Contrasting responses to drought stress in herbaceous perennial legumes

Background and aims Medicago sativa L. is widely grown in southern Australia, but is poorly adapted to dry, hot summers. This study aimed to identify perennial herbaceous legumes with greater resistance to drought stress and explore their adaptive strategies. Methods Ten herbaceous perennial legume species/accessions were grown in deep pots in a sandy, low-phosphorus field soil in a glasshouse. Drought stress was imposed by ceasing to water. A companion M. sativa plant in each pot minimised differences in leaf area and water consumption among species. Plants were harvested when stomatal conductance of stressed plants decreased to around 10% of well watered plants. Results A range of responses to drought stress were identified, including: reduced shoot growth; leaf curling; thicker pubescence on leaves and stems; an increased root:shoot ratio; an increase, decrease or no change in root distribution with depth; reductions in specific leaf area or leaf water potential; and osmotic adjustment. The suite of changes differed substantially among species and, less so, among accessions. Conclusions The inter- and intra-specific variability of responses to drought-stress in the plants examined suggests a wide range of strategies are available in perennial legumes to cope with drying conditions, and these could be harnessed in breeding/selection programs.

Variation in seedling growth of 11 perennial legumes in response to phosphorus supply

Phosphorus (P) deficiency is a major problem for Australian agriculture. Development of new perennial pasture legumes that acquire or use P more efficiently than the current major perennial pasture legume, lucerne (Medicago sativa L.), is urgent. A glasshouse experiment compared the response of ten perennial herbaceous legume species to a series of P supplies ranging from 0 to 384 µg g−1 soil, with lucerne as the control. Under low-P conditions, several legumes produced more biomass than lucerne. Four species (Lotononis bainesii Baker, Kennedia prorepens F.Muell, K. prostrata R.Br, Bituminaria bituminosa (L.) C.H.Stirt) achieved maximum growth at 12 µg P g−1 soil, while other species required 24 µg P g−1. In most tested legumes, biomass production was reduced when P supply was ≥192 µg g−1, due to P toxicity, while L. bainesii and K. prorepens showed reduced biomass when P was ≥24 µg g−1 and K. prostrata at ≥48 µg P g−1 soil. B. bituminosa and Glycine canescens F.J.Herm required less soil P to achieve 0.5 g dry mass than the other species did. Lucerne performed poorly with low P supply and our results suggest that some novel perennial legumes may perform better on low-P soils.

Variation in morphological and physiological parameters in herbaceous perennial legumes in response to phosphorus supply

Change in morphological and physiological parameters in response to phosphorus (P) supply was studied in 11 perennial herbaceous legume species, six Australian native (Lotus australis, Cullen australasicum, Kennedia prorepens, K. prostrata, Glycine canescens, C. tenax) and five exotic species (Medicago sativa, Lotononis bainesii, Bituminaria bituminosa var albomarginata, Lotus corniculatus, Macroptilium bracteatum). We aimed to identify mechanisms for P acquisition from soil. Plants were grown in sterilised washed river sand; eight levels of P as KH2PO4 ranging from 0 to 384 μg P g−1 soil were applied. Plant growth under low-P conditions strongly correlated with physiological P-use efficiency and/or P-uptake efficiency. Taking all species together, at 6 μg P g−1 soil there was a good correlation between P uptake and both root surface area and total root length. All species had higher amounts of carboxylates in the rhizosphere under a low level of P application. Six of the 11 species increased the fraction of rhizosphere citrate in response to low P, which was accompanied by a reduction in malonate, except L. corniculatus. In addition, species showed different plasticity in response to P-application levels and different strategies in response to P deficiency. Our results show that many of the 11 species have prospects for low-input agroecosystems based on their high P-uptake and P-use efficiency.

