The ecology of nectarivorous microbial communities remains virtually unknown, which precludes elucidating whether these organisms play some role in plant-pollinator mutualisms beyond minor commensalism. We simultaneously assessed microbial abundance and nectar composition at the individual nectary level in flowers of three southern Spanish bumble bee-pollinated plants (Helleborus foetidus, Aquilegia vulgaris, and Aquilegia pyrenaica cazorlensis). Yeasts were frequent and abundant in nectar of all species, and variation in yeast density was correlated with drastic changes in nectar sugar concentration and composition. Yeast communities built up in nectar from early to late floral stages, at which time all nectaries contained yeasts, often at densities between 10(4) and 10(5) cells/mm3. Total sugar concentration and percentage sucrose declined, and percentage fructose increased, with increasing density of yeast cells in nectar. Among-nectary variation in microbial density accounted for 65% (H. foetidus and A. vulgaris) and 35% (A. p. cazorlensis) of intraspecific variance in nectar sugar composition, and 60% (H. foetidus) and 38% (A. vulgaris) of variance in nectar concentration. Our results provide compelling evidence that nectar microbial communities can have detrimental effects on plants and/or pollinators via extensive nectar degradation and also call for a more careful interpretation of nectar traits in the future, if uncontrolled for yeasts.

Pattern-triggered immunity (PTI) is activated in plants upon recognition by pattern recognition receptors (PRRs) of damage-and microbe-associated molecular patterns (DAMPs and MAMPs) derived from plants or microorganisms, respectively. To understand better the plant mechanisms involved in the perception of carbohydrate-based structures recognized as DAMPs/MAMPs, we have studied the ability of mixed-linked b-1,3/1,4-glucans (MLGs), present in some plant and microbial cell walls, to trigger immune responses and disease resistance in plants. A range of MLG structures were tested for their capacity to induce PTI hallmarks, such as cytoplasmic Ca 2+ elevations, reactive oxygen species production, phosphorylation of mitogen-activated protein kinases and gene transcriptional reprogramming. These analyses revealed that MLG oligosaccharides are perceived by Arabidopsis thaliana and identified a trisaccharide, b-D-cellobiosyl-(1,3)-b-D-glucose (MLG43), as the smallest MLG structure triggering strong PTI responses. These MLG43-mediated PTI responses are partially dependent on LysM PRRs CERK1, LYK4 and LYK5, as they were weaker in cerk1 and lyk4 lyk5 mutants than in wild-type plants. Cross-elicitation experiments between MLG43 and the carbohydrate MAMP chitohexaose [b-1,4-D-(GlcNAc) 6 ], which is also perceived by these LysM PRRs, indicated that the mechanism of MLG43 recognition could differ from that of chitohexaose, which is fully impaired in cerk1 and lyk4 lyk5 plants. MLG43 treatment confers enhanced disease resistance in A. thaliana to the oomycete Hyaloperonospora arabidopsidis and in tomato and pepper to different bacterial and fungal pathogens. Our data support the classification of MLGs as a group of carbohydrate-based molecular patterns that are perceived by plants and trigger immune responses and disease resistance.

DNA cytosine methylation is a widespread epigenetic mechanism in eukaryotes, and plant genomes commonly are densely methylated. Genomic methylation can be associated with functional consequences such as mutational events, genomic instability or altered gene expression, but little is known on interspecific variation in global cytosine methylation in plants. In this paper, we compare global cytosine methylation estimates obtained by HPLC and use a phylogenetically-informed analytical approach to test for significance of evolutionary signatures of this trait across 54 angiosperm species in 25 families. We evaluate whether interspecific variation in global cytosine methylation is statistically related to phylogenetic distance and also whether it is evolutionarily correlated with genome size (C-value). Global cytosine methylation varied widely between species, ranging between 5.3% (Arabidopsis) and 39.2% (Narcissus). Differences between species were related to their evolutionary trajectories, as denoted by the strong phylogenetic signal underlying interspecific variation. Global cytosine methylation and genome size were evolutionarily correlated, as revealed by the significant relationship between the corresponding phylogenetically independent contrasts. On average, a ten-fold increase in genome size entailed an increase of about 10% in global cytosine methylation. Results show that global cytosine methylation is an evolving trait in angiosperms whose evolutionary trajectory is significantly linked to changes in genome size, and suggest that the evolutionary implications of epigenetic mechanisms are likely to vary between plant lineages.