Unsaturation in the air spaces of leaves and its implications.

Modern plant physiological theory stipulates that the resistance to water movement from plants to the atmosphere is overwhelmingly dominated by stomata. This conception necessitates a corollary assumption-that the air spaces in leaves must be nearly saturated with water vapour; that is, with a relative humidity that does not decline materially below unity. As this idea became progressively engrained in scientific discourse and textbooks over the last century, observations inconsistent with this corollary assumption were occasionally reported. Yet, evidence of unsaturation gained little traction, with acceptance of the prevailing framework motivated by three considerations: (1) leaf water potentials measured by either thermocouple psychrometry or the Scholander pressure chamber are largely consistent with the framework; (2) being able to assume near saturation of intercellular air spaces was transformational to leaf gas exchange analysis; and (3) there has been no obvious mechanism to explain a variable, liquid-phase resistance in the leaf mesophyll. Here, we review the evidence that refutes the assumption of universal, near saturation of air spaces in leaves. Refining the prevailing paradigm with respect to this assumption provides opportunities for identifying and developing mechanisms for increased plant productivity in the face of increasing evaporative demand imposed by global climate change.

Orthopaedic sport medicine surgeons and fellows value immersive virtual reality for improving surgical training, procedural planning, and distance learning.

OBJECTIVES: Overall, the potential utility of immersive virtual reality (iVR) technology in orthopaedic surgery is promising. The attitudes of medical students and surgical trainees on virtual reality simulated surgical training have been overwhelmingly positive. However, further research and understanding of the attitudes of practicing orthopaedic surgeons and fellows are needed to appreciate its benefits for clinical practice. The purpose of this study was to establish the face validity of iVR technology by assessing the attitudes of Canadian orthopaedic surgeons on the value of iVR for surgical training, clinical practice, and distance learning. METHODS: Forty-three orthopaedic surgeons and fellows attended an iVR demonstration at an annual orthopaedic meeting. The view and audio from the lead headset were cast to a large screen so the audience could follow the procedure in real time. Immediately after the presentation, the audience members were asked to complete a paper questionnaire assessing their perceptions and attitudes toward iVR for use in orthopaedic learning, clinical practice and distance education and mentoring. RESULTS: iVR was perceived to be valuable for the field of orthopaedic surgery providing face validity for the technology. All 13 questions were rated with mean Likert scores of five or greater, indicating a positive observed value for all 13 questions. The respondents indicated that iVR had value (score of 5 or greater) in each questionnaire domain, with agreement ranging from 78 to 98% for teaching and learning, 66-97% for clinical practice, and 88-100% for distance education and mentoring questions. CONCLUSION: This study has demonstrated that a group of Canadian sport medicine orthopaedic surgeons and fellows had favourable attitudes toward, and perceived that iVR has value in, orthopaedic surgical training, clinical practice, and distance learning and mentorship. The potential for utilizing iVR technology for distance learning, mentorship and global education appears promising. LEVEL OF EVIDENCE: II.

Linking water use efficiency with water use strategy from leaves to communities.

Limitations and utility of three measures of water use characteristics were evaluated: water use efficiency (WUE), intrinsic WUE and marginal water cost of carbon gain ( ∂ E / ∂ A ) estimated, respectively, as ratios of assimilation (A) to transpiration (E), of A to stomatal conductance (gs ) and of sensitivities of E and A with variation in gs . Only the measure ∂ E / ∂ A estimates water use strategy in a way that integrates carbon gain relative to water use under varying environmental conditions across scales from leaves to communities. This insight provides updated and simplified ways of estimating ∂ E / ∂ A and adds depth to understanding ways that plants balance water expenditure against carbon gain, uniquely providing a mechanistic means of predicting water use characteristics under changing environmental scenarios.

C4 maize and sorghum are more sensitive to rapid dehydration than C3 wheat and sunflower.

The high productive potential, heat resilience, and greater water use efficiency of C4 over C3 plants attract considerable interest in the face of global warming and increasing population, but C4 plants are often sensitive to dehydration, questioning the feasibility of their wider adoption. To resolve the primary effect of dehydration from slower from secondary leaf responses originating within leaves to combat stress, we conducted an innovative dehydration experiment. Four crops grown in hydroponics were forced to a rapid yet controlled decrease in leaf water potential by progressively raising roots of out of the solution while measuring leaf gas exchange. We show that, under rapid dehydration, assimilation decreased more steeply in C4 maize and sorghum than in C3 wheat and sunflower. This reduction was due to a rise of nonstomatal limitation at triple the rate in maize and sorghum than in wheat and sunflower. Rapid reductions in assimilation were previously measured in numerous C4 species across both laboratory and natural conditions. Hence, we deduce that high sensitivity to rapid dehydration might stem from the disturbance of an intrinsic aspect of C4 bicellular photosynthesis. We posit that an obstruction to metabolite transport between mesophyll and bundle sheath cells could be the cause.

