How does leaf succulence relate to plant drought resistance in woody shrubs?

Succulence describes the amount of water stored in cells or organs, regardless of plant life-form, including woody and herbaceous plants. In dry environments, plants with greater survival often have greater leaf succulence. However, it is unclear how leaf succulence relates to plant drought resistance strategies, including isohydry (closing stomata to maintain leaf water status) and anisohydry (adjusting cell turgor to tolerate low leaf water status), which exist on a continuum that can be quantified by hydroscape area (larger hydroscape area indicates more anisohydric). We evaluated 12 woody species with differing leaf succulence in a glasshouse dry-down experiment to determine relationships among leaf succulence (degree of leaf succulence, leaf succulent quotient and leaf thickness) and plant drought response (hydroscape area, plant water use, turgor loss point and predawn leaf water potential when transpiration ceased). Hydroscape areas ranged from 0.72 (Carpobrotus modestus S.T.Blake; crassulacean acid metabolism (CAM) plants) to 7.01 MPa2 (Rhagodia spinescens R.Br.; C3 plants), suggesting that C. modestus was more isohydric and R. spinescens was more anisohydric. More isohydric species C. modestus, Carpobrotus rossii (Haw.) Schwantes and Disphyma crassifolium (L.) L.Bolus (CAM plants) had greater leaf succulence, lower root allocation, used stored water and ceased transpiration at higher predawn leaf water potential, shortly after reaching their turgor loss point. The remaining nine species that are not CAM plants had larger hydroscape areas and ceased transpiration at lower predawn leaf water potential. Greater leaf succulence was not related to cumulative water loss until transpiration ceased in drying soils. All 12 species had high turgor loss points (-1.32 to -0.59 MPa), but turgor loss point was not related to hydroscape area or leaf succulence. Our data suggest that overall greater leaf succulence was related to isohydry, but this may have been influenced by the fact that these species were also CAM plants.

Diverse organ-specific localisation of a chemical defence, cyanogenic glycosides, in flowers of eleven species of Proteaceae.

Floral chemical defence strategies remain under-investigated, despite the significance of flowers to plant fitness. We used cyanogenic glycosides (CNglycs)-constitutive secondary metabolites that deter herbivores by releasing hydrogen cyanide, but also play other metabolic roles-to ask whether more apparent floral tissues and those most important for fitness are more defended as predicted by optimal defence theories, and what fine-scale CNglyc localisation reveals about function(s)? Florets of eleven species from the Proteaceae family were dissected to quantitatively compare the distribution of CNglycs within flowers and investigate whether distributions vary with other floral/plant traits. CNglycs were identified and their localisation in florets was revealed by matrix-assisted laser desorption ionisation mass spectrometry imaging (MALDI-MSI). We identified extremely high CNglyc content in floral tissues of several species (>1% CN), highly tissue-specific CNglyc distributions within florets, and substantial interspecific differences in content distributions, not all consistent with optimal defence hypotheses. Four patterns of within-flower CNglyc allocation were identified: greater tissue-specific allocations to (1) anthers, (2) pedicel (and gynophore), (3) pollen presenter, and (4) a more even distribution among tissues with higher content in pistils. Allocation patterns were not correlated with other floral traits (e.g. colour) or taxonomic relatedness. MALDI-MSI identified differential localisation of two tyrosine-derived CNglycs, demonstrating the importance of visualising metabolite localisation, with the diglycoside proteacin in vascular tissues, and monoglycoside dhurrin across floral tissues. High CNglyc content, and diverse, specific within-flower localisations indicate allocations are adaptive, highlighting the importance of further research into the ecological and metabolic roles of floral CNglycs.

Divergence in floral scent and morphology, but not thermogenic traits, associated with pollinator shift in two brood-site-mimicking Typhonium (Araceae) species.

