Biochemical and physiological mechanisms of legume nitrogen fixation under higher atmospheric CO2 concentrations

© 2019 Dr. Shahnaj ParvinAtmospheric CO2 concentration ([CO2]) is expected to rise from a current level of ~400 to 550 µmol mol-1 by 2050. It is well established that elevated [CO2] enhances plant growth and yield. However, the stimulation of plant growth at elevated [CO2] requires additional nitrogen (N) and prolonged exposure to elevated [CO2] potentially risks N limitation. Legumes can overcome such limitations by fixing aerial N. Previous studies under Free Air CO2 Enrichment (FACE) have shown that elevated [CO2] can stimulate N2 fixation, but it is unknown to what extent this applies to dryland Mediterranean environments or what impact environmental interactions have. Legumes grown in dryland environments frequently experience terminal drought accompanied by high temperature during reproductive phases. It has been suggested that elevated [CO2] delays the effect of drought by conserving soil water, maintaining N2 fixation mechanisms for longer under drought. This thesis addresses this gap by investigating the growth and N economy of three legumes (lentil, field pea and faba bean) in a FACE facility in a semi-arid environment where seasonal and experimentally controlled drought was imposed. In addition to N2 fixation itself, the supply and translocation of N compounds to the maturing grain is another point of interest, because it is crucial in maintaining grain N concentration. This thesis investigated N2 fixation, remobilization and grain quality of dryland legumes under predicted future e[CO2] atmosphere conditions, including interactions with drought, heat waves, and genotypes. Free Air CO2 Enrichment technology was used to simulate future growing conditions in the field with target [CO2] as expected by the middle of this century. Elevated [CO2] stimulated N2 fixation through increased nodule number, nodule biomass, and nodule activity to a greater extent under unstressed conditions. Soil water savings under elevated [CO2] were only temporary, so that drought reduced nodule activity due to lower C/sucrose supply and therefore decreased N2 fixation. Consequently, elevated [CO2] was found to stimulate N2 fixation of all three species of legumes, but this effect was smaller under drought or heat stress. The decrease of N2 fixation under drought caused depletion of grain N concentration under elevated [CO2]. In contrast, when soil water was sufficient, N2 fixation continued throughout the grain filling period, and grain N concentration was maintained under elevated [CO2]. Traits that allow N2 fixation for longer throughout the growing season, e. g. by exploiting potential water savings mechanisms under elevated [CO2], may confer benefits under future climatic conditions. Findings of this study are now available to underpin new strategies for improvement of the N2 fixation potential of legumes as atmospheric [CO2] continues to increase in the future

Potential Nitrogen Contributions by Tropical Legume Summer Cover Crops in Mediterranean-Type Cropping Systems

Legume cover crops in temperate cropping systems can fix substantial amounts of nitrogen (N) and reduce N fertiliser requirements for subsequent crops. However, little is known about potential biological N2 fixation by summer cover crop legumes in the short summer fallow in Mediterranean-type cropping systems. Six legume species (balansa clover, barrel medic, mung bean, sunn hemp, lablab and cowpea) were grown for 8–9 weeks in the field in semi-arid southern Australia during the summer fallow, and in a glasshouse experiment, to estimate N2 fixation using the 15N natural abundance method. Cowpea, sunn hemp and lablab produced 1.2–3.0 t ha−1 biomass in the field while balansa clover and barrel medic produced < 1.0 t ha−1. The percent of N derived from the atmosphere (%Ndfa) in the field ranged from 39% in barrel medic to 73% in sunn hemp, but only 15% (balansa clover) to 33% (sunn hemp) in the glasshouse experiment, likely due to higher soil mineral N availability in the glasshouse study. Biological N2 fixation of cowpea and sunn hemp in the field was 46–55 kg N ha−1, while N2 fixation in lablab and mung bean was lower (around 26 kg N ha−1). The N2 fixation in cowpea and sunn hemp of around 50 kg N ha−1 with supplementary irrigation in the field trial likely represents the upper limit of N contributions in the field in typically hot, dry summer conditions in Mediterranean-type climates. Given that any increase in summer cover crop biomass will have implications for water balances and subsequent cash crop growth, maximising N benefits of legume cover crops will rely on increasing the %Ndfa through improved rhizobium strains or inoculation technologies. This study provides the first known estimates of biological N2 fixation by legume cover crops in the summer fallow period in cropping systems in Mediterranean-type environments, providing a benchmark for further studies

Carbon sink strength of nodules but not other organs modulates photosynthesis of faba bean (Vicia faba) grown under elevated [CO2 ] and different water supply

