Ozone‐induced stomatal sluggishness changes stomatal parameters of Jarvis‐type model in white birch and deciduous oak

• Stomatal ozone flux is closely related to ozone injury to plants. Jarvis‐type multiplicative model has been recommended for estimating stomatal ozone flux in forest trees. Ozone can change stomatal conductance by both stomatal closure and less efficient stomatal control (stomatal sluggishness). However, current Jarvis‐type models do not account for these ozone effects on stomatal conductance in forest trees.
• We examined seasonal course of stomatal conductance in two common deciduous tree species native to northern Japan (white birch: Betula platyphylla var. japonica ; deciduous oak: Quercus mongolica var. crispula ) grown under free‐air ozone exposure. We innovatively considered stomatal sluggishness in the Jarvis‐type model using a simple parameter, s , relating to cumulative ozone uptake (defined as POD : phytotoxic ozone dose).
• We found that ozone decreased stomatal conductance of white birch leaves after full expansion (?28%). However, such a reduction of stomatal conductance by ozone fell in late summer (?10%). At the same time, ozone reduced stomatal sensitivity of white birch to VPD and increased stomatal conductance under low light conditions. In contrast, in deciduous oak, ozone did not clearly change the model parameters.
• The consideration of both ozone‐induced stomatal closure and stomatal sluggishness improved the model performance to estimate stomatal conductance and to explain the dose–response relationship on ozone‐induced decline of photosynthesis of white birch. Our results indicate that ozone effects on stomatal conductance (i.e . stomatal closure and stomatal sluggishness) are crucial for modelling studies to determine stomatal response in deciduous trees, especially in species sensitive to ozone.

     

Internal conductance does not scale with photosynthetic capacity: implications for carbon isotope discrimination and the economics of water and nitrogen use in photosynthesis

Central paradigms of ecophysiology are that there are recognizable and even explicit and predictable patterns among species, genera, and life forms in the economics of water and nitrogen use in photosynthesis and in carbon isotope discrimination (delta). However most previous examinations have implicitly assumed an infinite internal conductance (gi) and/or that internal conductance scales with the biochemical capacity for photosynthesis. Examination of published data for 54 species and a detailed examination for three well-characterized species--Eucalyptus globulus, Pseudotsuga menziesii and Phaseolus vulgaris--show these assumptions to be incorrect. The reduction in concentration of CO2 between the substomatal cavity (Ci) and the site of carbon fixation (Cc) varies greatly among species. Photosynthesis does not scale perfectly with gi and there is a general trend for plants with low gi to have a larger draw-down from Ci to Cc, further confounding efforts to scale photosynthesis and other attributes with gi. Variation in the gi-photosynthesis relationship contributes to variation in photosynthetic 'use' efficiency of N (PNUE) and water (WUE). Delta is an information-rich signal, but for many species only about two-thirds of this information relates to A/gs with the remaining one-third related to A/gi. Using data for three well-studied species we demonstrate that at common WUE, delta may vary by up to 3 per thousand. This is as large or larger than is commonly reported in many interspecific comparisons of delta, and adds to previous warnings about simplistic interpretations of WUE based on delta. A priority for future research should be elucidation of relationships between gi and gs and how these vary in response to environmental conditions (e.g. soil water, leaf-to-air vapour pressure deficit, temperature) and among species.     

Stomatal responses to changes in vapor pressure deficit reflect tissue‐specific differences in hydraulic conductance

The vapor pressure deficit (D) of the atmosphere can negatively affect plant growth as plants reduce stomatal conductance to water vapor (g_wv) in response to increasing D, limiting the ability of plants to assimilate carbon. The sensitivity of g_wv to changes in D varies among species and has been correlated with the hydraulic conductance of leaves (K_leaf), but the hydraulic conductance of other tissues has also been implicated in plant responses to changing D. Among the 19 grass species, we found that K_leaf was correlated with the hydraulic conductance of large longitudinal veins (K_lv, r² = 0.81), but was not related to K_root (r² = 0.01). Stomatal sensitivity to D was correlated with K_leaf relative to total leaf area (r² = 0.50), and did not differ between C₃ and C₄ species. Transpiration (E) increased in response to D, but 8 of the 19 plants showed a decline in E at high D, indicative of an ‘apparent feedforward’ response. For these individuals, E began to decline at lower values of D in plants with low K_root (r² = 0.72). These results show the significance of both leaf and root hydraulic conductance as drivers of plant responses to evaporative demand.     

