Shade delays flowering in Medicago sativa.

Shade-intolerant plants respond to the decrease in the red (R) to far-red (FR) light ratio (R:FR) occurring under shade by elongating stems and petioles and by re-positioning leaves, in a race to outcompete neighbors for the sunlight resource. In some annual species, the shade avoidance syndrome (SAS) is accompanied by the early induction of flowering. Anticipated flowering is viewed as a strategy to set seeds before the resources become severely limiting. Little is known about the molecular mechanisms of SAS in perennial forage crops like alfalfa (Medicago sativa). To study SAS in alfalfa, we exposed alfalfa plants to simulated shade by supplementing with FR light. Low R:FR light produced a classical SAS, with increased internode and petiole lengths, but unexpectedly also with delayed flowering. To understand the molecular mechanisms involved in uncoupling SAS from early flowering, we used a transcriptomic approach. The SAS is likely to be mediated by increased expression of msPIF3 and msHB2 in low R:FR light. Constitutive expression of these genes in Arabidopsis led to SAS, including early flowering, strongly suggesting that their roles are conserved. Delayed flowering was likely to be mediated by the downregulation of msSPL3, which promotes flowering in both Arabidopsis and alfalfa. Shade-delayed flowering in alfalfa may be important to extend the vegetative phase under suboptimal light conditions, and thus assure the accumulation of reserves necessary to resume growth after the next season.

Stable symbiotic nitrogen fixation under water-deficit field conditions by a stress-tolerant alfalfa microsymbiont and its complete genome sequence.

We here characterized the stress-tolerant alfalfa microsymbiont Sinorhizobium meliloti B401. B401-treated plants showed high nitrogen fixation rates under humid and semiarid environments. The production of glycine betaine in isolated bacteroids positively correlated with low precipitation levels, suggesting that this compound acts as a critical osmoprotectant under field conditions. Genome analysis revealed that strain B401 contains alternative pathways for the biosynthesis and uptake of glycine betaine and its precursors. Such genomic information will offer substantial insight into the environmental physiology of this biotechnologically valuable nitrogen-fixing bacterium.

Microevolution Rather than Large Genome Divergence Determines the Effectiveness of Legume-Rhizobia Symbiotic Interaction Under Field Conditions.

Despite the vast screening for natural nitrogen-fixing isolates by public and private consortia, no significant progresses in the production of improved nitrogen-fixing inoculants for alfalfa production have been made in the last years. Here, we present a comprehensive characterization of the nitrogen-fixing strain Ensifer meliloti B399 (originally named Rhizobium meliloti 102F34), probably the inoculant most widely used in alfalfa production since the 1960s. Complete nucleotide sequence and genome analysis of strain B399 showed that the three replicons present in this commercial strain and the model bacterium Ensifer meliloti 1021 are extremely similar to each other in terms of nucleotide identity and synteny conservation. In contrast to that observed in B399-treated plants, inoculation of plants with strain 1021 did not improve nitrogen content in different alfalfa cultivars under field conditions, suggesting that a small genomic divergence can drastically impact on the symbiotic phenotype. Therefore, in addition to the traditional screening of natural nitrogen-fixing isolates, the genome engineering of model strains could be an attractive strategy to improve nitrogen fixation in legume crops.

The sunflower transcription factor HaHB11 confers tolerance to water deficit and salinity to transgenic Arabidopsis and alfalfa plants.

Homeodomain-leucine zipper (HD-Zip) transcription factors are unique to the plant kingdom; members of subfamily I are known to be involved in abiotic stress responses. HaHB11 belongs to this subfamily and it was previously shown that it is able to confer improved yield and tolerance to flooding via a quiescent strategy. Here we show that HaHB11 expression is induced by ABA, NaCl and water deficit in sunflower seedlings and leaves. Arabidopsis transgenic plants expressing HaHB11, controlled either by its own promoter or by the constitutive 35S CaMV, presented rolled leaves and longer roots than WT when grown under standard conditions. In addition, these plants showed wider stems and more vascular bundles. To deal with drought, HaHB11 transgenic plants closed their stomata faster and lost less water than controls, triggering an enhanced tolerance to such stress condition and also to salinity stress. Concomitantly, ABA-synthesis and sensing related genes were differentially regulated in HaHB11 transgenic plants. Either under long-term salinity stress or mild drought stress, HaHB11 transgenic plants did not exhibit yield penalties. Moreover, alfalfa transgenic plants were generated which also showed enhanced drought tolerance. Altogether, the results indicated that HaHB11 was able to confer drought and salinity tolerance via a complex mechanism which involves morphological, physiological and molecular changes.