BACKGROUND: Flowers which imitate insect oviposition sites probably represent the most widespread form of floral mimicry, exhibit the most diverse floral signals and are visited by two of the most speciose and advanced taxa of insect - beetles and flies. Detailed comparative studies on brood-site mimics pollinated exclusively by each of these insect orders are lacking, limiting our understanding of floral trait adaptation to different pollinator groups in these deceptive systems. METHODS: Two closely related and apparent brood-site mimics, Typhonium angustilobum and T. wilbertii (Araceae) observed to trap these distinct beetle and fly pollinator groups were used to investigate potential divergence in floral signals and traits most likely to occur under pollinator-mediated selection. Trapped pollinators were identified and their relative abundances enumerated, and thermogenic, visual and chemical signals and morphological traits were examined using thermocouples and quantitative reverse transcription-PCR, reflectance, gas chromatography-mass spectrometry, floral measurements and microscopy. KEY RESULTS: Typhonium angustilobum and T. wilbertii were functionally specialized to trap saprophagous Coleoptera and Diptera, respectively. Both species shared similar colour and thermogenic traits, and contained two highly homologous AOX genes (AOX1a and AOX1b) most expressed in the thermogenic tissue and stage (unlike pUCP). Scent during the pistillate stage differed markedly - T. angustilobum emitted a complex blend of sesquiterpenes, and T. wilbertii, a dung mimic, emitted high relative amounts of skatole, p-cresol and irregular terpenes. The species differed significantly in floral morphology related to trapping mechanisms. CONCLUSIONS: Functional specialization and pollinator divergence were not associated with differences in anthesis rhythm and floral thermogenic or visual signals between species, but with significant differences in floral scent and morphological features, suggesting that these floral traits are critical for the attraction and filtering of beetle or fly pollinators in these two brood-site mimics.

Review of the Basics of Cognitive Error in Emergency Medicine: Still No Easy Answers.

Emergency physicians (EP) make clinical decisions multiple times daily. In some instances, medical errors occur due to flaws in the complex process of clinical reasoning and decision-making. Cognitive error can be difficult to identify and is equally difficult to prevent. To reduce the risk of patient harm resulting from errors in critical thinking, it has been proposed that we train physicians to understand and maintain awareness of their thought process, to identify error-prone clinical situations, to recognize predictable vulnerabilities in thinking, and to employ strategies to avert cognitive errors. The first step to this approach is to gain an understanding of how physicians make decisions and what conditions may predispose to faulty decision-making. We review the dual-process theory, which offers a framework to understand both intuitive and analytical reasoning, and to identify the necessary conditions to support optimal cognitive processing. We also discuss systematic deviations from normative reasoning known as cognitive biases, which were first described in cognitive psychology and have been identified as a contributing factor to errors in medicine. Training physicians in common biases and strategies to mitigate their effect is known as debiasing. A variety of debiasing techniques have been proposed for use by clinicians. We sought to review the current evidence supporting the effectiveness of these strategies in the clinical setting. This discussion of improving clinical reasoning is relevant to medical educators as well as practicing EPs engaged in continuing medical education.

Self-Directed Learning among Internal Medicine Residents in the Information Age.

OBJECTIVES: The revolution in information technology and a rapidly expanding evidence base are changing residency training. Understanding the habits and preferences of trainees' self-directed learning (SDL) has never been more important. Our goal was to provide a contemporary description of residents' SDL practices. METHODS: Internal medicine residents at four university-affiliated programs were surveyed in Spring 2017. Residents estimated the number of hours in their typical week spent in SDL on service and after hours when on inpatient and noninpatient rotations, how often they used specific educational resources for SDL, and the percentage of time that they used four different modes to access resources. RESULTS: Of 384 residents, a total of 254 (66%) responded. Residents spent more total hours in SDL on noninpatient services (median 11, interquartile range 8-17) than on inpatient services (median 7, interquartile range 4-10) and the same median number of hours in SDL on clinical duty as off hours for both inpatient (median 3 hours) and noninpatient (median 5 hours) rotations. Nearly all of the respondents (99%) reported using online point-of-care resources for SDL at least once per week. Most (77%) never used printed textbooks. Desktop/laptop was the most commonly used (47% of the time) medium to access resources. CONCLUSIONS: Although the resident learning environment and resource use are changing, residents engage in as much or more time in SDL as in previous studies, with a large proportion occurring during clinical service. Understanding residents' current SDL habits will better prepare educators to support and guide our trainees.

Unique and highly specific cyanogenic glycoside localization in stigmatic cells and pollen in the genus Lomatia (Proteaceae).