The reorganization of actin filaments is required for vacuolar fusion of guard cells during stomatal opening in Arabidopsis

The reorganization of actin filaments (AFs) and vacuoles in guard cells is involved in the regulation of stomatal movement. However, it remains unclear whether there is any interaction between the reorganization of AFs and vacuolar changes during stomatal movement. Here, we report the relationship between the reorganization of AFs and vacuolar fusion revealed in pharmacological experiments, and characterizing stomatal opening in actin‐related protein 2 (arp2) and arp3 mutants. Our results show that cytochalasin‐D‐induced depolymerization or phalloidin‐induced stabilization of AFs leads to an increase in small unfused vacuoles during stomatal opening in wild‐type (WT) Arabidopsis plants. Light‐induced stomatal opening is retarded and vacuolar fusion in guard cells is impaired in the mutants, in which the reorganization and the dynamic parameters of AFs are aberrant compared with those of the WT. In WT, AFs tightly surround the small separated vacuoles, forming a ring that encircles the boundary membranes of vacuoles partly fused during stomatal opening. In contrast, in the mutants, most AFs and actin patches accumulate abnormally around the nuclei of the guard cells, which probably further impair vacuolar fusion and retard stomatal opening. Our results suggest that the reorganization of AFs regulates vacuolar fusion in guard cells during stomatal opening.     

Ozone‐induced foliar damage and release of stress volatiles is highly dependent on stomatal openness and priming by low‐level ozone exposure in Phaseolus vulgaris

Acute ozone exposure triggers major emissions of volatile organic compounds (VOCs), but quantitatively, it is unclear how different ozone doses alter the start and the total amount of these emissions, and the induction rate of different stress volatiles. It is also unclear whether priming (i.e. pre‐exposure to lower O₃ concentrations) can modify the magnitude and kinetics of volatile emissions. We investigated photosynthetic characteristics and VOC emissions in Phaseolus vulgaris following acute ozone exposure (600 nmol mol^?1 for 30 min) under illumination and in darkness and after priming with 200 nmol mol^?1 O₃ for 30 min. Methanol and lipoxygenase (LOX) pathway product emissions were induced rapidly, followed by moderate emissions of methyl salicylate (MeSA). Stomatal conductance prior to acute exposure was lower in darkness and after low O₃ priming than in light and without priming. After low O₃ priming, no MeSA and lower LOX emissions were detected under acute exposure. Overall, maximum emission rates and the total amount of emitted LOX products and methanol were quantitatively correlated with total stomatal ozone uptake. These results indicate that different stress volatiles scale differently with ozone dose and highlight the key role of stomatal conductance in controlling ozone uptake, leaf injury and volatile release.     

Guard cell photosynthesis is critical for stomatal turgor production,yet does not directly mediate CO2‐ and ABA‐induced stomatal closing

Stomata mediate gas exchange between the inter‐cellular spaces of leaves and the atmosphere. CO₂ levels in leaves (Ci) are determined by respiration, photosynthesis, stomatal conductance and atmospheric [CO₂]. [CO₂] in leaves mediates stomatal movements. The role of guard cell photosynthesis in stomatal conductance responses is a matter of debate, and genetic approaches are needed. We have generated transgenic Arabidopsis plants that are chlorophyll‐deficient in guard cells only, expressing a constitutively active chlorophyllase in a guard cell specific enhancer trap line. Our data show that more than 90% of guard cells were chlorophyll‐deficient. Interestingly, approximately 45% of stomata had an unusual, previously not‐described, morphology of thin‐shaped chlorophyll‐less stomata. Nevertheless, stomatal size, stomatal index, plant morphology, and whole‐leaf photosynthetic parameters (PSII, qP, qN, F_V′/F_M′) were comparable with wild‐type plants. Time‐resolved intact leaf gas‐exchange analyses showed a reduction in stomatal conductance and CO₂‐assimilation rates of the transgenic plants. Normalization of CO₂ responses showed that stomata of transgenic plants respond to [CO₂] shifts. Detailed stomatal aperture measurements of normal kidney‐shaped stomata, which lack chlorophyll, showed stomatal closing responses to [CO₂] elevation and abscisic acid (ABA), while thin‐shaped stomata were continuously closed. Our present findings show that stomatal movement responses to [CO₂] and ABA are functional in guard cells that lack chlorophyll. These data suggest that guard cell CO₂ and ABA signal transduction are not directly modulated by guard cell photosynthesis/electron transport. Moreover, the finding that chlorophyll‐less stomata cause a ‘deflated’ thin‐shaped phenotype, suggests that photosynthesis in guard cells is critical for energization and guard cell turgor production.     