Tausz, M ORCiD: 0000-0001-8205-8561; Tausz-Posch, S ORCiD: 0000-0002-1213-7907Photosynthetic stimulation by elevated [CO2 ] (e[CO2 ]) may be limited by the capacity of sink organs to use photosynthates. In many legumes, N2 -fixing symbionts in root nodules provide an additional sink, so that legumes may be better able to profit from e[CO2 ]. However, drought not only constrains photosynthesis but also size and activity of sinks, and little is known about the interaction of e[CO2 ] and drought on carbon sink strength of nodules and other organs. To compare carbon sink strength, faba bean was grown under ambient (400 ppm) or elevated (700 ppm) atmospheric [CO2 ] and subjected to well-watered or drought treatments, and then exposed to 13 C pulse-labelling using custom-built chambers to track the fate of new photosynthates. Drought decreased 13 C uptake and nodule sink strength, and this effect was even greater under e[CO2 ], and associated with an accumulation of amino acids in nodules. This resulted in decreased N2 fixation, increased accumulation of new photosynthates (13 C/sugars) in leaves, which in turn can feed back on photosynthesis. Our study suggests that nodule C sink activity is key to avoid sink limitation in legumes under e[CO2 ], and legumes may only be able to achieve greater C gain if nodule activity is maintained

Elevated CO2 improves yield and N2 fixation but not grain N concentration of faba bean (Vicia faba L.) subjected to terminal drought

Tausz, M ORCiD: 0000-0001-8205-8561Legumes grown in Mediterranean environments frequently experience terminal drought which reduces yield and N2 fixation processes. Decreased N2 fixation during reproductive phases may constrain seed nitrogen concentrations ([N]), reducing protein concentration of grain legumes. Plants grown under elevated atmospheric CO2 concentrations ([CO2]) have greater water use efficiency. This may result in reduced use of conserved/stored soil water, potentially helping to reduce soil water deficits later during grain filling. The extent that this process applies to drought sensitive grain legumes, which are extensively cultivated in Mediterranean environments is unclear. The objectives of this study were to investigate yield, N2 fixation and seed N response of faba bean (Vicia faba L. cv. ‘Fiesta’) grown in a dryland Mediterranean-type environment under elevated [CO2]. Plants were grown in soil columns under ambient [CO2] (˜400 ppm) or elevated [CO2] (e[CO2], ˜550 ppm) in a Free-Air CO2 Enrichment (FACE) facility in the field. One sub-group was continuously well-watered (80% field capacity, FC), whereas a second sub-group was exposed to a drought treatment (water was withheld until 30% FC was reached, which was then maintained during the reproductive phases). Biomass, gas exchange, 13C isotopic discrimination, N2 fixation by the natural abundance 15N method, nodulation and soil water content were assessed throughout the crop developmental stages. Initially, plants grown under elevated [CO2] depleted soil water more slowly in the drought treatment than those under ambient [CO2], but as plants grown under elevated [CO2] produced more biomass they used soil water more rapidly, especially towards the critical pod-filling phase. Water savings during the first phase of the drought treatment, through flowering up to the start of pod-filling, were associated with increased yield (+25%) and N2 fixation (+15%) under drought. Elevated [CO2]-induced stimulation of nodulation and nodule density helped maintain N2 fixation under drought, even though nodule activity decreased under the combined effect of e[CO2] and drought from pod-filling onwards. This later stage decrease was associated with decreased carbohydrate and increased amino acid concentrations in nodules, indicating a down-regulation of N2 fixation. Associated with the decrease of N2 fixation during pod-filling, seed N concentration was lower under the combination of e[CO2] and drought. We propose a conceptual model to explain the importance of N2 fixation during the grain filling stage to maintain seed N concentration under e[CO2]. These findings suggest that e[CO2]-induced savings in soil water may mitigate negative effects of drought on yield and N2 fixation of faba bean, without fully compensating the effect of prolonged drought on seed N concentration. © 2019 Elsevier B.V

Analgesic, antibacterial and central nervous system depressant activities of Albizia procera leaves

Objective: To ascertain analgesic, antibacterial and central nervous system (CNS) depressant activities of ethyl acetate, dichloromethane and carbon tetrachloride fractions of methanol extract of Albizia procera (A. procera) leaves. Methods: Leaves extracts of A. procera were tested for analgesic activity by acetic acid induced and formalin test method in mice. The in vitro antibacterial activity was performed by agar well diffusion method. CNS depressant activity was evaluated by hole cross and open field tests. Results: All the extracts at 200 mg/kg exhibited significant (P<0.01) analgesic activity in acetic acid induced and formalin tests method in mice. Analgesic activity of the ethyl acetate fraction was almost same like as standard drug indomethacin in acetic acid induced method. The CNS depressant activity of the extracts at 500 mg/kg was comparable to the positive control diazepam as determined by hole cross and open field test method. The extracts exhibited moderate antimicrobial activity against all the tested microorganisms (Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Esherichia coli, Shigella soneii, Shigella boydii) at concentration of 0.8 mg/disc. The measured diameter of zone of inhibition for the extracts was within the range of 7 to 12 mm which was less than the standard kanamycin (16-24 mm). Conclusions: It is concluded that all the extracts possess potential analgesic and CNS depressants activity. This study also showed that different fractions of methanol extract could be potential sources of new antimicrobial agents