Commensalism in an agroecosystem: hydraulic redistribution by deep-rooted legumes improves survival of a droughted shallow-rooted legume companion

We investigated commensalism of water use among annual shallow-rooted and perennial deep-rooted pasture legumes by examining the effect of hydraulic lift by Cullen pallidum (N.T.Burb.) J.W.Grimes and Medicago sativa on growth, survival and nutrient uptake of Trifolium subterraneum L. A vertically split-root design allowed separate control of soil water in top and bottom soil. Thirty-five days after watering ceased in the top tube, but soil remained at field capacity in the bottom tube, an increase in shallow soil water content by hydraulic lift was 5.6 and 5.9 g kg−1 soil overnight for C. pallidum and M. sativa, respectively. Trifolium subterraneum in this treatment maintained higher leaf water potentials (with M. sativa) or exhibited a slower decline (with C. pallidum) than without companion perennial plants; and shoot biomass of T. subterraneum was 56% (with C. pallidum) and 67% (with M. sativa) of that when both top and bottom tubes were at field capacity. Uptake of rubidium (a potassium analog) and phosphorus by T. subterraneum was not facilitated by hydraulic lift. Interestingly, phosphorus content was threefold greater, and shoot biomass 1.5–3.3-fold greater when T. subterraneum was interplanted with C. pallidum compared with M. sativa, although dry weight of C. pallidum was much greater than that of M. sativa. This study showed that interplanting with deep-rooted perennial legumes has benefited the survival of T. subterraneum.

Cannabinoids and epilepsy

Cannabis has been used for centuries to treat seizures. Recent anecdotal reports, accumulating animal model data, and mechanistic insights have raised interest in cannabis-based antiepileptic therapies. In this study, we review current understanding of the endocannabinoid system, characterize the pro- and anticonvulsive effects of cannabinoids [e.g., Δ9-tetrahydrocannabinol and cannabidiol (CBD)], and high-light scientific evidence from pre-clinical and clinical trials of cannabinoids in epilepsy. These studies suggest that CBD avoids the psychoactive effects of the endocannabinoid system to provide a well-tolerated, promising therapeutic for the treatment of seizures, while whole-plant cannabis can both contribute to and reduce seizures. Finally, we discuss results from a new multicenter, open-label study using CBD in a population with treatment-resistant epilepsy. In all, we seek to evaluate our current understanding of cannabinoids in epilepsy and guide future basic science and clinical studies.

Molecular targets of cannabidiol in neurological disorders

Cannabis has a long history of anecdotal medicinal use and limited licensed medicinal use. Until recently, alleged clinical effects from anecdotal reports and the use of licensed cannabinoid medicines are most likely mediated by tetrahydrocannabinol by virtue of: 1) this cannabinoid being present in the most significant quantities in these preparations; and b) the proportion:potency relationship between tetrahydrocannabinol and other plant cannabinoids derived from cannabis. However, there has recently been considerable interest in the therapeutic potential for the plant cannabinoid, cannabidiol (CBD), in neurological disorders but the current evidence suggests that CBD does not directly interact with the endocannabinoid system except in vitro at supraphysiological concentrations. Thus, as further evidence for CBD’s beneficial effects in neurological disease emerges, there remains an urgent need to establish the molecular targets through which it exerts its therapeutic effects. Here, we conducted a systematic search of the extant literature for original articles describing the molecular phar- macology of CBD. We critically appraised the results for the validity of the molecular targets proposed. Thereafter, we considered whether the molecular targets of CBD identified hold therapeutic potential in relevant neurological diseases. The molecular targets identified include numerous classical ion channels, receptors, transporters, and enzymes. Some CBD effects at these targets in in vitro assays only manifest at high concentrations, which may be difficult to achieve in vivo, particularly given CBD’s relatively poor bioavailability. Moreover, several targets were asserted through experimental designs that demonstrate only correlation with a given target rather than a causal proof. When the molecular targets of CBD that were physiologically plausible were considered for their potential for exploitation in neurological therapeu- tics, the results were variable. In some cases, the targets identified had little or no established link to the diseases considered. In others, molecular targets of CBD were entirely consistent with those already actively exploited in relevant, clinically used, neurological treatments. Finally, CBD was found to act upon a number of targets that are linked to neurological therapeutics but that its actions were not consistent with modulation of such targets that would derive a therapeutically beneficial outcome. Overall, we find that while >65 discrete molecular targets have been reported in the literature for CBD, a relatively limited number represent plausible targets for the drug’s action in neurological disorders when judged by the criteria we set. We conclude that CBD is very unlikely to exert effects in neurological diseases through modulation of the endocannabinoid system. Moreover, a number of other molecular targets of CBD reported in the literature are unlikely to be of relevance owing to effects only being observed at supraphysiological concentrations. Of interest and after excluding unlikely and implausible targets, the remaining molecular targets of CBD with plausible evidence for involvement in therapeutic effects in neurological disorders (e.g., voltage-dependent anion channel 1, G protein-coupled receptor 55, CaV3.x, etc.) are associated with either the regulation of, or responses to changes in, intracellular calcium levels. While no causal proof yet exists for CBD’s effects at these targets, they represent the most probable for such investigations and should be prioritized in further studies of CBD’s therapeutic mechanism of action.

