment of indole-3-acetic acid (IAA), IBA, and their conjugates in transgenic plants ectopically expressing UGT74E2 indicated that the catalytic specificity was maintained in planta. In these transgenic plants, not only were IBA-Glc concentrations increased, but also free IBA levels were elevated and the conjugated IAA pattern was modified. This perturbed IBA and IAA homeostasis was associated with architectural changes, including increased shoot branching and altered rosette shape, and resulted in significantly improved survival during drought and salt stress treatments. Hence, our results reveal that IBA and IBA-Glc are important regulators of morphological and physiological stress adaptation mechanisms and provide molecular evidence for the interplay between hydrogen peroxide and auxin homeostasis through the action of an IBA UGT.

Using spontaneous photon emission to image lipid oxidation patterns in plant tissues.

Plants, like almost all living organisms, spontaneously emit photons of visible light. We used a highly sensitive, low-noise cooled charge coupled device camera to image spontaneous photon emission (autoluminescence) of plants. Oxidative stress and wounding induced a long-lasting enhancement of plant autoluminescence, the origin of which is investigated here. This long-lived phenomenon can be distinguished from the short-lived chlorophyll luminescence resulting from charge recombinations within the photosystems by pre-adapting the plant to darkness for about 2 h. Lipids in solvent were found to emit a persistent luminescence after oxidation in vitro, which exhibited the same time and temperature dependence as plant autoluminescence. Other biological molecules, such as DNA or proteins, either did not produce measurable light upon oxidation or they did produce a chemiluminescence that decayed rapidly, which excludes their significant contribution to the in vivo light emission signal. Selective manipulation of the lipid oxidation levels in Arabidopsis mutants affected in lipid hydroperoxide metabolism revealed a causal link between leaf autoluminescence and lipid oxidation. Addition of chlorophyll to oxidized lipids enhanced light emission. Both oxidized lipids and plants predominantly emit light at wavelengths higher than 600 nm; the emission spectrum of plant autoluminescence was shifted towards even higher wavelengths, a phenomenon ascribable to chlorophyll molecules acting as luminescence enhancers in vivo. Taken together, the presented results show that spontaneous photon emission imaged in plants mainly emanates from oxidized lipids. Imaging of this signal thus provides a simple and sensitive non-invasive method to selectively visualize and map patterns of lipid oxidation in plants.

The cytosolic/nuclear HSC70 and HSP90 molecular chaperones are important for stomatal closure and modulate abscisic acid-dependent physiological responses in Arabidopsis.

Cytosolic/nuclear molecular chaperones of the heat shock protein families HSP90 and HSC70 are conserved and essential proteins in eukaryotes. These proteins have essentially been implicated in the innate immunity and abiotic stress tolerance in higher plants. Here, we demonstrate that both chaperones are recruited in Arabidopsis (Arabidopsis thaliana) for stomatal closure induced by several environmental signals. Plants overexpressing HSC70-1 or with reduced HSP90.2 activity are compromised in the dark-, CO(2)-, flagellin 22 peptide-, and abscisic acid (ABA)-induced stomatal closure. HSC70-1 and HSP90 proteins are needed to establish basal expression levels of several ABA-responsive genes, suggesting that these chaperones might also be involved in ABA signaling events. Plants overexpressing HSC70-1 or with reduced HSP90.2 activity are hypersensitive to ABA in seed germination assays, suggesting that several chaperone complexes with distinct substrates might tune tissue-specific responses to ABA and the other biotic and abiotic stimuli studied. This study demonstrates that the HSC70/HSP90 machinery is important for stomatal closure and serves essential functions in plants to integrate signals from their biotic and abiotic environments.

Variable mesophyll conductance revisited: theoretical background and experimental implications.

The CO(2) concentration at the site of carboxylation inside the chloroplast stroma depends not only on the stomatal conductance, but also on the conductance of CO(2) between substomatal cavities and the site of CO(2) fixation. This conductance, commonly termed mesophyll conductance (g(m) ), significantly constrains the rate of photosynthesis. Here we show that estimates of g(m) are influenced by the amount of respiratory and photorespiratory CO(2) from the mitochondria diffusing towards the chloroplasts. This results in an apparent CO(2) and oxygen sensitivity of g(m) that does not imply a change in intrinsic diffusion properties of the mesophyll, but depends on the ratio of mitochondrial CO(2) release to chloroplast CO(2) uptake. We show that this effect (1) can bias the estimation of the CO(2) photocompensation point and non-photorespiratory respiration in the light; (2) can affect the estimates of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) kinetic constants in vivo; and (3) results in an apparent obligatory correlation between stomatal conductance and g(m) . We further show that the amount of photo(respiratory) CO(2) that is refixed by Rubisco can be directly estimated through measurements of g(m) .

