Impairment of sugar transport in the vascular system acts on nitrogen remobilization and nitrogen use efficiency in Arabidopsis.

Carbon (C) and nitrogen (N) metabolisms have long been known to be coupled, and this is required for adjusting nitrogen use efficiency (NUE). Despite this intricate relationship, it is still unclear how deregulation of sugar transport impacts N allocation. Here, we investigated in Arabidopsis the consequences of the simultaneous downregulation of the genes coding for the sugar transporters SWEET11, SWEET12, SWEET16, and SWEET17 on various anatomical and physiological traits ranging from the stem's vascular system development to plant biomass production, seed yield, and N remobilization and use efficiency. Our results show that intracellular sugar exchanges mediated by SWEET16 and SWEET17 proteins specifically impact vascular development but do not play a significant role in the distribution of N. Most importantly, we showed that the double mutant swt11 swt12, which has an impacted vascular development, displays an improved NUE and nitrogen remobilization to the seeds. In addition, a significant negative correlation between sugar and amino acids contents and the inflorescence stem radial growth exists, highlighting the complex interaction between the maintenance of C/N homeostasis and the inflorescence stem development. Our results thus deepen the link between sugar transport, C/N allocation, and vascular system development.

A vacuolar hexose transport is required for xylem development in the inflorescence stem.

In Angiosperms, the development of the vascular system is controlled by a complex network of transcription factors. However, how nutrient availability in the vascular cells affects their development remains to be addressed. At the cellular level, cytosolic sugar availability is regulated mainly by sugar exchanges at the tonoplast through active and/or facilitated transport. In Arabidopsis (Arabidopsis thaliana), among the genes encoding tonoplastic transporters, SUGAR WILL EVENTUALLY BE EXPORTED TRANSPORTER 16 (SWEET16) and SWEET17 expression has been previously detected in the vascular system. Here, using a reverse genetics approach, we propose that sugar exchanges at the tonoplast, regulated by SWEET16, are important for xylem cell division as revealed in particular by the decreased number of xylem cells in the swt16 mutant and the accumulation of SWEET16 at the procambium-xylem boundary. In addition, we demonstrate that transport of hexoses mediated by SWEET16 and/or SWEET17 is required to sustain the formation of the xylem secondary cell wall. This result is in line with a defect in the xylem cell wall composition as measured by Fourier-transformed infrared spectroscopy in the swt16swt17 double mutant and by upregulation of several genes involved in secondary cell wall synthesis. Our work therefore supports a model in which xylem development partially depends on the exchange of hexoses at the tonoplast of xylem-forming cells.

The Physcomitrium (Physcomitrella) patens PpKAI2L receptors for strigolactones and related compounds function via MAX2-dependent and -independent pathways.

In angiosperms, the α/ß hydrolase DWARF14 (D14), along with the F-box protein MORE AXILLARY GROWTH2 (MAX2), perceives strigolactones (SL) to regulate developmental processes. The key SL biosynthetic enzyme CAROTENOID CLEAVAGE DIOXYGENASE8 (CCD8) is present in the moss Physcomitrium patens, and PpCCD8-derived compounds regulate moss extension. The PpMAX2 homolog is not involved in the SL response, but 13 PpKAI2LIKE (PpKAI2L) genes homologous to the D14 ancestral paralog KARRIKIN INSENSITIVE2 (KAI2) encode candidate SL receptors. In Arabidopsis thaliana, AtKAI2 perceives karrikins and the elusive endogenous KAI2-Ligand (KL). Here, germination assays of the parasitic plant Phelipanche ramosa suggested that PpCCD8-derived compounds are likely noncanonical SLs. (+)-GR24 SL analog is a good mimic for PpCCD8-derived compounds in P. patens, while the effects of its enantiomer (-)-GR24, a KL mimic in angiosperms, are minimal. Interaction and binding assays of seven PpKAI2L proteins pointed to the stereoselectivity toward (-)-GR24 for a single clade of PpKAI2L (eu-KAI2). Enzyme assays highlighted the peculiar behavior of PpKAI2L-H. Phenotypic characterization of Ppkai2l mutants showed that eu-KAI2 genes are not involved in the perception of PpCCD8-derived compounds but act in a PpMAX2-dependent pathway. In contrast, mutations in PpKAI2L-G, and -J genes abolished the response to the (+)-GR24 enantiomer, suggesting that PpKAI2L-G, and -J proteins are receptors for moss SLs.

Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling.

Strigolactones (SLs) are key hormonal regulators of flowering plant development and are widely distributed amongst streptophytes. In Arabidopsis, SLs signal via the F-box protein MORE AXILLARY GROWTH2 (MAX2), affecting multiple aspects of development including shoot branching, root architecture and drought tolerance. Previous characterization of a Physcomitrella patens moss mutant with defective SL synthesis supports an ancient role for SLs in land plants, but the origin and evolution of signalling pathway components are unknown. Here we investigate the function of a moss homologue of MAX2, PpMAX2, and characterize its role in SL signalling pathway evolution by genetic analysis. We report that the moss Ppmax2 mutant shows very distinct phenotypes from the moss SL-deficient mutant. In addition, the Ppmax2 mutant remains sensitive to SLs, showing a clear transcriptional SL response in dark conditions, and the response to red light is also altered. These data suggest divergent evolutionary trajectories for SL signalling pathway evolution in mosses and vascular plants. In P. patens, the primary roles for MAX2 are in photomorphogenesis and moss early development rather than in SL response, which may require other, as yet unidentified, factors.

Protein labeling strategies for liquid-state NMR spectroscopy using cell-free synthesis.

Preparation of a protein sample for liquid-state nuclear magnetic resonance (NMR) spectroscopy analysis requires optimization of many parameters. This review describes labeling strategies for obtaining assignments of protein resonances. Particular emphasis is placed on the advantages of cell-free protein production, which enables exclusive labeling of the protein of interest, thereby simplifying downstream processing steps and increasing the availability of different labeling strategies for a target protein. Furthermore, proteins can be synthesized in milligram yields, and the open nature of the cell-free system allows the addition of stabilizers, scrambling inhibitors or hydrophobic solubilization environments directly during the protein synthesis, which is especially beneficial for membrane proteins. Selective amino acid labeling of the protein of interest, the possibility of addressing scrambling issues and avoiding the need for labile amino acid precursors have been key factors in enabling the introduction of new assignment strategies based on different labeling schemes as well as on new pulse sequences. Combinatorial selective labeling methods have been developed to reduce the number of protein samples necessary to achieve a complete backbone assignment. Furthermore, selective labeling helps to decrease spectral overlap and overcome size limitations for solution NMR analysis of larger complexes, oligomers, intrinsically disordered proteins and membrane proteins.

Analyzing native membrane protein assembly in nanodiscs by combined non-covalent mass spectrometry and synthetic biology.

Membrane proteins frequently assemble into higher order homo- or hetero-oligomers within their natural lipid environment. This complex formation can modulate their folding, activity as well as substrate selectivity. Non-disruptive methods avoiding critical steps, such as membrane disintegration, transfer into artificial environments or chemical modifications are therefore essential to analyze molecular mechanisms of native membrane protein assemblies. The combination of cell-free synthetic biology, nanodisc-technology and non-covalent mass spectrometry provides excellent synergies for the analysis of membrane protein oligomerization within defined membranes. We exemplify our strategy by oligomeric state characterization of various membrane proteins including ion channels, transporters and membrane-integrated enzymes assembling up to hexameric complexes. We further indicate a lipid-dependent dimer formation of MraY translocase correlating with the enzymatic activity. The detergent-free synthesis of membrane protein/nanodisc samples and the analysis by LILBID mass spectrometry provide a versatile platform for the analysis of membrane proteins in a native environment.

From Nanodiscs to Isotropic Bicelles: A Procedure for Solution Nuclear Magnetic Resonance Studies of Detergent-Sensitive Integral Membrane Proteins.

Nanodiscs and isotropic bicelles are promising membrane mimetics in the field of solution nuclear magnetic resonance (NMR) spectroscopy of integral membrane proteins (IMPs). Despite varied challenges to solution NMR studies of IMPs, we attribute the paucity of solution NMR structures in these environments to the inability of diverse IMPs to withstand detergent treatment during standard nanodisc and bicelle preparations. Here, we present a strategy that creates small isotropic bicelles from IMPs co-translationally embedded in large nanodiscs using cell-free expression. Our results demonstrate appreciable gains in NMR spectral quality while preserving lipid-IMP contacts. We validate the approach on the detergent-sensitive LspA, which finally allowed us to perform high-quality triple-resonance NMR experiments for structural studies. Our strategy of producing bicelles from nanodiscs comprehensively avoids detergent during expression and preparation and is suitable for solution NMR spectroscopy of lipid-IMP complexes.

