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BackgroundImprovement of freezing tolerance of red clover (Trifolium pratense L.) would increase its persistence under cold climate. In this study, we assessed the freezing tolerance and compared the proteome composition of non-acclimated and cold-acclimated plants of two initial cultivars of red clover: Endure (E-TF0) and Christie (C-TF0) and of populations issued from these cultivars after three (TF3) and four (TF4) cycles of phenotypic recurrent selection for superior freezing tolerance. Through this approach, we wanted to identify proteins that are associated with the improvement of freezing tolerance in red clover.ResultsFreezing tolerance expressed as the lethal temperature for 50 % of the plants (LT50) increased markedly from approximately −2 to −16 °C following cold acclimation. Recurrent selection allowed a significant 2 to 3 °C increase of the LT50 after four cycles of recurrent selection. Two-dimensional difference gel electrophoresis (2D-DIGE) was used to study variations in protein abundance. Principal component analysis based on 2D-DIGE revealed that the largest variability in the protein data set was attributable to the cold acclimation treatment and that the two genetic backgrounds had differential protein composition in the acclimated state only. Vegetative storage proteins (VSP), which are essential nitrogen reserves for plant regrowth, and dehydrins were among the most striking changes in proteome composition of cold acclimated crowns of red clovers. A subset of proteins varied in abundance in response to selection including a dehydrin that increased in abundance in TF3 and TF4 populations as compared to TF0 in the Endure background.ConclusionRecurrent selection performed indoor is an effective approach to improve the freezing tolerance of red clover. Significant improvement of freezing tolerance by recurrent selection was associated with differential accumulation of a small number of cold-regulated proteins that may play an important role in the determination of the level of freezing tolerance.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0751-2) contains supplementary material, which is available to authorized users.

The aim of this study was to evaluate the effects of fresh and composted paper sludge on macroaggregate stability of a silt loam under field conditions, and to assess the possible role of carbohydrate fractions and humic substances. The treatments included fresh paper mill sludge (PMS) and its compost (CPMS) applied at a rate of 40 Mg ha−1 with or without a mineral N fertilizer (120 kg N ha−1), N fertilizer only (recommended rate of 160 kg N ha−1), and an unamended control. Measurements of total and amino sugars and humic substances were made on slaking‐resistant aggregates 2 yr after the last of three successive annual applications of the treatments. Compared with the treatments that received no organic amendment, the PMS and CPMS applications increased macroaggregate stability by an average of 45%. The effects of fresh vs. composted amendments on soil macroaggregates and their organic C contents were similar but differences in C composition were observed. Humic acid content of aggregates >2 mm was significantly higher (50%) with CPMS than PMS, although part of this effect could be attributed to the slightly greater C application rate with CPMS. Conversely, glucosamine content, an indicator of fungi abundance, was significantly greater following PMS than CPMS application. We concluded that microorganisms, in particular fungi, were a more important factor of stable macroaggregation in the soil amended with fresh sludge, while humic substances played a greater role in compost‐amended soil. These effects were long lasting in the field since they were still noticeable 2 yr after the last application.

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Legume crops leave N‐rich residues and improve soil properties that can boost the yield of subsequent crops. This study conducted at two sites in Québec, eastern Canada, identified the most appropriate preceding legume crops for subsequent corn (Zea mays L.) and wheat (Triticum aestivum L.) yield and N nutrition. Legumes were established in 2011, in monoculture or mixed with grain crops, for a total of 13 treatments: common bean (Phaseolus vulgaris L.), soybean (Glycine max L.), dry pea (Pisum sativum L.), hairy vetch (Vicia villosa Roth), alfalfa (Medicago sativa L.), and crimson clover (Trifolium incarnatum L.), hairy vetch/wheat, crimson clover/wheat, field pea/wheat, alfalfa/corn, hairy vetch/corn, crimson clover/corn) and a non‐N fixing crop (corn) as the control. In 2012, each plot was split and five N fertilizer rates applied to corn and wheat. Four legume systems (alfalfa, hairy vetch, crimson clover, and hairy vetch/wheat) significantly increased the soil structure stability, alkaline phosphatase and dehydrogenase activities at warmer St‐Mathieu‐de‐Beloeil location but not at the cooler St‐Lambert‐de‐Lauzon site. These legumes also significantly increased yields and N nutrition of corn and wheat at St Mathieu‐de‐Beloeil and of wheat only at St‐Lambert‐de‐Lauzon. Although legume N credit was found low (∼30 kg N ha−1), the N fertilizer replacement value was 51 to 77 kg N ha−1 for corn and up to 37 kg N ha−1 for wheat, depending on the preceding legume crop. This suggests that indirect effects related to improved soil properties impacted positively corn and wheat yield and N nutrition.

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