Jennifer R. Watling

Contribution of the Alternative Pathway to Respiration during Thermogenesis in Flowers of the Sacred Lotus

We report results from in vivo measurements, using oxygen isotope discrimination techniques, of fluxes through the alternative and cytochrome respiratory pathways in thermogenic plant tissue, the floral receptacle of the sacred lotus (Nelumbo nucifera). Fluxes through both pathways were measured in thermoregulating flowers undergoing varying degrees of thermogenesis in response to ambient temperature. Significant increases in alternative pathway flux were found in lotus receptacles with temperatures 16°C to 20°C above ambient, but not in those with lesser amounts of heating. Alternative pathway flux in the hottest receptacles was 75% of the total respiratory flux. In contrast, fluxes through the cytochrome pathway did not change significantly during thermogenesis. These data support the hypothesis that increased flux through the alternative pathway is responsible for heating in the lotus and that it is unlikely that uncoupling proteins, which would have produced increased fluxes through the cytochrome pathway, contribute significantly to heating in this tissue. Comparisons of actual flux, with capacity determined using inhibitors, suggested that the alternative pathway was operating at close to maximum capacity in heating tissues of lotus. However, in nonheating tissues the inhibitor data significantly overestimated the alternative pathway flux. This confirms that isotopic measurements are necessary for accurate determination of fluxes through the two pathways.

Synchronicity of thermogenic activity, alternative pathway respiratory flux, AOX protein content, and carbohydrates in receptacle tissues of sacred lotus during floral development

The relationships between heat production, alternative oxidase (AOX) pathway flux, AOX protein, and carbohydrates during floral development in Nelumbo nucifera (Gaertn.) were investigated. Three distinct physiological phases were identified: pre-thermogenic, thermogenic, and post-thermogenic. The shift to thermogenic activity was associated with a rapid, 10-fold increase in AOX protein. Similarly, a rapid decrease in AOX protein occurred post-thermogenesis. This synchronicity between AOX protein and thermogenic activity contrasts with other thermogenic plants where AOX protein increases some days prior to heating. AOX protein in thermogenic receptacles was significantly higher than in post-thermogenic and leaf tissues. Stable oxygen isotope measurements confirmed that the increased respiratory flux supporting thermogenesis was largely via the AOX, with little or no contribution from the cytochrome oxidase pathway. During the thermogenic phase, no significant relationship was found between AOX protein content and either heating or AOX flux, suggesting that regulation is likely to be post-translational. Further, no evidence of substrate limitation was found; starch accumulated during the early stages of floral development, peaking in thermogenic receptacles, before declining by 89% in post-thermogenic receptacles. Whilst coarse regulation of AOX flux occurs via protein synthesis, the ability to thermoregulate probably involves precise regulation of AOX protein, most probably by effectors such as alpha-keto acids.

Two Cys or Not Two Cys? That Is the Question; Alternative Oxidase in the Thermogenic Plant Sacred Lotus

Sacred lotus (Nelumbo nucifera) regulates temperature in its floral chamber to 32°C to 35°C across ambient temperatures of 8°C to 40°C with heating achieved through high alternative pathway fluxes. In most alternative oxidase (AOX) isoforms, two cysteine residues, Cys1 and Cys2, are highly conserved and play a role in posttranslational regulation of AOX. Further control occurs via interaction of reduced Cys1 with α-keto acids, such as pyruvate. Here, we report on the in vitro regulation of AOX isolated from thermogenic receptacle tissues of sacred lotus. AOX protein was mostly present in the reduced form, and only a small fraction could be oxidized with diamide. Cyanide-resistant respiration in isolated mitochondria was stimulated 4-fold by succinate but not pyruvate or glyoxylate. Insensitivity of the alternative pathway of respiration to pyruvate and the inability of AOX protein to be oxidized by diamide suggested that AOX in these tissues may lack Cys1. Subsequently, we isolated two novel cDNAs for AOX from thermogenic tissues of sacred lotus, designated as NnAOX1a and NnAOX1b. Deduced amino acid sequences of both confirmed that Cys1 had been replaced by serine; however, Cys2 was present. This contrasts with AOXs from thermogenic Aroids, which contain both Cys1 and Cys2. An additional cysteine was present at position 193 in NnAOX1b. The significance of the sequence data for regulation of the AOX protein in thermogenic sacred lotus is discussed and compared with AOXs from other thermogenic and nonthermogenic species.

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.

PHOTOINHIBITION AND PHOTOACCLIMATION OF TURF ALGAL COMMUNITIES ON A TEMPERATE REEF, AFTER IN SITU TRANSPLANTATION EXPERIMENTS1

The photophysiology of turf algal communities was studied in situ on a temperate reef off the coast of South Australia. Algal communities were grown on artificial substrate at depths of 2, 4, and 10 m. To investigate the response of the algal communities to changing light environments in both the short and long term, reciprocal transplantation experiments were conducted among these depths on a seasonal basis. The extent of photoinhibition was assessed every 3 h for the first 2 days following transplantation and then on a daily basis for 16 days after transplantation. Photosynthetic acclimation was assessed using photosynthesis–light curves obtained from transplanted and non‐transplanted turfs after the acclimation period. Transplanted turfs responded very quickly to the light shift. Algae acclimated to low light (10 m depth) were highly susceptible to photoinhibition and photodamage, having greater decreases in maximum and effective quantum yields than turfs from shallower depths. Yield recovery and acclimation usually occurred very rapidly in algae from all depths (3–5 days), but were faster in spring and summer compared with winter. Changes in photosynthetic capacity (across seasons, depths, and after transplantation to a different depth) were accompanied by changes in respiration, so that the ratio of net to gross photosynthetic capacity (Pmnet : Pmgross) remained high and constant over the whole range of light levels. We discuss the possible acclimation strategies of turfs, taking into account the balance between photoacclimation, production, and growth strategy.