Drought-inhibition of photosynthesis in C3 plants: stomatal and non-stomatal limitations revisited

There is a long-standing controversy as to whether drought limits photosynthetic CO2 assimilation through stomatal closure or by metabolic impairment in C3 plants. Comparing results from different studies is difficult due to interspecific differences in the response of photosynthesis to leaf water potential and/or relative water content (RWC), the most commonly used parameters to assess the severity of drought. Therefore, we have used stomatal conductance (g) as a basis for comparison of metabolic processes in different studies. The logic is that, as there is a strong link between g and photosynthesis (perhaps co-regulation between them), so different relationships between RWC or water potential and photosynthetic rate and changes in metabolism in different species and studies may be 'normalized' by relating them to g. Re-analysing data from the literature using light-saturated g as a parameter indicative of water deficits in plants shows that there is good correspondence between the onset of drought-induced inhibition of different photosynthetic sub-processes and g. Contents of ribulose bisphosphate (RuBP) and adenosine triphosphate (ATP) decrease early in drought development, at still relatively high g (higher than 150 mmol H20 m(-2) s(-1)). This suggests that RuBP regeneration and ATP synthesis are impaired. Decreased photochemistry and Rubisco activity typically occur at lower g (<100 mmol H20 m(-2) s(-1)), whereas permanent photoinhibition is only occasional, occurring at very low g (<50 mmol H20 m(-2) s(-1)). Sub-stomatal CO2 concentration decreases as g becomes smaller, but increases again at small g. The analysis suggests that stomatal closure is the earliest response to drought and the dominant limitation to photosynthesis at mild to moderate drought. However, in parallel, progressive down-regulation or inhibition of metabolic processes leads to decreased RuBP content, which becomes the dominant limitation at severe drought, and thereby inhibits photosynthetic CO2 assimilation.     

Baobab trees (Adansonia) in Madagascar use stored water to flush new leaves but not to support stomatal opening before the rainy season

Baobab trees (Adansonia, Bombacaceae) are widely thought to store water in their stems for use when water availability is low. We tested this hypothesis by assessing the role of stored water during the dry season in three baobab species in Madagascar. In the dry season, leaves are present only during and after leaf flush. We quantified the relative contributions of stem and soil water during this period through measures of stem water content, sap flow and stomatal conductance. Rates of sap flow at the base of the trunk were near zero, indicating that leaf flushing was almost entirely dependent on stem water. Stem water content declined by up to 12% during this period, yet stomatal conductance and branch sap flow rates remained very low. Stem water reserves were used to support new leaf growth and cuticular transpiration, but not to support stomatal opening before the rainy season. Stomatal opening coincided with the onset of sap flow at the base of the trunk and occurred only after significant rainfall.     

Association genetics,geography and ecophysiology link stomatal patterning in Populus trichocarpa with carbon gain and disease resistance trade‐offs

Stomata are essential for diffusive entry of gases to support photosynthesis, but may also expose internal leaf tissues to pathogens. To uncover trade‐offs in range‐wide adaptation relating to stomata, we investigated the underlying genetics of stomatal traits and linked variability in these traits with geoclimate, ecophysiology, condensed foliar tannins and pathogen susceptibility in black cottonwood (Populus trichocarpa). Upper (adaxial) and lower (abaxial) leaf stomatal traits were measured from 454 accessions collected throughout much of the species range. We calculated broad‐sense heritability (H²) of stomatal traits and, using SNP data from a 34K Populus SNP array, performed a genome‐wide association studies (GWAS) to uncover genes underlying stomatal trait variation. H² values for stomatal traits were moderate (average H² = 0.33). GWAS identified genes associated primarily with adaxial stomata, including polarity genes (PHABULOSA), stomatal development genes (BRASSINOSTEROID‐INSENSITIVE 2) and disease/wound‐response genes (GLUTAMATE‐CYSTEINE LIGASE). Stomatal traits correlated with latitude, gas exchange, condensed tannins and leaf rust (Melampsora) infection. Latitudinal trends of greater adaxial stomata numbers and guard cell pore size corresponded with higher stomatal conductance (g_s) and photosynthesis (A_max), faster shoot elongation, lower foliar tannins and greater Melampsora susceptibility. This suggests an evolutionary trade‐off related to differing selection pressures across the species range. In northern environments, more adaxial stomata and larger pore sizes reflect selection for rapid carbon gain and growth. By contrast, southern genotypes have fewer adaxial stomata, smaller pore sizes and higher levels of condensed tannins, possibly linked to greater pressure from natural leaf pathogens, which are less significant in northern ecosystems.     