The impact of soil microorganisms on the global budget of delta18O in atmospheric CO2.

Improved global estimates of terrestrial photosynthesis and respiration are critical for predicting the rate of change in atmospheric CO(2). The oxygen isotopic composition of atmospheric CO(2) can be used to estimate these fluxes because oxygen isotopic exchange between CO(2) and water creates distinct isotopic flux signatures. The enzyme carbonic anhydrase (CA) is known to accelerate this exchange in leaves, but the possibility of CA activity in soils is commonly neglected. Here, we report widespread accelerated soil CO(2) hydration. Exchange was 10-300 times faster than the uncatalyzed rate, consistent with typical population sizes for CA-containing soil microorganisms. Including accelerated soil hydration in global model simulations modifies contributions from soil and foliage to the global CO(18)O budget and eliminates persistent discrepancies existing between model and atmospheric observations. This enhanced soil hydration also increases the differences between the isotopic signatures of photosynthesis and respiration, particularly in the tropics, increasing the precision of CO(2) gross fluxes obtained by using the delta(18)O of atmospheric CO(2) by 50%.

Elevated expression of PGR5 and NDH-H in bundle sheath chloroplasts in C4 flaveria species.

Cyclic electron transport around PSI has been proposed to supply the additional ATP required for C(4) photosynthesis. To investigate the nature of cyclic electron pathways involved in C(4) photosynthesis, we analyzed tissue-specific expression of PGR5 (PROTON GRADIENT REGULATION 5), which is involved in the antimycin A-sensitive pathway, and NDH-H, a subunit of the plastidial NAD(P)H dehydrogenase complex, in four Flaveria species comprising NADP-malic enzyme (ME)-type C(4), C(3)-C(4) intermediate and C(3) species. PGR5 was highly expressed in the C(4) species and enriched in bundle sheath chloroplasts together with NDH-H, suggesting that electron transport of both PGR5-dependent and NDH-dependent cyclic pathways is promoted to drive C(4) photosynthesis.

Resistances along the CO2 diffusion pathway inside leaves.

CO(2) faces a series of resistances while diffusing between the substomatal cavities and the sites of carboxylation within chloroplasts. The absence of techniques to measure the resistance of individual steps makes it difficult to define their relative importance. Resistance to diffusion through intercellular airspace differs between leaves, but is usually of minor importance. Leaves with high photosynthetic capacity per unit leaf area reduce mesophyll resistance by increasing the surface area of chloroplasts exposed to intercellular airspace per unit leaf area, S(c). Cell walls impose a significant resistance. Assuming an effective porosity of the cell wall of 0.1 or 0.05, then cell walls could account for 25% or 50% of the total mesophyll resistance, respectively. Since the fraction of apoplastic water that is unbound and available for unhindered CO(2) diffusion is unknown, it is possible that the effective porosity is <0.05. Effective porosity could also vary in response to changes in pH or cation concentration. Consequently, cell walls could account for >50% of the total resistance and a variable proportion. Most of the remaining resistance is imposed by one or more of the three membranes as mesophyll resistance can be altered by varying the expression of cooporins. The CO(2) permeability of vesicles prepared from chloroplast envelopes has been reduced by RNA interference (RNAi) expression of NtAQP1, but not those prepared from the plasma membrane. Carbonic anhydrase activity also influences mesophyll resistance. Mesophyll resistance is relatively insensitive to the manipulation of any step in the pathway because it represents only part of the total and may also be countered by pleiotropic compensatory changes. The parameters in greatest need of additional measurements are S(c), mesophyll cell wall thickness, and the permeabilities of the plasma membrane and chloroplast envelope.

Estimating mesophyll conductance to CO2: methodology, potential errors, and recommendations.

