Release of Medicago truncatula Gaertn. and Pisum sativum subsp. elatius (M. Bieb.) Asch. et Graebn. Seed Dormancy Tested in Soil Conditions

Medicago truncatula (barrel medic) and Pisum sativum subsp. elatius (wild pea) accessions originating from variable environmental conditions in the Mediterranean basin were used to study physical seed dormancy (PY) release. The effect of soil burial on PY release was tested on 112 accessions of medic and 46 accessions of pea over the period of 3 months in situ at three common gardens (Hungary, Spain and Greece) from 2017 through 2019. PY release after soil exhumation followed by experimental laboratory germination of remaining dormant seeds (wet, 25 °C, 21 days) were related to the environmental conditions of the common garden and macroclimatic variables of the site of origin of the accessions. Higher PY release was observed in buried seeds under humid rather than under dry and hot environments. Exposure of remaining dormant seeds to experimental laboratory conditions increased total PY release up to 70% and 80% in barrel medic and wild pea, respectively. Wild pea showed higher phenotypic plasticity on PY release than barrel medic, which had higher bet-hedging within-season. Wild pea showed lower bet-hedging among-season (PY < 10%) in relation to precipitation than barrel medic, which was more conservative (PY ≈ 20%). Observed variability suggests that these species have the capability to cope with ongoing climate change.

Evaluation of virus-induced gene silencing methods for forage legumes including alfalfa, sainfoin, and fenugreek

Virus-induced gene silencing (VIGS) is a rapid reverse genetics tool that has been developed in a wide variety of plant species for assessing gene functions. However, while VIGS has been utilized successfully in the diploid model leguminous species Medicago truncatula (Gaertn.) (barrel medic), such a platform has yet to be established in forage legume crop species. Therefore, we evaluated the effectiveness of this method in forage legumes using a previously developed PEBV (pea early browning virus) system whereby a fragment of the pea (Pisum sativum L.) PHYTOENE DESATURASE (PDS) gene was transferred into a range of alfalfa (Medicago sativa L.), sainfoin (Onobrychis viciifolia Scop.), and fenugreek (Trigonella foenum-graecum L.) cultivars using leaf infiltration and apical meristem injection. Barrel medic was used as a positive control. Gene silencing was observed after 10–15 d through the presence of a leaf bleaching phenotype, and was confirmed using quantitative real-time RT-PCR. Silencing of PDS was achieved in a selection of cultivars in all species assessed, with the highest silencing efficiency apparent in fenugreek. The introduction of a highly homologous gene fragment from a heterologous plant species to target endogenous genes for transient VIGS-based silencing in a range of species of interest represents a potentially useful strategy for the rapid functional characterization of candidate genes in forages.

Evaluation of Aluminum Sensitivity in Barrel Medic Germplasm

Aluminum (Al) toxicity in acid soils is a major constraint to global agricultural production, affecting ≈30% of the world’s arable land area. To study Al tolerance in barrel medic (Medicago truncatula), we assessed responses to excess Al in 91 accessions collected from different geographic regions. Root elongations were used to characterize the sensitivity of each accession. Seedlings were grown in an agarose medium that contained three levels of Al (50, 100, and 200 µm), and root elongation was measured at 72 hours after exposure to Al. The ratio of root elongation in the presence and absence of Al [relative root growth (RRG)] differed among accessions. At 50 µm Al, we observed the greatest range of intraspecific variation. Aluminum sensitivity of 30 accessions was tested further by hematoxylin staining. Relative root growth was regressed linearly against the visual staining score, and a significant, negative, linear relationship was found between RRG at 50 or 100 µm Al and the intensity of staining scores. Twelve selected accessions differing in their resistance were grown in Al-toxic soil to confirm their Al response. Such information could be useful in breeding or selecting for improved Al tolerance in barrel medic, as well as other crop species.