The diversity of arbuscular mycorrhizas of selected Australian Fabaceae

Members of the Australian native perennial Fabaceae have been little explored with regard to their root biology and the role played by arbuscular mycorrhizal (AM) fungi in their establishment, nutrition and long-term health. The ultimate goal of our research is to determine the dependency of native perennial legumes on their co-evolved AM fungi and conversely, the impact of AM fungal species in agricultural fields on the productivity of sown native perennial legume pastures. In this paper we investigate the colonisation morphology in roots and the AMF, identified by spores extracted from rhizosphere soil, from three replicate plots of each of the native legumes, Cullen australasicum, C. tenax and Lotus australis and the exotic legumes L. pedunculatus and Medicago sativa. The plants were grown in an agricultural field. The level and density of colonisation by AM fungi, and the frequency of intraradical and extraradical hyphae, arbuscules, intraradical spores and hyphal coils all differed between host plants and did not consistently differ between native and exotic species. However, there were strong similarities between species in the same genus. The three dominant species of AM fungi in rhizosphere soil also differed with host plant, but one fungus (Glomus mosseae) was always the most dominant. Sub-dominant AM species were the same between species in the same genus. No consistent differences in dominant spores were observed between the exotic and native Fabaceae species. Our results suggest that plant host influences the mycorrhizal community in the rhizosphere soil and that structural and functional differences in the symbiosis may occur at the plant genus level, not the species level or due to provenance.

Perception of bitterness, sweetness and liking of different genotypes of lettuce

Lettuce is an important leafy vegetable, consumed across the world, containing bitter sesquiterpenoid lactone (SL) compounds that may negatively affect consumer acceptance and consumption. We assessed liking of samples with differing absolute abundance and different ratios of bitter:sweet compounds by analysing recombinant inbred lines (RILs) from an interspecific lettuce mapping population derived from a cross between a wild (L. serriola acc. UC96US23) and domesticated lettuce, (L. sativa, cv. Salinas). We found that the ratio of bitter:sweet compounds was a key determinant of bitterness perception and liking. We were able to demonstrate that SLs such as 8-deoxylactucin-15-sulphate contribute most strongly to bitterness perception, whilst 15-p-hydroxylphenylacetyllactucin-8-sulphate does not contribute to bitter taste. Glucose was the sugar most highly correlated with sweetness perception. There is a genetic basis to the biochemical composition of lettuce. This information will be useful in lettuce breeding programmes in order to produce leaves with more favourable taste profiles.

