Direct imaging of glycans in Arabidopsis roots via click labeling of metabolically incorporated azido-monosaccharides

Background: Carbohydrates, also called glycans, play a crucial but not fully understood role in plant health and development. The non-template driven formation of glycans makes it impossible to image them in vivo with genetically encoded fluorescent tags and related molecular biology approaches. A solution to this problem is the use of tailor-made glycan analogs that are metabolically incorporated by the plant into its glycans. These metabolically incorporated probes can be visualized, but techniques documented so far use toxic copper-catalyzed labeling. To further expand our knowledge of plant glycobiology by direct imaging of its glycans via this method, there is need for novel click-compatible glycan analogs for plants that can be bioorthogonally labelled via copper-free techniques. Results: Arabidopsis seedlings were incubated with azido-containing monosaccharide analogs of N-acetylglucosamine, N-acetylgalactosamine, l-fucose, and l-arabinofuranose. These azido-monosaccharides were metabolically incorporated in plant cell wall glycans of Arabidopsis seedlings. Control experiments indicated active metabolic incorporation of the azido-monosaccharide analogs into glycans rather than through non-specific absorption of the glycan analogs onto the plant cell wall. Successful copper-free labeling reactions were performed, namely an inverse-electron demand Diels-Alder cycloaddition reaction using an incorporated N-acetylglucosamine analog, and a strain-promoted azide-alkyne click reaction. All evaluated azido-monosaccharide analogs were observed to be non-toxic at the used concentrations under normal growth conditions. Conclusions: Our results for the metabolic incorporation and fluorescent labeling of these azido-monosaccharide analogs expand the possibilities for studying plant glycans by direct imaging. Overall we successfully evaluated five azido-monosaccharide analogs for their ability to be metabolically incorporated in Arabidopsis roots and their imaging after fluorescent labeling. This expands the molecular toolbox for direct glycan imaging in plants, from three to eight glycan analogs, which enables more extensive future studies of spatiotemporal glycan dynamics in a wide variety of plant tissues and species. We also show, for the first time in metabolic labeling and imaging of plant glycans, the potential of two copper-free click chemistry methods that are bio-orthogonal and lead to more uniform labeling. These improved labeling methods can be generalized and extended to already existing and future click chemistry-enabled monosaccharide analogs in Arabidopsis

A new bernissartiid crocodyliform from the Lower Cretaceous Wessex Formation (Wealden Group, Barremian) of the Isle of Wight, southern England

Concentration-dependent Ac4GalNAz incorporation (24 h incubation). (DOCX 322 kb

Draft genome sequence of the nitrogen-fixing Rhizobium sullae type strain IS123^T focusing on the key genes for symbiosis with its host Hedysarum coronarium L.

© 2017 Sablok, Rosselli, Seeman, van Velzen, Polone, Giacomini, La Porta, Geurts, Muresu and Squartini. The prominent feature of rhizobia is their molecular dialogue with plant hosts. Such interaction is enabled by the presence of a series of symbiotic genes encoding for the synthesis and export of signals triggering organogenetic and physiological responses in the plant. The genome of the Rhizobium sullae type strain IS123T nodulating the legume Hedysarum coronarium, was sequenced and resulted in 317 scaffolds for a total assembled size of 7,889,576 bp. Its features were compared with those of genomes from rhizobia representing an increasing gradient of taxonomical distance, from a conspecific isolate (Rhizobium sullae WSM1592), to two congeneric cases (Rhizobium leguminosarum bv. viciae and Rhizobium etli) and up to different genera within the legume-nodulating taxa. The host plant is of agricultural importance, but, unlike the majority of other domesticated plant species, it is able to survive quite well in the wild. Data showed that that the type strain of R. sullae, isolated from a wild host specimen, is endowed with a richer array of symbiotic genes in comparison to other strains, species or genera of rhizobia that were rescued from domesticated plant ecotypes. The analysis revealed that the bacterium by itself is incapable of surviving in the extreme conditions that its host plant can tolerate. When exposed to drought or alkaline condition, the bacterium depends on its host to survive. Data are consistent with the view of the plant phenotype as the primary factor enabling symbiotic nitrogen fixing bacteria to survive in otherwise limiting environments