BACKGROUND AND AIMS: Floral chemical defence strategies remain understudied despite the significance of flowers to plant fitness, and the fact that many flowers contain secondary metabolites that confer resistance to herbivores. Optimal defence and apparency theories predict that the most apparent plant parts and/or those most important to fitness should be most defended. To test whether within-flower distributions of chemical defence are consistent with these theories we used cyanogenic glycosides (CNglycs), which are constitutive defence metabolites that deter herbivores by releasing hydrogen cyanide upon hydrolysis. METHODS: We used cyanogenic florets of the genus Lomatia to investigate at what scale there may be strategic allocation of CNglycs in flowers, what their localization reveals about function, and whether levels of floral CNglycs differ between eight congeneric species across a climatic gradient. Within-flower distributions of CNglycs during development were quantified, CNglycs were identified and their localization was visualized in cryosectioned florets using matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). KEY RESULTS: Florets of all congeneric species studied were cyanogenic, and concentrations differed between species. Within florets there was substantial variation in CNglyc concentrations, with extremely high concentrations (up to 14.6 mg CN g-1 d. wt) in pollen and loose, specialized surface cells on the pollen presenter, among the highest concentrations reported in plant tissues. Two tyrosine-derived CNglycs, the monoglycoside dhurrin and diglycoside proteacin, were identified. MALDI-MSI revealed their varying ratios in different floral tissues; proteacin was primarily localized to anthers and ovules, and dhurrin to specialized cells on the pollen presenter. The mix of transient specialized cells and pollen of L. fraxinifolia was ~11 % dhurrin and ~1.1 % proteacin by mass. CONCLUSIONS: Tissue-specific distributions of two CNglycs and substantial variation in their concentrations within florets suggests their allocation is under strong selection. Localized, high CNglyc concentrations in transient cells challenge the predictions of defence theories, and highlight the importance of fine-scale metabolite visualization, and the need for further investigation into the ecological and metabolic roles of CNglycs in floral tissues.

Divergent responses of above- and below-ground chemical defence to nitrogen and phosphorus supply in waratahs (Telopea speciosissima).

Plant nutrition can affect the allocation of resources to plant chemical defences, yet little is known about how phosphorus (P) supply, and relative nitrogen (N) and P supply, affect chemical defences, especially in species with intrinsically conservative nutrient use adapted to P-impoverished soils. Waratah (Telopea speciosissima (Sm.) R.Br.), like other Proteaceae, is adapted nutrient-poor soils. It was identified as having cyanogenic glycosides (CNglycs) throughout the plant. T. speciosissima seedlings were grown for 15 weeks under two N and P concentrations. CNglycs (N-based defence) and nutrients were quantified in above- and below-ground organs; foliar carbon (C)-based phenolics and tannins were also quantified. CNglyc concentrations in roots were on average 51-fold higher than in above-ground tissues and were affected by both N and P supply, whereas foliar CNglyc concentrations only responded to N supply. Leaves had high concentrations of C-based defences, which increased under low N, and were not correlated with N-based defences. Greater root chemical defence against herbivores and pathogens may be important in a non-mycorrhizal species that relies on basal resprouting following disturbance. The differing responses of secondary chemistry in above- and below-ground organs to P and N demonstrate the importance of broadening the predominantly foliar focus of plant defence studies.

Photoprotection enhanced by red cell wall pigments in three East Antarctic mosses.

BACKGROUND: Antarctic bryophytes (mosses and liverworts) are resilient to physiologically extreme environmental conditions including elevated levels of ultraviolet (UV) radiation due to depletion of stratospheric ozone. Many Antarctic bryophytes synthesise UV-B-absorbing compounds (UVAC) that are localised in their cells and cell walls, a location that is rarely investigated for UVAC in plants. This study compares the concentrations and localisation of intracellular and cell wall UVAC in Antarctic Ceratodon purpureus, Bryum pseudotriquetrum and Schistidium antarctici from the Windmill Islands, East Antarctica. RESULTS: Multiple stresses, including desiccation and naturally high UV and visible light, seemed to enhance the incorporation of total UVAC including red pigments in the cell walls of all three Antarctic species analysed. The red growth form of C. purpureus had significantly higher levels of cell wall bound and lower intracellular UVAC concentrations than its nearby green form. Microscopic and spectroscopic analyses showed that the red colouration in this species was associated with the cell wall and that these red cell walls contained less pectin and phenolic esters than the green form. All three moss species showed a natural increase in cell wall UVAC content during the growing season and a decline in these compounds in new tissue grown under less stressful conditions in the laboratory. CONCLUSIONS: UVAC and red pigments are tightly bound to the cell wall and likely have a long-term protective role in Antarctic bryophytes. Although the identity of these red pigments remains unknown, our study demonstrates the importance of investigating cell wall UVAC in plants and contributes to our current understanding of UV-protective strategies employed by particular Antarctic bryophytes. Studies such as these provide clues to how these plants survive in such extreme habitats and are helpful in predicting future survival of the species studied.