Perennial legumes native to Australia: a preliminary investigation of nutritive value and response to cutting

Six Australian native herbaceous perennial legumes (Lotus australis, Swainsona colutoides, Swainsona swainsonioides, Cullen tenax, Glycine tabacina and Kennedia prorepens) were assessed in the glasshouse for nutritive value, soluble condensed tannins and production of herbage in response to three cutting treatments (regrowth harvested every 4 and 6 weeks and plants left uncut for 12 weeks). The Mediterranean perennial legumes Medicago sativa and Lotus corniculatus were also included. Dry matter (DM) yield of some native legumes was comparable to L. corniculatus, but M. sativa produced more DM than all species except S. swainsonioides after 12 weeks of regrowth. Dry matter yield of all native legumes decreased with increased cutting frequency, indicating a susceptibility to frequent defoliation. Shoot in vitro dry matter digestibility (DMD) was high (>70%) in most native legumes, except G. tabacina (65%) and K. prorepens (55%). Crude protein ranged from 21-28% for all legumes except K. prorepens (12%). More frequent cutting resulted in higher DMD and crude protein in all species, except for the DMD of C. tenax and L. australis, which did not change. Concentrations of soluble condensed tannins were 2-9 g/kg DM in the Lotus spp., 10-18 g/kg DM in K. prorepens and negligible (<1 g/kg) in the other legumes. Of the native species, C. tenax, S. swainsonioides and L. australis showed the most promise for use as forage plants and further evaluation under field conditions is now warranted.

Root distributions of Australian herbaceous perennial legumes in response to phosphorus placement

Many Australian plant species have specific root adaptations for growth in phosphorus-impoverished soils, and are often sensitive to high external P concentrations. The growth responses of native Australian legumes in agricultural soils with elevated P availability in the surface horizons are unknown. The aim of these experiments was to test the hypothesis that increased P concentration in surface soil would reduce root proliferation at depth in native legumes. The effect of P placement on root distribution was assessed for two Australian legumes, Kennedia prorepens F. Muell. and Lotus australis Andrews, and the exotic Medicago sativa L. Three treatments were established in a low-P loam soil: amendment of 0.15 g mono-calcium phosphate in either (i) the top 50 mm (120 µg P g–1) or (ii) the top 500 mm (12 µg P g–1) of soil, and an unamended control. In the unamended soil M. sativa was shallow rooted, with 58% of the root length of in the top 50 mm. K. prorepens and L. australis had a more even distribution down the pot length, with only 4 and 22% of their roots in the 0–50 mm pot section, respectively. When exposed to amendment of P in the top 50 mm, root length in the top 50 mm increased 4-fold for K. prorepens and 10-fold for M. sativa, although the pattern of root distribution did not change for M. sativa. L. australis was relatively unresponsive to P additions and had an even distribution of roots down the pot. Shoot P concentrations differed according to species but not treatment (K. prorepens 2.1 mg g–1, L. australis 2.4 mg g–1, M. sativa 3.2 mg g–1). Total shoot P content was higher for K. prorepens than for the other species in all treatments. In a second experiment, mono-ester phosphatases were analysed from 1-mm slices of soil collected directly adjacent to the rhizosphere. All species exuded phosphatases into the rhizosphere, but addition of P to soil reduced phosphatase activity only for K. prorepens. Overall, high P concentration in the surface soil altered root distribution, but did not reduce root proliferation at depth. Furthermore, the Australian herbaceous perennial legumes had root distributions that enhanced P acquisition from low-P soils.