The Chlamydomonas reinhardtii chloroplast envelope protein LCIA transports bicarbonate in planta.

LCIA (low CO2-inducible protein A) is a chloroplast envelope protein associated with the CO2-concentrating mechanism of the green alga Chlamydomonas reinhardtii. LCIA is postulated to be a HCO3- channel, but previous studies were unable to show that LCIA was actively transporting bicarbonate in planta. Therefore, LCIA activity was investigated more directly in two heterologous systems: an Escherichia coli mutant (DCAKO) lacking both native carbonic anhydrases and an Arabidopsis mutant (ßca5) missing the plastid carbonic anhydrase ßCA5. Neither DCAKO nor ßca5 can grow in ambient CO2 conditions, as they lack carbonic anhydrase-catalyzed production of the necessary HCO3- concentration for lipid and nucleic acid biosynthesis. Expression of LCIA restored growth in both systems in ambient CO2 conditions, which strongly suggests that LCIA is facilitating HCO3- uptake in each system. To our knowledge, this is the first direct evidence that LCIA moves HCO3- across membranes in bacteria and plants. Furthermore, the ßca5 plant bioassay used in this study is the first system for testing HCO3- transport activity in planta, an experimental breakthrough that will be valuable for future studies aimed at improving the photosynthetic efficiency of crop plants using components from algal CO2-concentrating mechanisms.

Assessing the CO2 concentration at the surface of photosynthetic mesophyll cells.

We present a robust estimation of the CO2 concentration at the surface of photosynthetic mesophyll cells (cw ), applicable under reasonable assumptions of assimilation distribution within the leaf. We used Capsicum annuum, Helianthus annuus and Gossypium hirsutumas model plants for our experiments. We introduce calculations to estimate cw using independent adaxial and abaxial gas exchange measurements, and accounting for the mesophyll airspace resistances. The cw was lower than adaxial and abaxial estimated intercellular CO2 concentrations (ci ). Differences between cw and the ci of each surface were usually larger than 10 µmol mol-1 . Differences between adaxial and abaxial ci ranged from a few µmol mol-1 to almost 50 µmol mol-1 , where the largest differences were found at high air saturation deficits (ASD). Differences between adaxial and abaxial ci and the ci estimated by mixing both fluxes ranged from -30 to +20 µmol mol-1 , where the largest differences were found under high ASD or high ambient CO2 concentrations. Accounting for cw improves the information that can be extracted from gas exchange experiments, allowing a more detailed description of the CO2 and water vapor gradients within the leaf.

Humidity gradients in the air spaces of leaves.

Stomata are orifices that connect the drier atmosphere with the interconnected network of more humid air spaces that surround the cells within a leaf. Accurate values of the humidities inside the substomatal cavity, wi, and in the air, wa, are needed to estimate stomatal conductance and the CO2 concentration in the internal air spaces of leaves. Both are vital factors in the understanding of plant physiology and climate, ecological and crop systems. However, there is no easy way to measure wi directly. Out of necessity, wi has been taken as the saturation water vapour concentration at leaf temperature, wsat, and applied to the whole leaf intercellular air spaces. We explored the occurrence of unsaturation by examining gas exchange of leaves exposed to various magnitudes of wsat - wa, or Δw, using a double-sided, clamp-on chamber, and estimated degrees of unsaturation from the gradient of CO2 across the leaf that was required to sustain the rate of CO2 assimilation through the upper surface. The relative humidity in the substomatal cavities dropped to about 97% under mild Δw and as dry as around 80% when Δw was large. Measurements of the diffusion of noble gases across the leaf indicated that there were still regions of near 100% humidity distal from the stomatal pores. We suggest that as Δw increases, the saturation edge retreats into the intercellular air spaces, accompanied by the progressive closure of mesophyll aquaporins to maintain the cytosolic water potential.

Multienvironment QTL analysis delineates a major locus associated with homoeologous exchanges for water-use efficiency and seed yield in canola.