Simple and Efficient Targeting of Multiple Genes Through CRISPR-Cas9 in Physcomitrella patens.

Powerful genome editing technologies are needed for efficient gene function analysis. The CRISPR-Cas9 system has been adapted as an efficient gene-knock-out technology in a variety of species. However, in a number of situations, knocking out or modifying a single gene is not sufficient; this is particularly true for genes belonging to a common family, or for genes showing redundant functions. Like many plants, the model organism Physcomitrella patens has experienced multiple events of polyploidization during evolution that has resulted in a number of families of duplicated genes. Here, we report a robust CRISPR-Cas9 system, based on the codelivery of a CAS9 expressing cassette, multiple sgRNA vectors, and a cassette for transient transformation selection, for gene knock-out in multiple gene families. We demonstrate that CRISPR-Cas9-mediated targeting of five different genes allows the selection of a quintuple mutant, and all possible subcombinations of mutants, in one experiment, with no mutations detected in potential off-target sequences. Furthermore, we confirmed the observation that the presence of repeats in the vicinity of the cutting region favors deletion due to the alternative end joining pathway, for which induced frameshift mutations can be potentially predicted. Because the number of multiple gene families in Physcomitrella is substantial, this tool opens new perspectives to study the role of expanded gene families in the colonization of land by plants.

Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression.

Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein. We demonstrate the feasibility of using this approach for the K(+) channels KcsA and MVP and the amino acid transporter LeuT. We determine the crystal structure of the KcsA channel obtained by CFPS and in vitro folding to show the structural similarity to the cellular expressed KcsA channel and to establish the feasibility of using this two-step approach for membrane protein production for structural studies. Our studies show that the correct folding of these membrane proteins with complex topologies can take place in vitro without the involvement of the cellular machinery for membrane protein biogenesis. This indicates that the folding instructions for these complex membrane proteins are contained entirely within the protein sequence.

Structural modelling and transcriptional responses highlight a clade of PpKAI2-LIKE genes as candidate receptors for strigolactones in Physcomitrella patens.

MAIN CONCLUSION: A set of PpKAI2 - LIKE paralogs that may encode strigolactone receptors in Physcomitrella patens were identified through evolutionary, structural, and transcriptional analyses, suggesting that strigolactone perception may have evolved independently in basal land plants in a similar manner as spermatophytes. Carotenoid-derived compounds known as strigolactones are a new class of plant hormones that modulate development and interactions with parasitic plants and arbuscular mycorrhizal fungi. The strigolactone receptor protein DWARF14 (D14) belongs to the α/ß hydrolase family. D14 is closely related to KARRIKIN INSENSITIVE2 (KAI2), a receptor of smoke-derived germination stimulants called karrikins. Strigolactone and karrikin structures share a butenolide ring that is necessary for bioactivity. Charophyte algae and basal land plants produce strigolactones that influence their development. However phylogenetic studies suggest that D14 is absent from algae, moss, and liverwort genomes, raising the question of how these basal plants perceive strigolactones. Strigolactone perception during seed germination putatively evolved in parasitic plants through gene duplication and neofunctionalization of KAI2 paralogs. The moss Physcomitrella patens shows an increase in KAI2 gene copy number, similar to parasitic plants. In this study we investigated whether P. patens KAI2-LIKE (PpKAI2L) genes may contribute to strigolactone perception. Based on phylogenetic analyses and homology modelling, we predict that a clade of PpKAI2L proteins have enlarged ligand-binding cavities, similar to D14. We observed that some PpKAI2L genes have transcriptional responses to the synthetic strigolactone GR24 racemate or its enantiomers. These responses were influenced by light and dark conditions. Moreover, (+)-GR24 seems to be the active enantiomer that induces the transcriptional responses of PpKAI2L genes. We hypothesize that members of specific PpKAI2L clades are candidate strigolactone receptors in moss.

Strigolactones inhibit caulonema elongation and cell division in the moss Physcomitrella patens.