Grain mineral quality of dryland legumes as affected by elevated CO2 and drought: A FACE study on lentil (Lens culinaris) and faba bean (Vicia faba)

Tausz, M ORCiD: 0000-0001-8205-8561Stimulation of grain yield under elevated [CO] grown plants is often associated with the deterioration of grain quality. This effect may be further complicated by the frequent occurrence of drought, as predicted in most of the climate change scenarios. Lentil (Lens culinaris Medik.) and faba bean (Vicia faba L.) were grown in the Australian Grains Free Air CO Enrichment facility under either ambient CO concentration ([CO], ∼400 mol mol-1) or elevated [CO] (e[CO], ∼550 mol mol-1), and with two contrasting watering regimes (for faba bean) or over two consecutive seasons contrasting in rainfall (for lentil), to investigate the interactive effect of e[CO] and drought on concentrations of selected grain minerals (Fe, Zn, Ca, Mg, P, K, S, Cu, Mn, Na). Grain mineral concentration (Fe, Zn, Ca, K, S, Cu) increased and grain mineral yield (i.e. g mineral per plot surface area) decreased in dry growing environments, and vice versa in wet growing environments. Elevated [CO] decreased Fe, Zn, P and S concentrations in both crops however, the relative decrease was greater under dry (20-25) than wet (4-10) growing conditions. Principal component analysis showed that greater grain yield stimulation under e[CO] was associated with a reduction in Fe and Zn concentrations, indicating a yield dilution effect, but this was not consistently observed for other minerals. Even if energy intake is kept constant to adjust for lower yields, decreased legume micronutrients densities under e[CO] may have negative consequences for human nutrition, especially under drier conditions and in areas with less access to food. © 2019 CSIRO

Free air CO2 enrichment (FACE) improves water use efficiency and moderates drought effect on N 2 fixation of Pisum sativum L.

Tausz, M ORCiD: 0000-0001-8205-8561Background and aims: Legume N 2 fixation is highly sensitive to drought. Elevated [CO 2 ] (e[CO 2 ]) decreases stomatal conductance (g s ) and improves water use efficiency (WUE), which may result in soil water conservation and allow N 2 fixation to continue longer under drought. Using a Free-Air CO 2 Enrichment (FACE) approach, this study aimed to elucidate whether e[CO 2 ] improves N 2 fixation of Pisum sativum L. under drought. Methods: In a FACE system, plants were grown in ambient [CO 2 ] (~400 ppm) or e[CO 2 ] (~550 ppm) and subjected to either terminal drought or well-watered treatments. Measurements were taken of photosynthesis, soil water dynamics, water soluble carbohydrates (WSC), amino acids (AA) and N 2 fixation. Results: Lower g s under e[CO 2 ] increased water use efficiency at leaf and plant level, and this translated to slower soil water depletion during drought. Elevated [CO 2 ] increased WSC and decreased total AA concentrations in nodules, and increased nodule activity under drought. N 2 fixation was stimulated (+51%) by e[CO 2 ] in proportion to biomass changes. Under e[CO 2 ] a greater proportion of plant total N was derived from fixed N 2 and a smaller proportion from soil N uptake compared to a[CO 2 ]. Conclusion: This study suggests that e[CO 2 ] increased WUE and this resulted in slower soil water depletion, allowing pea plants to maintain greater nodule activity under drought and resulting in appreciable increases in N 2 fixation. Our results suggest that growth under e[CO 2 ] can mitigate drought effects on N 2 fixation and reduce dependency on soil N resources especially in water-limited agro-ecosystems. © 2019, Springer Nature Switzerland AG

The water use dynamics of canola cultivars grown under elevated CO2 are linked to their leaf area development

Tausz, M ORCiD: 0000-0001-8205-8561The ‘CO2 fertilisation effect’ is often predicted to be greater under drier than wetter conditions, mainly due to hypothesised early season water savings under elevated [CO2] (e[CO2]). However, water savings largely depend on the balance between CO2-induced improvement of leaf-level water use efficiency and CO2-stimulation of transpiring leaf area. The dynamics of water use during the growing season can therefore vary depending on leaf area development. Two canola (Brassica napus L.) cultivars of contrasting growth and vigour (vigorous hybrid cv. Hyola 50 and non-hybrid cv. Thumper) were grown under ambient [CO2] (a[CO2], ∼400 μmol mol−1) or e[CO2] (∼700 μmol mol−1) with two water treatments (well-watered and mild drought) in a glasshouse to investigate the interdependence of leaf area development and water use. Dynamics of water use during the growing season varied depending on [CO2] and cultivars. Early stimulation of leaf growth under e[CO2], which also depended on cultivar, overcompensated for the effect of increased leaf-level water use efficiency, so that weekly water use was greater and water depletion from soil greater under e[CO2] than a[CO2]. This result shows that the balance between leaf area and water use efficiency stimulation by e[CO2] can tip towards early depletion of available soil water, so that e[CO2] does not lead to water savings, and the ‘CO2 fertilisation effect’ is not greater under drier conditions. © 2018 Elsevier Gmb