Canola varieties exhibit variation in drought avoidance and drought escape traits, reflecting adaptation to water-deficit environments. Our understanding of underlying genes and their interaction across environments in improving crop productivity is limited. A doubled haploid population was analysed to identify quantitative trait loci (QTL) associated with water-use efficiency (WUE) related traits. High WUE in the vegetative phase was associated with low seed yield. Based on the resequenced parental genome data, we developed sequence-capture-based markers and validated their linkage with carbon isotope discrimination (Δ13 C) in an F2 population. RNA sequencing was performed to determine the expression of candidate genes underlying Δ13 C QTL. QTL contributing to main and QTL × environment interaction effects for Δ13 C and yield were identified. One multiple-trait QTL for Δ13 C, days to flower, plant height, and seed yield was identified on chromosome A09. Interestingly, this QTL region overlapped with a homoeologous exchange (HE) event, suggesting its association with the multiple traits. Transcriptome analysis revealed 121 significantly differentially expressed genes underlying Δ13 C QTL on A09 and C09, including in HE regions. Sorting out the negative relationship between vegetative WUE and seed yield is a priority. Genetic and genomic resources and knowledge so developed could improve canola WUE and yield.

Do 2 H and 18 O in leaf water reflect environmental drivers differently?

We compiled hydrogen and oxygen stable isotope compositions (δ2 H and δ18 O) of leaf water from multiple biomes to examine variations with environmental drivers. Leaf water δ2 H was more closely correlated with δ2 H of xylem water or atmospheric vapour, whereas leaf water δ18 O was more closely correlated with air relative humidity. This resulted from the larger proportional range for δ2 H of meteoric waters relative to the extent of leaf water evaporative enrichment compared with δ18 O. We next expressed leaf water as isotopic enrichment above xylem water (Δ2 H and Δ18 O) to remove the impact of xylem water isotopic variation. For Δ2 H, leaf water still correlated with atmospheric vapour, whereas Δ18 O showed no such correlation. This was explained by covariance between air relative humidity and the Δ18 O of atmospheric vapour. This is consistent with a previously observed diurnal correlation between air relative humidity and the deuterium excess of atmospheric vapour across a range of ecosystems. We conclude that 2 H and 18 O in leaf water do indeed reflect the balance of environmental drivers differently; our results have implications for understanding isotopic effects associated with water cycling in terrestrial ecosystems and for inferring environmental change from isotopic biomarkers that act as proxies for leaf water.

Cuticular conductance of adaxial and abaxial leaf surfaces and its relation to minimum leaf surface conductance.

Cuticular conductance to water (gcw ) is difficult to quantify for stomatous surfaces due to the complexity of separating cuticular and stomatal transpiration, and additional complications arise for determining adaxial and abaxial gcw . This has led to the neglect of gcw as a separate parameter in most common gas exchange measurements. Here, we describe a simple technique to simultaneously estimate adaxial and abaxial values of gcw , tested in two amphistomatous plant species. What we term the 'Red-Light method' is used to estimate gcw from gas exchange measurements and a known CO2 concentration inside the leaf during photosynthetic induction under red light. We provide an easy-to-use web application to assist with the calculation of gcw . While adaxial and abaxial gcw varies significantly between leaves of the same species we found that the ratio of adaxial/abaxial gcw (γn ) is stable within a plant species. This has implications for use of generic values of gcw when analysing gas exchange data. The Red-Light method can be used to estimate total cuticular conductance (gcw-T ) accurately with the most common setup of gas exchange instruments, i.e. a chamber mixing the adaxial and abaxial gases, allowing for a wide application of this technique.

An improved theory for calculating leaf gas exchange more precisely accounting for small fluxes.

The widely used theory for gas exchange proposed by von Caemmerer and Farquhar (vCF) integrates molar fluxes, mole fraction gradients and ternary effects but does not account for cuticular fluxes, for separation of the leaf surface conditions or for ternary effects within the boundary layer. The magnitude of cuticular conductance to water (gcw) is a key factor for determining plant survival in drought but is difficult to measure and often neglected in routine gas exchange studies. The vCF ternary effect is applied to the total flux without the recognition of different pathways that are affected by it. These simplifications lead to errors in estimations of stomatal conductance, intercellular carbon dioxide concentration (Ci) and other gas exchange parameters. The theory presented here is a more precise physical approach to the electrical resistance analogy for gas exchange, resulting in a more accurate calculation of gas exchange parameters. Additionally, we extend our theory, using physiological concepts, to create a model that allows us to calculate cuticular conductance to water.

Revisiting carbon isotope discrimination in C3 plants shows respiration rules when photosynthesis is low.