The influence of the hemiparasitic angiosperm Cassytha pubescens on photosynthesis of its host Cytisus scoparius

Infection with Cassytha pubescens R.Br, an Australian native hemiparasitic plant, can lead to death of the invasive shrub, Cytisus scoparius L. Link (Scotch broom). We examined the influence of C. pubescens on photosynthetic physiology of C. scoparius to determine whether this might contribute to death of infected plants. Infected C. scoparius had significantly lower photosynthetic rates, stomatal conductance and transpiration, and higher Ci (internal [CO2]), than uninfected plants. Rapid light response curves, determined using chlorophyll fluorescence, indicated significantly lower light-saturated electron transport rates and lower quantum yields for infected plants relative to uninfected plants. However, Rubisco content did not differ between infected and uninfected plants, suggesting the lower photosynthetic rates were most likely due to stomatal closure, rather than lower photosynthetic capacity. As a consequence of lower assimilation rates, PSII efficiency was lower in infected plants than uninfected plants across the diurnal cycle. Infected plants also had significantly lower pre-dawn Fv/Fm values and slower recovery from exposure to high light than uninfected plants. Our results suggest that infected C. scoparius are more susceptible to photodamage than uninfected plants. Combined with lower carbon fixation rates, this could contribute to the poor performance and even death of infected plants.

Mechanisms of thermoregulation in plants

Endothermic heating of floral tissues and even thermoregulation is known to occur in a number of plant species across a wide taxonomic range. The mechanisms by which flowers heat, however, are only just beginning to be understood, and even less is known about how heating is regulated in response to changes in ambient temperature. We have recently demonstrated that the alternative pathway of respiration, in which the alternative oxidase (AOX) rather than cytochrome C (COX) acts as terminal electron acceptor, is responsible for heat generation in one thermoregulating species, the sacred lotus (Nelumbo nucifera). In the March issue of the Journal of Experimental Botany we further demonstrated that AOX-mediated heat production in this species is regulated at both the level of gene expression and also post-translationally. Similarly, AOX has also been implicated in heat production in other thermogenic species. In this addendum we discuss the central role of AOX in heat production and how post-translational mechanisms may provide the fine control necessary for thermoregulation. Addendum to: Grant NM, Miller RE, Watling JR, Robinson SA. Synchronicity of thermogenic activity, alternative pathway respiratory flux, AOX protein content, and carbohydrates in receptacle tissues of sacred lotus during floral development. J Exp Bot 2008; 59:705-14.

The potential for deep groundwater use by Acacia papyrocarpa (Western myall) in a water-limited environment

Emma K. Steggles, Kate L. Holland, David J. Chittleborough, Samantha L. Doudle. Laurence J. Clarke, Jennifer R. Watling, Jose M. Facelli

Temperature influences stomatal density and maximum potential water loss through stomata of Dodonaea viscosa subsp. angustissima along a latitude gradient in southern Australia

It is well known that physical leaf traits influence leaf functions, and that these traits vary across environmental gradients. Stomata can influence leaf function, with changes in density and size affecting potential water loss, CO2 uptake, and also leaf cooling. Plasticity in stomatal traits occurs in response to environmental factors; however, identifying which factors have the greatest influence is often difficult. We investigated variation in leaf size, stomatal density and size, and potential water loss from open stomata (gwmax), in the Australian native shrub Dodonaea viscosa subsp. angustissima, across a range of environmental factors including temperature, rainfall and CO2. We used herbarium specimens collected across a latitudinal gradient, and also sampled along an elevation gradient in southern Australia. There were significant relationships between mean summer maximum temperature and stomatal density, and gwmax. We found no significant relationships between rainfall or CO2 and the leaf traits we studied. Increased stomatal density at warmer locations may result in an increase in the potential for transpiration, as a means for evaporative cooling. Alternatively, it may enable increased CO2 and nutrient uptake during the short, winter-growing season.

Functional transition in the floral receptacle of the sacred lotus (Nelumbo nucifera): from thermogenesis to photosynthesis

The receptacle of the sacred lotus is the main source of heat during the thermogenic stage of floral development. Following anthesis, it enlarges, greens and becomes a fully functional photosynthetic organ. We investigated development of photosynthetic traits during this unusual functional transition. There were two distinct phases of pigment accumulation in receptacles. Lutein and photoprotective xanthophyll cycle pigments accumulated first with 64 and 95% of the maximum, respectively, present before anthesis. Lutein epoxide comprised 32% of total carotenoids in yellow receptacles, but declined with development. By contrast, more than 85% of maximum total chlorophyll, β-carotene and Rubisco were produced after anthesis, and were associated with significant increases in maximum electron transport rates (ETR) and photochemical efficiency (Fv/Fm). Leaves and mature receptacles had similar Rubisco content and ETRs (>200 μmol m-2 s-1), although total chlorophyll and total carotenoid contents of leaves were significantly higher than those of green receptacles. Receptacle δ13C before anthesis was similar to that of leaves; consistent with leaf photosynthesis being the source of C for these tissues. In contrast, mature receptacles had significantly lower δ13C than leaves, suggesting that 14-24% of C in mature receptacles is the result of refixation of respired CO2.