Drought limitation of photosynthesis differs between C3 and C4 grass species in a comparative experiment

Phylogenetic analyses show that C₄ grasses typically occupy drier habitats than their C₃ relatives, but recent experiments comparing the physiology of closely related C₃ and C₄ species have shown that advantages of C₄ photosynthesis can be lost under drought. We tested the generality of these paradoxical findings in grass species representing the known evolutionary diversity of C₄ NADP‐me and C₃ photosynthetic types. Our experiment investigated the effects of drought on leaf photosynthesis, water potential, nitrogen, chlorophyll content and mortality. C₄ grasses in control treatments were characterized by higher CO₂ assimilation rates and water potential, but lower stomatal conductance and nitrogen content. Under drought, stomatal conductance declined more dramatically in C₃ than C₄ species, and photosynthetic water‐use and nitrogen‐use efficiency advantages held by C₄ species under control conditions were each diminished by 40%. Leaf mortality was slightly higher in C₄ than C₃ grasses, but leaf condition under drought otherwise showed no dependence on photosynthetic‐type. This phylogenetically controlled experiment suggested that a drought‐induced reduction in the photosynthetic performance advantages of C₄ NADP‐me relative to C₃ grasses is a general phenomenon.     

Chloride as a macronutrient increases water‐use efficiency by anatomically driven reduced stomatal conductance and increased mesophyll diffusion to CO2

Chloride (Cl^?) has been recently described as a beneficial macronutrient, playing specific roles in promoting plant growth and water‐use efficiency (WUE). However, it is still unclear how Cl^? could be beneficial, especially in comparison with nitrate (NO₃^?), an essential source of nitrogen that shares with Cl^? similar physical and osmotic properties, as well as common transport mechanisms. In tobacco plants, macronutrient levels of Cl^? specifically reduce stomatal conductance (g_s) without a concomitant reduction in the net photosynthesis rate (A_N). As stomata‐mediated water loss through transpiration is inherent in the need of C₃ plants to capture CO₂, simultaneous increase in photosynthesis and WUE is of great relevance to achieve a sustainable increase in C₃ crop productivity. Our results showed that Cl^?‐mediated stimulation of larger leaf cells leads to a reduction in stomatal density, which in turn reduces g_s and water consumption. Conversely, Cl^? improves mesophyll diffusion conductance to CO₂ (g_m) and photosynthetic performance due to a higher surface area of chloroplasts exposed to the intercellular airspace of mesophyll cells, possibly as a consequence of the stimulation of chloroplast biogenesis. A key finding of this study is the simultaneous improvement of A_N and WUE due to macronutrient Cl^? nutrition. This work identifies relevant and specific functions in which Cl^? participates as a beneficial macronutrient for higher plants, uncovering a sustainable approach to improve crop yield.     

Causes of reduced leaf‐level photosynthesis during strong El Niño drought in a Central Amazon forest