Stable isotopes are commonly used to study the diffusion of CO2 within photosynthetic plant tissues. The standard method used to interpret the observed preference for the lighter carbon isotope in C3 photosynthesis involves the model of Farquhar et al., which relates carbon isotope discrimination to physical and biochemical processes within the leaf. However, under many conditions the model returns unreasonable results for mesophyll conductance to CO2 diffusion (gm), especially when rates of photosynthesis are low. Here, we re-derive the carbon isotope discrimination model using modified assumptions related to the isotope effect of mitochondrial respiration. In particular, we treat the carbon pool associated with respiration as separate from the pool of primary assimilates. We experimentally test the model by comparing gm values measured with different CO2 source gases varying in their isotopic composition, and show that our new model returns matching gm values that are much more reasonable than those obtained with the previous model. We use our results to discuss CO2 diffusion properties within the mesophyll.

Two-Source δ18O Method to Validate the CO18O-Photosynthetic Discrimination Model: Implications for Mesophyll Conductance.

Theoretical models of photosynthetic isotopic discrimination of CO2 (13C and 18O) are commonly used to estimate mesophyll conductance (g m). This requires making simplifying assumptions and assigning parameter values so that g m can be solved for as the residual term. Uncertainties in g m estimation occur due to measurement noise and assumptions not holding, including parameter uncertainty and model parametrization. Uncertainties in the 13C model have been explored previously, but there has been little testing undertaken to determine the reliability of g m estimates from the 18O model (g m18). In this study, we exploited the action of carbonic anhydrase in equilibrating CO2 with leaf water and manipulated the observed photosynthetic discrimination (Δ18O) by changing the oxygen isotopic composition of the source gas CO2 and water vapor. We developed a two-source δ18O method, whereby two measurements of Δ18O were obtained for a leaf with its gas-exchange characteristics otherwise unchanged. Measurements were performed in broad bean (Vicia faba) and Algerian oak (Quercus canariensis) in response to light and vapor pressure deficit. Despite manipulating the Δ18O by over 100‰, in most cases we observed consistency in the calculated g m18, providing confidence in the measurements and model theory. Where there were differences in g m18 estimates between source-gas measurements, we explored uncertainty associated with two model assumptions (the isotopic composition of water at the sites of CO2-water exchange, and the humidity of the leaf internal airspace) and found evidence for both. Finally, we provide experimental guidelines to minimize the sensitivity of g m18 estimates to measurement errors. The two-source δ18O method offers a flexible tool for model parameterization and provides an opportunity to refine our understanding of leaf water and CO2 fluxes.

Stomatal conductance responses to evaporative demand conferred by rice drought-yield quantitative trait locus qDTY12.1.

Rice quantitative trait locus (QTL) qDTY12.1 is a major-effect drought yield QTL that was identified from a cross of Vandana (recipient parent) and Way Rarem (donor parent) through breeding efforts to improve rice yield under upland drought stress conditions. The two main physiological effects previously observed to be related to the presence of qDTY12.1 were (i) increased lateral root growth, and (ii) increased transpiration efficiency. Since relatively more progress has thus far been made on characterising the lateral root growth response related to qDTY12.1, the present study focussed on characterising how qDTY12.1 confers higher transpiration efficiency under upland drought stress in the Vandana background. In a series of field experiments in which stomatal conductance was measured across different times of day in four qDTY12.1 near isogenic lines (NILs), the NILs and Way Rarem showed consistently higher stomatal conductance than Vandana under conditions of low vapour pressure deficit (VPD) and low photosynthetically active radiation (PAR), and consistently lower stomatal conductance than Vandana under high VPD and high PAR. Leaf δ18O was higher in the qDTY12.1 NIL than in Vandana, and although this trend was previously observed for leaf δ13C it appeared to be more consistent across measurement dates and treatments for leaf δ18O. The qDTY12.1 NILs and Way Rarem tended to show greater large vein to small vein interveinal distance and mesophyll area than Vandana, also consistent across treatments. In terms of aquaporin-related plant hydraulics, variation among NILs in terms of aquaporin inhibition of root hydraulic conductivity (Lpr) was observed, with the highest-yielding NIL showing a lack of Lpr inhibition similar to Way Rarem. The results reported here suggest that the effects of qDTY12.1 are in response not only to soil moisture, but also to atmospheric conditions. An interaction among multiple mechanisms including leaf anatomy and aquaporin function appear to confer the transpiration efficiency effect of qDTY12.1.

On the contributions of photorespiration and compartmentation to the contrasting intramolecular 2H profiles of C3 and C4 plant sugars.