Genome-wide characterization and phylogenetic and expression analyses of the caleosin gene family in soybean, common bean and barrel medic

Caleosin are a class of calcium-binding proteins embedded in the phospholipid monolayer of lipid droplets. In addition to maintaining thestructure of lipid droplets, caleosin proteins areinvolved in dormancy and lipid signaling, and areassociatedwith the stress response via their histidine-dependent peroxygenase activity. To date, caleosins have been studied in Arabidopsis thaliana. However, little is known about these genes in legumes,including the most cultivated oilseed crop, soybean. In this paper,20 caleosin genes in soybean, common bean and barrel medic werestudied. Among these, 13 caleosin genes, including 3 in Glycine max, 5 in Phaseolus vulgarisand 5 in Medicago truncatula, are identified for the first time. The structures, characteristics and evolution of the 20 caleosin proteins are analyzed. Expansion patterns show that tandem duplication was the main reason for the caleosin family expansion in the legume. Expression profiles indicate that L-caleosin in soybean and common bean are more important than H-caleosin, which is just the opposite in Arabidopsis thaliana. GmaCLO2, PvuCLO1, PvuCLO3and MtrCLO3may play important roles, while GmaCLO6, GmaCLO10and MtrCLO4may lose their function in the examined tissues. In addition, according to the results of cis-element analyses, we propose potential functions for the more important caleosin genes in leguminous plants. Our work provides helpful information for further evolution and function analyses of the caleosin gene family in soybean, common bean and barrel medic.

Response of soil nitrous oxide flux to nitrogen fertiliser application and legume rotation in a semi-arid climate, identified by smoothing spline models

Although large areas of semi-arid land are extensively cropped, few studies have investigated the effect of nitrogen (N) fertiliser on nitrous oxide (N2O) emissions in these regions (Galbally et al. 2010). These emissions need to be measured in order to estimate N losses and calculate national greenhouse gas inventories. We examined the effect of different agronomic management practices applied to wheat (Triticum aestivum) grown on an alkaline Vertosol in south-eastern Australia on N2O emissions. In 2007, N2O emissions were measured over 12 months, during which N fertiliser (urea) was applied at sowing or N fertiliser plus supplementary irrigation (50 mm) was applied during the vegetative stage and compared with a treatment of no N fertiliser or irrigation. In a second experiment (2008), the effect of source of N on N2O emissions was examined. Wheat was grown on plots where either a pulse (field peas, Pisum sativum) or pasture legume (barrel medic, Medicago truncatula) crop had been sown in the previous season compared with a non-legume crop (canola, Brassica napus). To account for the N supplied by the legume phase, N fertiliser (50 kg N ha–1 as urea) was applied only to the wheat in the plots previously sown to canola. Fluxes of N2O were measured on a sub-daily basis (up to 16 measurements per chamber) by using automated chamber enclosures and a tuneable diode laser, and treatment differences were evaluated by a linear mixed model including cubic smoothing splines. Fluxes were low and highly variable, ranging from –3 to 28 ng N2O-N m–2 s–1. The application of N fertiliser at sowing increased N2O emissions for ~2 months after the fertiliser was applied. Applying irrigation (50 mm) during the vegetative growth stage produced a temporary (~1-week) but non-significant increase in N2O emissions compared with plots that received N fertiliser at sowing but were not irrigated. Including a legume in the rotation significantly increased soil inorganic N at sowing of the following wheat crop by 38 kg N ha–1 (field peas) or 57 kg ha–1 (barrel medic) compared with a canola crop. However, N2O emissions were greater in wheat plots where N fertiliser was applied than where wheat was sown into legume plots where no N fertiliser was applied. Over the 2 years of the field study, N2O emissions attributed to fertiliser ranged from 41 to 111 g N2O-N ha–1, and averaged of 75 g N2O-N ha–1 or 0.15% of the applied N fertiliser. Our findings confirm that the proportion of N fertiliser emitted as N2O from rainfed grain crops grown in Australian semi-arid regions is less than the international average of 1.0%.