Trees use more non-structural carbohydrate reserves during epicormic than basal resprouting.

Non-structural carbohydrates (NSCs) are crucial to support tree resprouting after disturbances that damage the crown or stem. Epicormic resprouting (from stem) could demand more from NSC reserves than basal resprouting (following top-kill), since epicormically resprouting trees need to maintain a greater living biomass. Yet, little is known about NSC use during epicormic resprouting, particularly the relative importance of stem and below-ground NSC reserves. We compared the distribution and magnitude of NSC decreases during epicormic and basal resprouting by experimentally removing crowns or stems of 14-year-old Eucalyptus obliqua L'Hér. trees in native forest, then harvesting these trees over a 10-month period (start, sprouts emerged, sprouts expanded) to measure changes in NSC concentration and mass by organ (stem, lignotuber, roots). We hypothesized that (i) NSC depletion during resprouting is primarily due to decreases in starch rather than soluble sugars concentrations; (ii) during epicormic resprouting, stem NSC concentrations are decreased irrespective of any decreases in roots; and (iii) absolute decreases in NSC mass are greater for epicormic than basal resprouting during the leafless period due to the carbon demands associated with maintaining greater living biomass. Results confirmed our hypotheses; starch was the primary storage carbohydrate, stems were an important source of starch during epicormic resprouting and carbon demands of maintenance functions were greater for epicormic resprouting, leading to greater decreases in NSC reserves. Roots were a more important starch storage organ than the lignotuber for both epicormic and basal resprouting. The proportional decrease in starch was severe for both modes of resprouting due to a long leafless period, after which trees resprouting epicormically relied on starch reserves for longer than those resprouting basally. It remains to be seen how the timing of disturbance affects the timing and vigour of resprouting, and how long-term NSC recovery differs for epicormic and basal resprouting.

Whole-tree distribution and temporal variation of non-structural carbohydrates in broadleaf evergreen trees.

Non-structural carbohydrates (NSCs) form a fundamental yet poorly quantified carbon pool in trees. Studies of NSC seasonality in forest trees have seldom measured whole-tree NSC stocks and allocation among organs, and are not representative of all tree functional types. Non-structural carbohydrate research has primarily focussed on broadleaf deciduous and coniferous evergreen trees with distinct growing seasons, while broadleaf evergreen trees remain under-studied despite their different growth phenology. We measured whole-tree NSC allocation and temporal variation in Eucalyptus obliqua L'Hér., a broadleaf evergreen tree species typically occurring in mixed-age temperate forests, which has year-round growth and the capacity to resprout after fire. Our overarching objective was to improve the empirical basis for understanding the functional importance of NSC allocation and stock changes at the tree- and organ-level in this tree functional type. Starch was the principal storage carbohydrate and was primarily stored in the stem and roots of young (14-year-old) trees rather than the lignotuber, which did not appear to be a specialized starch storage organ. Whole-tree NSC stocks were depleted during spring and summer due to significant decreases in starch mass in the roots and stem, seemingly to support root and crown growth but potentially exacerbated by water stress in summer. Seasonality of stem NSCs differed between young and mature trees, and was not synchronized with stem basal area increments in mature trees. Our results suggest that the relative magnitude of seasonal NSC stock changes could vary with tree growth stage, and that the main drivers of NSC fluctuations in broadleaf evergreen trees in temperate biomes could be periodic disturbances such as summer drought and fire, rather than growth phenology. These results have implications for understanding post-fire tree recovery via resprouting, and for incorporating NSC pools into carbon models of mixed-age forests.

Photoprotection enhanced by red cell wall pigments in three East Antarctic mosses

BACKGROUND: Antarctic bryophytes (mosses and liverworts) are resilient to physiologically extreme environmental conditions including elevated levels of ultraviolet (UV) radiation due to depletion of stratospheric ozone. Many Antarctic bryophytes synthesise UV-B-absorbing compounds (UVAC) that are localised in their cells and cell walls, a location that is rarely investigated for UVAC in plants. This study compares the concentrations and localisation of intracellular and cell wall UVAC in Antarctic Ceratodon purpureus, Bryum pseudotriquetrum and Schistidium antarctici from the Windmill Islands, East Antarctica. RESULTS: Multiple stresses, including desiccation and naturally high UV and visible light, seemed to enhance the incorporation of total UVAC including red pigments in the cell walls of all three Antarctic species analysed. The red growth form of C. purpureus had significantly higher levels of cell wall bound and lower intracellular UVAC concentrations than its nearby green form. Microscopic and spectroscopic analyses showed that the red colouration in this species was associated with the cell wall and that these red cell walls contained less pectin and phenolic esters than the green form. All three moss species showed a natural increase in cell wall UVAC content during the growing season and a decline in these compounds in new tissue grown under less stressful conditions in the laboratory. CONCLUSIONS: UVAC and red pigments are tightly bound to the cell wall and likely have a long-term protective role in Antarctic bryophytes. Although the identity of these red pigments remains unknown, our study demonstrates the importance of investigating cell wall UVAC in plants and contributes to our current understanding of UV-protective strategies employed by particular Antarctic bryophytes. Studies such as these provide clues to how these plants survive in such extreme habitats and are helpful in predicting future survival of the species studied.