Compartmentation of C4 photosynthetic biochemistry into bundle sheath (BS) and mesophyll (M) cells, and photorespiration in C3 plants is predicted to have hydrogen isotopic consequences for metabolites at both molecular and site-specific levels. Molecular-level evidence was recently reported (Zhou et al., 2016), but evidence at the site-specific level is still lacking. We propose that such evidence exists in the contrasting 2H distribution profiles of glucose samples from naturally grown C3, C4 and CAM plants: photorespiration contributes to the relative 2H enrichment in H5 and relative 2H depletion in H1 & H6 (the average of the two pro-chiral Hs and in particular H6,pro-R) in C3 glucose, while 2H-enriched C3 mesophyll cellular (chloroplastic) water most likely contributes to the enrichment at H4; export of (transferable hydrogen atoms of) NADPH from C4 mesophyll cells to bundle sheath cells (via the malate shuttle) and incorporation of 2H-relatively unenriched BS cellular water contribute to the relative depletion of H4 & H5 respectively; shuttling of triose-phosphates (PGA: phosphoglycerate dand DHAP: dihydroacetone phosphate) between C4 bundle sheath and mesophyll cells contributes to the relative enrichment in H1 & H6 (in particular H6,pro-R) in C4 glucose.

Hydrogen isotopic differences between C3 and C4 land plant lipids: consequences of compartmentation in C4 photosynthetic chemistry and C3 photorespiration.

The 2 H/1 H ratio of carbon-bound H in biolipids holds potential for probing plant lipid biosynthesis and metabolism. The biochemical mechanism underlying the isotopic differences between lipids from C3 and C4 plants is still poorly understood. GC-pyrolysis-IRMS (gas chromatography-pyrolysis-isotope ratio mass spectrometry) measurement of the 2 H/1 H ratio of leaf lipids from controlled and field grown plants indicates that the biochemical isotopic fractionation (ε2 Hlipid_biochem ) differed between C3 and C4 plants in a pathway-dependent manner: ε2 HC4 > ε2 HC3 for the acetogenic pathway, ε2 HC4 < ε2 HC3 for the mevalonic acid pathway and the 1-deoxy-D-xylulose 5-phosphate pathway across all species examined. It is proposed that compartmentation of photosynthetic CO2 fixation into C4 mesophyll (M) and bundle sheath (BS) cells and suppression of photorespiration in C4 M and BS cells both result in C4 M chloroplastic pyruvate - the precursor for acetogenic pathway - being more depleted in 2 H relative to pyruvate in C3 cells. In addition, compartmentation in C4 plants also results in (i) the transferable H of NADPH being enriched in 2 H in C4 M chloroplasts compared with that in C3 chloroplasts for the 1-deoxy-D-xylulose 5-phosphate pathway pathway and (ii) pyruvate relatively 2 H-enriched being used for the mevalonic acid pathway in the cytosol of BS cells in comparison with that in C3 cells.

Leaf vein fraction influences the Péclet effect and 18 O enrichment in leaf water.

The process of evaporation results in the fractionation of water isotopes such that the lighter 16 O isotope preferentially escapes the gas phase leaving the heavier 18 O isotope to accumulate at the sites of evaporation. This applies to transpiration from a leaf with the degree of fractionation dependent on a number of environmental and physiological factors that are well understood. Nevertheless, the 18 O enrichment of bulk leaf water is often less than that predicted for the sites of evaporation. The advection of less enriched water in the transpiration stream has been suggested to limit the back diffusion of enriched evaporative site water (Péclet effect); however, evidence for this effect has been varied. In sampling across a range of species with different vein densities and saturated water contents, we demonstrate the importance of accounting for the relative 'pool' sizes of the vascular and mesophyll water for the interpretation of a Péclet effect. Further, we provide strong evidence for a Péclet signal within the xylem that if unaccounted for can lead to confounding of the estimated enrichment within the mesophyll water. This has important implications for understanding variation in the effective path length of the mesophyll and hence potentially the δ18 O of organic matter.

Physical injury and workplace assault in UK mental health trusts: An analysis of formal reports.

Workplace violence is a significant problem for health service personnel, with National Health Service (NHS) workers subject to 68 683 physical assaults between 2013 and 2014. Almost 70% of assaults occur in the mental health sector, and although serious, non-fatal injury is rare, the individual and economic impact can be substantial. In the present study, we analysed mandatory incident reports from a national database to examine whether there were identifiable precursors to incidents leading to staff injury, and whether staff characteristics were associated with injury. In line with previous descriptions, we found injury occurred either as a direct result of patient assault or during physical interventions employed by staff to contain aggression. Importantly, we found little evidence from staff reports that patients' symptoms were driving aggression, and we found less evidence of patient perspectives among reports. We make several recommendations regarding the reporting of these events that could inform policy and interventions aimed at minimizing the likelihood of injury.