Salt stress alters DNA methylation levels in alfalfa (Medicago spp).

Modification of DNA methylation status is one of the mechanisms used by plants to adjust gene expression at both the transcriptional and posttranscriptional levels when plants are exposed to suboptimal conditions. Under abiotic stress, different cultivars often show heritable phenotypic variation accompanied by epigenetic polymorphisms at the DNA methylation level. This variation may provide the raw materials for plant breeding programs that aim to enhance abiotic stress tolerance, including salt tolerance. In this study, methylation-sensitive amplified polymorphism (MSAP) analysis was used to assess cytosine methylation levels in alfalfa (Medicago spp) roots exposed to increasing NaCl concentrations (0.0, 8.0, 12.0, and 20.0 dS/m). Eleven indigenous landraces were analyzed, in addition to a salt-tolerant cultivar that was used as a control. There was a slight increase in DNA methylation upon exposure to high levels of soil salinity. Phylogenetic analysis using MSAP showed epigenetic variation within and between the alfalfa landraces when exposed to saline conditions. Based on MSAP and enzyme-linked immunosorbent assay results, we found that salinity increased global DNA methylation status, particularly in plants exposed to the highest level of salinity (20 dS/m). Quantitative reverse transcription-polymerase chain reaction indicated that this might be mediated by the overexpression of methyltransferase homolog genes after exposure to saline conditions. DNA demethylation using 5-azacytidine reduced seedling lengths and dry and fresh weights, indicating a possible decrease in salinity tolerance. These results suggest that salinity affects DNA methylation flexibility.

The symbiotic defect in a Sinorhizobium meliloti lipopolysaccharide mutant can be overcome by expression of other surface polysaccharides.

In this work we have examined the extent of functional complementation in symbiosis among different Sinorhizobium meliloti surface polysaccharides including lipopolysaccharide (LPS). We show that a symbiotic deficiency associated with an LPS defect can be reversed by appropriate expression of other surface polysaccharides such as galactoglucan (EPSII) and a particular form of capsular polysaccharide (KdoPS). It is noteworthy that, while succinoglycan EPSI and LPS cannot functionally substitute for each other, they can both be replaced by the same common set of polysaccharides (i.e., EPSII/KdoPS). The complex pattern of functional complementation in symbiosis among S. meliloti surface polysaccharides was shown to be different in Medicago truncatula compared to that previously reported for M. sativa.

Removal of azinphos methyl by alfalfa plants (Medicago sativa L.) in a soil-free system.

The objective of this study was to investigate the removal of azinphos methyl assisted by alfalfa plants, with special emphasis on the effects of this compound on some plant's physiological parameters. Hydroponic cultures of alfalfa (Medicago sativa L., var Romagnola) were employed as a model system. These cultures were exposed to a nutrient medium containing 10 mg/l of azinphos methyl. A first-order kinetic approach was used to describe the removal of azinphos methyl from the solution. After 20 days of culture, the initial amount of azinphos methyl was reduced to non-detectable levels in the presence of plants. In the absence of plants, 20% of azinphos methyl remained in the solution after 30 days of treatment. The half-life of the pesticide was reduced from 10.8 to 3.4 days in the presence of plants. The growth index of alfalfa plants exposed to azinphos methyl was negatively affected. Chlorophyll contents were reduced after 24 h of treatment and thereafter the levels were comparable to that of control plants. The peroxidase activity of alfalfa roots was not affected by the presence of azinphos methyl. In conclusion, alfalfa plants were able to survive when exposed to an effective concentration of 10 mg/l of azinphos methyl in the root zone, with some alterations on their physiological parameters.