‘Symptomless’ infection by Botrytis cinerea

The study was carried out to clarify the nature of symptomless infection by Botrytis cinerea and to what extent it differs from aggressive necrotic infection in Lactuca sativa (lettuce) and Arabidopsis thaliana. Symptomless plants were produced by dry spore inoculation in plants growing in controlled environmental conditions or in glasshouses. Plating out of surface-disinfected and non-surface-disinfected samples of inoculated, apparently healthy, plants on selective medium revealed that the fungus was spreading from the initial inoculation site to newly developing plant organs both internally and externally. Similar findings were obtained in microscope experiments in which host plants were inoculated with GFP labelled B. cinerea and symptomless spreading was monitored under confocal laser scanning microscope. Spore germination on leaf surface was followed by development of sub-cuticular vesicles and plant cell damage in the infected epidermal cell and a few nearby cells. Sparsely branched long hyphae arose from the vesicles and spread on the leaf surface; spread was mostly on the outer surface of the epidermal layer but occasionally below the cuticle or epidermal cells. In the late symptomless phase, mycelium arising from single vesicles formed several mycelial networks on leaves. Experiments were carried out to compare the extent of gene expression in symptomless and necrotic infections, using RT-qPCR. Expression of selected genes was quantified in tissue samples based on the amount of mRNA of the respective genes found. In both host species, the mRNA concentration of signalling genes bcg1, bmp1 and calcineurin, and the pathogenicity genes bcsod1 and bcpg1 were similar to or slightly greater in symptomless samples than in necrotic samples. The mRNA of the signalling gene bac and pathogenicity genes bcbot1 and bcnep1, were not detected or detected in lower abundance than in necrosis. In lettuce, the leaves developing distant from the site of inoculation showed similar results to A. thaliana, but in healthy leaves close to the site of inoculation mRNA concentrations of bac and bcnep1 were similar to necrotic samples. Thus, in both host species, the fungus grew along with the plant and moved to newly growing plant parts without producing symptoms; during this growth some pathogenicity genes were less expressed than in necrotic infection.

The effect of simulated flooding post-anthesis on pre-harvest sprouting and subsequent seed longevity in contrasting rice cultivars

Unpredictable flooding is a major constraint to rice production. It can occur at any growth stage. The effect of simulated flooding post-anthesis on yield and subsequent seed quality of pot-grown rice (Oryza sativa L.) plants was investigated in glasshouses and controlled-environment growth cabinets. Submergence post-anthesis (9-40 DAA) for 3 or 5 days reduced seed weight of japonica rice cv. Gleva, with considerable pre-harvest sprouting (up to 53%). The latter was greater the later in seed development and maturation that flooding occurred. Sprouted seed had poor ability to survive desiccation or germinate normally upon rehydration, whereas the effects of flooding on the subsequent air-dry seed storage longevity (p50) of the non-sprouted seed fraction was negligible. The indica rice cvs IR64 and IR64Sub1 (introgression of submergence tolerance gene Submergence1A-1) were both far more tolerant to flooding post-anthesis than cv. Gleva: four days’ submergence of these two near-isogenic cultivars at 10-40 DAA resulted less than 1% sprouted seeds. The presence of the Sub1A-1 allele in cv. IR64Sub1 was verified by gel electrophoresis and DNA sequencing. It had no harmful effect on loss in seed viability during storage compared with IR64 in both control and flooded environments. Moreover, the germinability and changes in dormancy during seed development and maturation were very similar to IR64. The efficiency of using chemical spray to increase seed dormancy was investigated in the pre-harvest sprouting susceptible rice cv. Gleva. Foliar application of molybdenum at 100 mg L-1 reduced sprouted seeds by 15-21% following 4 days’ submergence at 20-30 DAA. Analyses confirmed that the treatment did result in molybdenum uptake by the plants, and also tended to increase seed abscisic acid concentration. The latter was reduced by submergence and declined exponentially during grain ripening. The selection of submergence-tolerant varieties was more successful than application of molybdenum in reducing pre-harvest sprouting.