Interactive effects of temperature and drought on cassava growth and toxicity: implications for food security?

Cassava is an important dietary component for over 1 billion people, and its ability to yield under drought has led to it being promoted as an important crop for food security under climate change. Despite its known photosynthetic plasticity in response to temperature, little is known about how temperature affects plant toxicity or about interactions between temperature and drought, which is important because cassava tissues contain high levels of toxic cyanogenic glucosides, a major health and food safety concern. In a controlled glasshouse experiment, plants were grown at 2 daytime temperatures (23 °C and 34 °C), and either well-watered or subject to a 1 month drought prior to harvest at 6 months. The objective was to determine the separate and interactive effects of temperature and drought on growth and toxicity. Both temperature and drought affected cassava physiology and chemistry. While temperature alone drove differences in plant height and above-ground biomass, drought and temperature × drought interactions most affected tuber yield, as well as foliar and tuber chemistry, including C : N, nitrogen and cyanide potential (CNp; total cyanide released from cyanogenic glucosides). Conditions that most stimulated growth and yield (well-watered × high temperature) effected a reduction in tuber toxicity, whereas drought inhibited growth and yield, and was associated with increased foliar and tuber toxicity. The magnitude of drought effects on tuber yield and toxicity were greater at high temperature; thus, increases in tuber CNp were not merely a consequence of reduced tuber biomass. Findings confirm that cassava is adaptable to forecast temperature increases, particularly in areas of adequate or increasing rainfall; however, in regions forecast for increased incidence of drought, the effects of drought on both food quality (tuber toxicity) and yield are a greater threat to future food security and indicate an increasing necessity for processing of cassava to reduce toxicity.

Age versus stage: does ontogeny modify the effect of phosphorus and arbuscular mycorrhizas on above- and below-ground defence in forage sorghum?

Arbuscular mycorrhizas (AM) can increase plant acquisition of P and N. No published studies have investigated the impact of P and AM on the allocation of N to the plant defence, cyanogenic glucosides. We investigated the effects of soil P and AM on cyanogenic glucoside (dhurrin) concentration in roots and shoots of two forage sorghum lines differing in cyanogenic potential (HCNp). Two harvest times allowed plants grown at high and low P to be compared at the same age and the same size, to take account of known ontogenetic changes in shoot HCNp. P responses were dependent on ontogeny and tissue type. At the same age, P-limited plants were smaller and had higher shoot HCNp but lower root HCNp. Ontogenetically controlled comparisons showed a P effect of lesser magnitude, and that there was also an increase in the allocation of N to dhurrin in shoots of P-limited plants. Colonization by AM had little effect on shoot HCNp, but increased root HCNp and the allocation of N to dhurrin in roots. Divergent responses of roots and shoots to P, AM and with ontogeny demonstrate the importance of broadening the predominantly foliar focus of plant defence studies/theory, and of ontogenetically controlled comparisons.

Reports on the distribution of aromatic cyanogenic glycosides in Australian tropical rainforest tree species of the Lauraceae and Sapindaceae.

The aromatic cyanogenic glycosides taxiphyllin [(R)-4-hydroxymandelonitrile ß-D-glucoside] and prunasin [(R)-mandelonitrile ß-D-glucoside] were identified as the main cyanogenic compounds in tissues of Australian endemic tropical rainforest tree taxa in the Lauraceae and Sapindaceae families, respectively. The tyrosine-derived taxiphyllin was the main cyanogenic glycoside in foliage of Beilschmiedia collina. This is the first reported cyanogenic compound from the Lauraceae. In addition, substantial quantitative variation in the capacity for cyanogenesis was detected in leaves from 40 individuals, with taxiphyllin concentrations ranging from 23 to 1263 µg CN g(-1) dry wt. No acyanogenic individuals were detected. Concentrations of taxiphyllin were, on average, 2.2-fold greater in young leaves than in old leaves. Prunasin was the dominant cyanogenic compound in tissues of Mischocarpus grandissimus (leaves) and Mischocarpus exangulatus (leaves and seed capsule). Better known for cyanolipids in seed oils, this is the first time a phenylalanine-derived cyanogenic glycoside has been reported in the Sapindaceae. The concentrations of prunasin varied widely, over an order of magnitude, among individuals and different tissue types in these species, with the higher concentrations found in seed capsules and young leaves.