Red clover (Trifolium pratense L.) draft genome provides a platform for trait improvement

Red clover (Trifolium pratense L.) is a globally significant forage legume in pastoral livestock farming systems. It is an attractive component of grassland farming, because of its high yield and protein content, nutritional value and ability to fix atmospheric nitrogen. Enhancing its role further in sustainable agriculture requires genetic improvement of persistency, disease resistance, and tolerance to grazing. To help address these challenges, we have assembled a chromosome-scale reference genome for red clover. We observed large blocks of conserved synteny with Medicago truncatula and estimated that the two species diverged ~23 million years ago. Among the 40,868 annotated genes, we identified gene clusters involved in biochemical pathways of importance for forage quality and livestock nutrition. Genotyping by sequencing of a synthetic population of 86 genotypes show that the number of markers required for genomics-based breeding approaches is tractable, making red clover a suitable candidate for association studies and genomic selection

Specificity in legume nodule symbiosis

Some plant species establish mutualistic cooperation with nitrogen-fixing bacteria to overcome nitrogen shortage. The interaction between legume plants and rhizobial bacteria is the canonical example of such mutualism. It leads to the formation of root nodules, which provide the environment for the bacteria to convert atmospheric dinitrogen into ammonia. Nodule formation is initiated by rhizobium-secreted compounds called nodulation (Nod) factors. Perception of Nod factors by receptors in the plant root cells initiates nodule organogenesis and is essential for bacterial infection (1). Several legumes evolved a narrow host range such that only one or a few rhizobium species can initiate nodulation (2). This specificity is largely determined by the Nod factor receptors. On page 663 of this issue, Bozsoki et al. (3) provide structural characterization of the binding site of a Nod factor receptor in legumes. The structural basis of rhizobial Nod factor recognition is a key to understanding the evolution of specificity in symbioses

What Does It Take to Evolve A Nitrogen-Fixing Endosymbiosis?

Plant rhizo- and phyllospheres are exposed to a plethora of nitrogen-fixing bacteria, providing opportunities for the establishment of symbiotic associations. Nitrogen-fixing endosymbioses are most profitable and have evolved more than ten times in the angiosperms. This suggests that the evolutionary trajectory towards endosymbiosis is not complex. Here, we argue that microbe-induced cell divisions are a prerequisite for the entrance of diazotrophic prokaryotes into living plant cells. For rhizobia and Frankia bacteria, this is achieved by adapting the readout of the common symbiosis signalling pathway, such that cell divisions are induced. The common symbiosis signalling pathway is conserved in the plant kingdom and is required to establish an endosymbiosis with mycorrhizal fungi. We also discuss the adaptations that may have occurred that allowed nitrogen-fixing root nodule endosymbiosis. Studies in legumes, Parasponia, and actinorhizal plants provided insights into the genetic constraints that guided the evolution of nitrogen-fixing root nodules with rhizobium or Frankia bacteria.Oscillation of the nuclear calcium concentration is a hallmark of symbiotic signaling in legumes and nonlegumes.Calcium oscillations can be triggered not only by lipochitooligosaccharides (LCOs), but, in several root nodule-forming plant species, also by nonLCO signal molecules.Ectopic expression of either wild-type or dominant active alleles of seven genes of the common symbiosis signalling pathway trigger spontaneous nodule formation in legumes.</p

A Resurrected Scenario : Single Gain and Massive Loss of Nitrogen-Fixing Nodulation

Root nodule endosymbiosis with nitrogen-fixing bacteria provides plants with unlimited access to fixed nitrogen, but at a significant energetic cost. Nodulation is generally considered to have originated in parallel in different lineages, but this hypothesis downplays the genetic complexity of nodulation and requires independent recruitment of many common features across lineages. Recent phylogenomic studies revealed that genes that function in establishing or maintaining nitrogen-fixing nodules are independently lost in non-nodulating relatives of nitrogen-fixing plants. In our opinion, these data are best explained by a scenario of a single gain followed by massively parallel loss of nitrogen-fixing root nodules triggered by events at geological scale.</p