Sustained drought and concomitant high temperature may reduce photosynthesis and cause tree mortality. Possible causes of reduced photosynthesis include stomatal closure and biochemical inhibition, but their relative roles are unknown in Amazon trees during strong drought events. We assessed the effects of the recent (2015) strong El Niño drought on leaf‐level photosynthesis of Central Amazon trees via these two mechanisms. Through four seasons of 2015, we measured leaf gas exchange, chlorophyll a fluorescence parameters, chlorophyll concentration, and nutrient content in leaves of 57 upper canopy and understory trees of a lowland terra firme forest on well‐drained infertile oxisol. Photosynthesis decreased 28% in the upper canopy and 17% in understory trees during the extreme dry season of 2015, relative to other 2015 seasons and was also lower than the climatically normal dry season of the following non‐El Niño year. Photosynthesis reduction under extreme drought and high temperature in the 2015 dry season was related only to stomatal closure in both upper canopy and understory trees, and not to chlorophyll a fluorescence parameters, chlorophyll, or leaf nutrient concentration. The distinction is important because stomatal closure is a transient regulatory response that can reverse when water becomes available, whereas the other responses reflect more permanent changes or damage to the photosynthetic apparatus. Photosynthesis decrease due to stomatal closure during the 2015 extreme dry season was followed 2 months later by an increase in photosynthesis as rains returned, indicating a margin of resilience to one‐off extreme climatic events in Amazonian forests.     

Seasonal and within‐canopy variation in shoot‐scale resource‐use efficiency trade‐offs in a Norway spruce stand

Previous leaf‐scale studies of carbon assimilation describe short‐term resource‐use efficiency (RUE) trade‐offs where high use efficiency of one resource requires low RUE of another. However, varying resource availabilities may cause long‐term RUE trade‐offs to differ from the short‐term patterns. This may have important implications for understanding canopy‐scale resource use and allocation. We used continuous gas exchange measurements collected at five levels within a Norway spruce, Picea abies (L.) karst., canopy over 3 years to assess seasonal differences in the interactions between shoot‐scale resource availability (light, water and nitrogen), net photosynthesis (A_n) and the use efficiencies of light (LUE), water (WUE) and nitrogen (NUE) for carbon assimilation. The continuous data set was used to develop and evaluate multiple regression models for predicting monthly shoot‐scale A_n. These models showed that shoot‐scale A_n was strongly dependent on light availability and was generally well described with simple one‐ or two‐parameter models. WUE peaked in spring, NUE in summer and LUE in autumn. However, the relative importance of LUE for carbon assimilation increased with canopy depth at all times. Our results suggest that accounting for seasonal and within‐canopy trade‐offs may be important for RUE‐based modelling of canopy carbon uptake.     

Gas exchange and water‐use efficiency in plant canopies

In this review, I first address the basics of gas exchange, water‐use efficiency and carbon isotope discrimination in C₃ plant canopies. I then present a case study of water‐use efficiency in northern Australian tree species. In general, C₃ plants face a trade‐off whereby increasing stomatal conductance for a given set of conditions will result in a higher CO₂ assimilation rate, but a lower photosynthetic water‐use efficiency. A common garden experiment suggested that tree species which are able to establish and grow in drier parts of northern Australia have a capacity to use water rapidly when it is available through high stomatal conductance, but that they do so at the expense of low water‐use efficiency. This may explain why community‐level carbon isotope discrimination does not decrease as steeply with decreasing rainfall on the North Australian Tropical Transect as has been observed on some other precipitation gradients. Next, I discuss changes in water‐use efficiency that take place during leaf expansion in C₃ plant leaves. Leaf phenology has recently been recognised as a significant driver of canopy gas exchange in evergreen forest canopies, and leaf expansion involves changes in both photosynthetic capacity and water‐use efficiency. Following this, I discuss the role of woody tissue respiration in canopy gas exchange and how photosynthetic refixation of respired CO₂ can increase whole‐plant water‐use efficiency. Finally, I discuss the role of water‐use efficiency in driving terrestrial plant responses to global change, especially the rising concentration of atmospheric CO₂. In coming decades, increases in plant water‐use efficiency caused by rising CO₂ are likely to partially mitigate impacts on plants of drought stress caused by global warming.     

Moderate water stress causes different stomatal and non‐stomatal changes in the photosynthetic functioning of Phaseolus vulgaris L. genotypes