The three most commonly used methods for estimating mesophyll conductance (g(m)) are described. They are based on gas exchange measurements either (i) by themselves; (ii) in combination with chlorophyll fluorescence quenching analysis; or (iii) in combination with discrimination against (13)CO(2). To obtain reliable estimates of g(m), the highest possible accuracy of gas exchange is required, particularly when using small leaf chambers. While there may be problems in achieving a high accuracy with leaf chambers that clamp onto a leaf with gaskets, guidelines are provided for making necessary corrections that increase reliability. All methods also rely on models for the calculation of g(m) and are sensitive to variation in the values of the model parameters. The sensitivity to these factors and to measurement error is analysed and ways to obtain the most reliable g(m) values are discussed. Small leaf areas can best be measured using one of the fluorescence methods. When larger leaf areas can be measured in larger chambers, the online isotopic methods are preferred. Using the large CO(2) draw-down provided by big chambers, and the isotopic method, is particularly important when measuring leaves with high g(m) that have a small difference in [CO(2)] between the substomatal cavity and the site of carboxylation in the chloroplast (C(i)-C(c) gradient). However, equipment for the fluorescence methods is more easily accessible. Carbon isotope discrimination can also be measured in recently synthesized carbohydrates, which has its advantages under field conditions when large number of samples must be processed. The curve-fitting method that uses gas exchange measurements only is not preferred and should only be used when no alternative is available. Since all methods have their weaknesses, the use of two methods for the estimation of g(m), which are as independent as possible, is recommended.

Effect of PGR5 impairment on photosynthesis and growth in Arabidopsis thaliana.

PGR5 has been reported as an important factor for the activity of the ferredoxin-dependent cyclic electron transport around PSI. To elucidate the role of PGR5 in C(3) photosynthesis, we characterized the photosynthetic electron transport rate (ETR), CO(2) assimilation and growth in the Arabidopsis thaliana pgr5 mutant at various irradiances and with CO(2) regimes. In low-light-grown pgr5, the CO(2) assimilation rate and ETR were similar to the those of the wild type at low irradiance, but decreased at saturating irradiance under photorespiratory conditions as well as non-photorespiratory conditions. Although non-photochemical quenching of chlorophyll fluorescence (NPQ) was not induced in the pgr5 mutant under steady-state photosynthesis, we show that it was induced under dark to light transition at low CO(2) concentration. Under low light conditions in air, pgr5 showed the same growth as the wild type, but a significant growth reduction compared with the wild type at >150 mumol photons m(-2) s(-1). This growth impairment was largely suppressed under high CO(2) concentrations. Based on the intercellular CO(2) concentration dependency of CO(2) assimilation, ETR and P700 oxidation measurements, we conclude that reduction of photosynthesis and growth result from (i) ATP deficiency and (ii) inactivation of PSI. We discuss these data in relation to the role of PGR5-dependent regulatory mechanisms in tuning the ATP/NADPH ratio and preventing inactivation of PSI, especially under conditions of high irradiance or enhanced photorespiration.

Autoluminescence imaging: a non-invasive tool for mapping oxidative stress.

Living organisms spontaneously emit faint light, and this autoluminescence is stimulated in response to many stresses. This phenomenon is attributable to the endogenous production of excited states during oxidative reactions, particularly during peroxidation of lipids, which generates light-emitting molecules such as triplet carbonyls and singlet oxygen. Using highly sensitive cameras, it is now possible to remotely image spontaneous luminescence with a good spatial resolution, providing a new non-invasive tool for mapping oxidative stress and lipid peroxidation in plants.

Increased zinc content in transplastomic tobacco plants expressing a polyhistidine-tagged Rubisco large subunit.

Rubisco is a hexadecameric enzyme composed of two subunits: a small subunit (SSU) encoded by a nuclear gene (rbcS), and a large subunit (LSU) encoded by a plastid gene (rbcL). Due to its high abundance, Rubisco represents an interesting target to express peptides or small proteins as fusion products at high levels. In an attempt to modify the plant metal content, a polyhistidine sequence was fused to Rubisco, the most abundant protein of plants. Plastid transformation was used to express a polyhistidine (6x) fused to the C-terminal extremity of the tobacco LSU. Transplastomic tobacco plants were generated by cotransformation of polyethylene glycol-treated protoplasts using two vectors: one containing the 16SrDNA marker gene, conferring spectinomycin resistance, and the other the polyhistidine-tagged rbcL gene. Homoplasmic plants containing L8-(His)6S8 as a single enzyme species were obtained. These plants contained normal Rubisco amounts and activity and displayed normal photosynthetic properties and growth. Interestingly, transplastomic plants accumulated higher zinc amounts than the wild-type when grown on zinc-enriched media. The highest zinc increase observed exceeded the estimated chelating ability of the polyhistidine sequence, indicating a perturbation in intracellular zinc homeostasis. We discuss the possibility of using Rubisco to express foreign peptides as fusion products and to confer new properties to higher plants.