The rare cyanogen proteacin, and dhurrin, from foliage of Polyscias australiana, a tropical Araliaceae.

The tyrosine-derived cyanogenic di-glucoside proteacin and related mono-glucoside dhurrin were identified as the cyanogens in foliage of the tropical tree species Polyscias australiana, present in the approximate ratio 9:1. To date cyanogenic glycosides have not been characterised from the Araliaceae or the Apiales. Concentrations of cyanogenic glycosides varied significantly between plant parts and with leaf age, with the highest concentrations in young emerging leaves (mean 2217.1 µg CN g(-1) dry wt), petioles (rachis; 1487.1 µg CN g(-1) dry wt) and floral buds (265.8 µg CN g(-1) dry wt). Between 2% and 10% of nitrogen in emerging leaves and petioles was present as cyanogenic glycosides. With the exception of floral buds, all tissues apparently lack a specific cyanogenic ß-glucosidase to catalyse the first step in the breakdown of these cyanogenic glycosides. Only with the addition of emulsin, an exogenous non-specific ß-glucosidase from almonds, were high concentrations of cyanogenic glycosides detected, as much as 20-fold greater than the low to negligible cyanogenic glycoside concentrations determined in the absence of exogenous enzyme. High concentrations of cyanogens in young tissues may confer protection, but may also be a nitrogen source during leaf expansion.

Drought adversely affects tuber development and nutritional quality of the staple crop cassava (Manihot esculenta Crantz).

Cassava (Manihot esculenta Crantz) is the staple food source for over 850million people worldwide. Cassava contains cyanogenic glucosides and can be toxic to humans, causing paralysing diseases such as konzo, and even death if not properly processed. Konzo epidemics are often associated with times of drought. This may be due to a greater reliance on cassava as it is drought tolerant, but it may also be due to an increase in cyanogenic glucosides. Episodic droughts are forecast to become more common in many cassava-growing regions. We therefore sought to quantify the effect of water-stress on both yield and cyanogenic glucoside concentration (CNc) in the developing tubers of cassava. Five-month-old plants were grown in a glasshouse and either well watered or droughted for 28 days. A subset of droughted plants was re-watered half way through the experiment. Droughted plants had 45% fewer leaves and lower tuber yield, by 83%, compared with well-watered plants. CNc was 2.9-fold higher in the young leaves of droughted plants, whereas CNc in tubers from droughted plants was 4-fold greater than in tubers from well-watered plants. Re-watered plants had a similar biomass to control plants, and lower CNc than droughted plants. These findings highlight the important link between food quality and episodic drought.

In the heat of the night--alternative pathway respiration drives thermogenesis in Philodendron bipinnatifidum.

• Philodendron bipinnatifidum inflorescences heat up to 42 °C and thermoregulate. We investigated whether they generate heat via the cytochrome oxidase pathway uncoupled by uncoupling proteins (pUCPs), or the alternative oxidase (AOX). • Contribution of AOX and pUCPs to heating in fertile (FM) and sterile (SM) male florets was determined using a combination of oxygen isotope discrimination, protein and substrate analyses. • Both FM and SM florets thermoregulated independently for up to 30 h ex planta. In both floret types, AOX contributed > 90% of respiratory flux during peak heating. The AOX protein increased fivefold with the onset of thermogenesis in both floret types, whereas pUCP remained low throughout development. These data indicate that AOX is primarily responsible for heating, despite FM and SM florets potentially using different substrates, carbohydrates or lipids, respectively. Measurements of discrimination between O2 isotopes in strongly respiring SM florets were affected by diffusion; however, this diffusional limitation was largely overcome using elevated O2. • The first in vivo respiratory flux measurements in an arum show AOX contributes the bulk of heating in P. bipinnatifidum. Fine-scale regulation of AOX activity is post-translational. We also demonstrate that elevated O2 can aid measurement of respiratory pathway fluxes in dense tissues.