The impact of moderate water deficit on the photosynthetic apparatus of three Phaseolus vulgaris L. cultivars, Plovdiv 10 (P10), Dobrudjanski Ran (DR) and Prelom (Prel), was investigated. Water shortage had less impact on leaf hydration, RWC (predawn and midday) and predawn water potential in Prel. RWC and Ψ_p were more reduced in P10, while there was no osmotic adjustment in any cultivar. Although drought drastically reduced stomatal opening in P10 and DR, reduced A_max indicated non‐stomatal limitations that contributed to the negligible P_n. These limitations were on potential thylakoid electron transport rates of PSI and II, pointing to photosystem functioning as a major limiting step in photosynthesis. This agrees with decreases in actual photochemical efficiency of PSII (F_v′/F_m′), quantum yield of photosynthetic non‐cyclic electron transport (?_e) and energy‐driven photochemical events (q_P), although the impact on these parameters would also include down‐regulation processes. When compared to DR, Prel retained a higher functional state of the photosynthetic machinery, justifying reduced need for photoprotective mechanisms (non‐photochemical quenching, zeaxanthin, lutein, β‐carotene) and maintenance of the balance between energy capture and dissipative pigments. The highest increases in fructose, glucose, arabinose and sorbitol in Prel might be related to tolerance to a lower oxidative state. All cultivars had reduced A_max due to daytime stomatal closure in well‐watered conditions. Under moderate drought, Prel had highest tolerance, higher leaf hydration and maintenance of important photochemical use of energy. However, water shortage caused appreciable non‐stomatal limitations to photosynthesis linked to regulation/imbalance at the metabolic level (and growth) in all cultivars. This included damage, as reflected in decreased potential photosystem functioning, pointing to higher sensitivity of photosynthesis to drought than is commonly assumed.     

Seasonal differences in photosynthesis between the C3 and C4 subspecies of Alloteropsis semialata are offset by frost and drought

The regional abundance of C₄ grasses is strongly controlled by temperature, however, the role of precipitation is less clear. Progress in elucidating the direct effects of photosynthetic pathway on these climate relationships is hindered by the significant genetic divergence between major C₃ and C₄ grass lineages. We addressed this problem by examining seasonal climate responses of photosynthesis in Alloteropsis semialata , a unique grass species with both C₃ and C₄ subspecies. Experimental manipulation of rainfall in a common garden in South Africa tested the hypotheses that: (1) photosynthesis is greater in the C₄ than C₃ subspecies under high summer temperatures, but this pattern is reversed at low winter temperatures; and (2) the photosynthetic advantage of C₄ plants is enhanced during drought events. Measurements of leaf gas exchange over 2 years showed a significant photosynthetic advantage for the C₄ subspecies under irrigated conditions from spring through autumn. However, the C₄ leaves were killed by winter frost, while photosynthesis continued in the C₃ plants. Unexpectedly, the C₄ subspecies also lost its photosynthetic advantage during natural drought events, despite greater water-use efficiency under irrigated conditions. This study highlights previously unrecognized roles for climatic extremes in determining the ecological success of C₃ and C₄ grasses.     

Lower responsiveness of canopy evapotranspiration rate than of leaf stomatal conductance to open‐air CO2 elevation in rice

An elevated atmospheric CO₂ concentration ([CO₂]) can reduce stomatal conductance of leaves for most plant species, including rice (Oryza sativa L.). However, few studies have quantified seasonal changes in the effects of elevated [CO₂] on canopy evapotranspiration, which integrates the response of stomatal conductance of individual leaves with other responses, such as leaf area expansion, changes in leaf surface temperature, and changes in developmental stages, in field conditions. We conducted a field experiment to measure seasonal changes in stomatal conductance of the uppermost leaves and in the evapotranspiration, transpiration, and evaporation rates using a lysimeter method. The study was conducted for flooded rice under open‐air CO₂ elevation. Stomatal conductance decreased by 27% under elevated [CO₂], averaged throughout the growing season, and evapotranspiration decreased by an average of 5% during the same period. The decrease in daily evapotranspiration caused by elevated [CO₂] was more significantly correlated with air temperature and leaf area index (LAI) rather than with other parameters of solar radiation, days after transplanting, vapor‐pressure deficit and FAO reference evapotranspiration. This indicates that higher air temperatures, within the range from 16 to 27 °C, and a larger LAI, within the range from 0 to 4 m² m^?2, can increase the magnitude of the decrease in evapotranspiration rate caused by elevated [CO₂]. The crop coefficient (i.e. the evapotranspiration rate divided by the FAO reference evapotranspiration rate) was 1.24 at ambient [CO₂] and 1.17 at elevated [CO₂]. This study provides the first direct measurement of the effects of elevated [CO₂] on rice canopy evapotranspiration under open‐air conditions using the lysimeter method, and the results will improve future predictions of water use in rice fields.