EVALUATION OF THE INTERACTION OF NUTRIENTS IN THE ZINC APPLICATION IN SEEDS OF THE RICE

The efficient fertilizer use contend micronutrient depends, also, of the interactions that occur with some nutrients. The objective was to study the interaction of zinc with the excessively nutrient ones in function of the application of different doses and sources of Zn saw seed in the culture of the rice. The experiment was carried through in conditions of vegetation house, in the FCAV/Unesp. The used experimental delineation was entirely cazualized, with three repetitions. The treatments had been five doses: 0; 1,0; 2,0; 4,0 and 8,0 g of Zn for kg of seed; e two zinc sources; sulphate of zinc (22% of Zn) and zinc oxide (50% of Zn). The experimental unit was a translucent polyethylene tray, filled with 5 washed thick sand, where 50 seeds of rice had been sown (to var. Caiapo). To the 30 days after the sowing, effected the cut of the plants, separating them in aerial part and roots. From the results of the dry substance and text of nutrients of the aerial part and root of the rice, the accumulation of the nutrients in the respective agencies of the plants was calculated. Becoming fullfilled it variance analysis and the when necessary unfoldings. It had effect of the interactions in distinguishing way between root and aerial part of rice, with regard to the doses and sources.

Storage proteins and cell wall mobilisation in seeds of Sesbania virgata (Cav.) Pers. (Leguminosae)

The endosperm of seeds of Sesbania virgata (Cav.) Pers. accumulates galactomannan as a cell wall storage polysaccharide. It is hydrolysed by three enzymes, one of them being alpha-galactosidase. A great amount of protein bodies is found in the cytoplasm of endospermic cells, which are thought to play the major role as a nitrogen reserve in this seed. The present work aimed at understanding how the production of enzymes that degrade storage compounds is controlled. We performed experiments with addition of inhibitors of transcription (actinomycin-d and alpha-amanitin) and translation (cycloheximide) during and after germination. In order to follow the performance of storage mobilisation, we measured fresh mass, protein contents and alpha-galactosidase activity. All the inhibitors tested had little effect on seed germination and seedling development. Actinomycin-d and cycloheximide provoked a slight inhibition of the storage protein degradation and concomitantly lead to an elevation of the alpha-galactosidase activity. Although alpha-amanitin showed some effect on seedling development at latter stages, it presented the former effect and did not change galactomannan degradation performance. Our data suggest that some of the proteases may be synthesised de novo, whereas alpha-galactosidase seems to be present in the endosperm cells probably as an inactive polypeptide in the protein bodies, being probably activated by proteolysis when the latter organelle is disassembled. These evidences suggest the existence of a connection between storage proteins and carbohydrates mobilisation in seeds of S. virgata, which would play a role by assuring a balanced afflux of the carbon and nitrogen to the seedling development.

Characterisation, immunolocalisation and antifungal activity of a lipid transfer protein from chili pepper (Capsicum annuum) seeds with novel alpha-amylase inhibitory properties

Lipid transfer proteins (LTPs) were thus named because they facilitate the transfer of lipids between membranes in vitro. This study was triggered by the characterization of a 9-kDa LTP from Capsicum annuum seeds that we call Ca-LTP(1). Ca-LTP(1) was repurified, and in the last chromatographic purification step, propanol was used as the solvent in place of acetonitrile to maintain the protein`s biological activity. Bidimensional electrophoresis of the 9-kDa band, which corresponds to the purified Ca-LTP(1), showed the presence of three isoforms with isoelectric points (pIs) of 6.0, 8.5 and 9.5. Circular dichroism (CD) analysis suggested a predominance of alpha-helices, as expected for the structure of an LTP family member. LTPs immunorelated to Ca-LTP(1) from C. annuum were also detected by western blotting in exudates released from C. annuum seeds and also in other Capsicum species. The tissue and subcellular localization of Ca-LTP(1) indicated that it was mainly localized within dense vesicles. In addition, isolated Ca-LTP(1) exhibited antifungal activity against Colletotrichum lindemunthianum, and especially against Candida tropicalis, causing several morphological changes to the cells including the formation of pseudohyphae. Ca-LTP(1) also caused the yeast plasma membrane to be permeable to the dye SYTOX green, as verified by fluorescence microscopy. We also found that Ca-LTP(1) is able to inhibit mammalian alpha-amylase activity in vitro.