In vascular plants, strigolactones (SLs) are known for their hormonal role and for their role as signal molecules in the rhizosphere. SLs are also produced by the moss Physcomitrella patens, in which they act as signaling factors for controlling filament extension and possibly interaction with neighboring individuals. To gain a better understanding of SL action at the cellular level, we investigated the effect of exogenously added molecules (SLs or analogs) in moss growth media. We used the previously characterized Ppccd8 mutant that is deficient in SL synthesis and showed that SLs affect moss protonema extension by reducing caulonema cell elongation and mainly cell division rate, both in light and dark conditions. Based on this effect, we set up bioassays to examine chemical structure requirements for SL activity in moss. The results suggest that compounds GR24, GR5, and 5-deoxystrigol are active in moss (as in pea), while other analogs that are highly active in the control of pea branching show little activity in moss. Interestingly, the karrikinolide KAR1, which shares molecular features with SLs, did not have any effect on filament growth, even though the moss genome contains several genes homologous to KAI2 (encoding the KAR1 receptor) and no canonical homologue to D14 (encoding the SL receptor). Further studies should investigate whether SL signaling pathways have been conserved during land plant evolution.

Strigolactones regulate protonema branching and act as a quorum sensing-like signal in the moss Physcomitrella patens.

Strigolactones are a novel class of plant hormones controlling shoot branching in seed plants. They also signal host root proximity during symbiotic and parasitic interactions. To gain a better understanding of the origin of strigolactone functions, we characterised a moss mutant strongly affected in strigolactone biosynthesis following deletion of the CAROTENOID CLEAVAGE DIOXYGENASE 8 (CCD8) gene. Here, we show that wild-type Physcomitrella patens produces and releases strigolactones into the medium where they control branching of protonemal filaments and colony extension. We further show that Ppccd8 mutant colonies fail to sense the proximity of neighbouring colonies, which in wild-type plants causes the arrest of colony extension. The mutant phenotype is rescued when grown in the proximity of wild-type colonies, by exogenous supply of synthetic strigolactones or by ectopic expression of seed plant CCD8. Thus, our data demonstrate for the first time that Bryophytes (P. patens) produce strigolactones that act as signalling factors controlling developmental and potentially ecophysiological processes. We propose that in P. patens, strigolactones are reminiscent of quorum-sensing molecules used by bacteria to communicate with one another.

A hypothesis on the identification of the editing enzyme in plant organelles.

RNA editing in plant organelles is an enigmatic process leading to conversion of cytidines into uridines. Editing specificity is determined by proteins; both those known so far are pentatricopeptide repeat (PPR) proteins. The enzyme catalysing RNA editing in plants is still totally unknown. We propose that the DYW domain found in many higher plant PPR proteins is the missing catalytic domain. This hypothesis is based on two compelling observations: (i) the DYW domain contains invariant residues that match the active site of cytidine deaminases; (ii) the phylogenetic distribution of the DYW domain is strictly correlated with RNA editing.

Dual targeting is the rule for organellar aminoacyl-tRNA synthetases in Arabidopsis thaliana.

In plants, protein synthesis occurs in the cytosol, mitochondria, and plastids. Each compartment requires a full set of tRNAs and aminoacyl-tRNA synthetases. We have undertaken a systematic analysis of the targeting of organellar aminoacyl-tRNA synthetases in the model plant Arabidopsis thaliana. Dual targeting appeared to be a general rule. Among the 24 identified organellar aminoacyl-tRNA synthetases (aaRSs), 15 (and probably 17) are shared between mitochondria and plastids, and 5 are shared between cytosol and mitochondria (one of these aaRSs being present also in chloroplasts). Only two were shown to be uniquely chloroplastic and none to be uniquely mitochondrial. Moreover, there are no examples where the three aaRS genes originating from the three ancestral genomes still coexist. These results indicate that extensive exchange of aaRSs has occurred during evolution and that many are now shared between two or even three compartments. The findings have important implications for studies of the translation machinery in plants and on protein targeting and gene transfer in general.

Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis.

The complete sequence of the Arabidopsis thaliana genome revealed thousands of previously unsuspected genes, many of which cannot be ascribed even putative functions. One of the largest and most enigmatic gene families discovered in this way is characterized by tandem arrays of pentatricopeptide repeats (PPRs). We describe a detailed bioinformatic analysis of 441 members of the Arabidopsis PPR family plus genomic and genetic data on the expression (microarray data), localization (green fluorescent protein and red fluorescent protein fusions), and general function (insertion mutants and RNA binding assays) of many family members. The basic picture that arises from these studies is that PPR proteins play constitutive, often essential roles in mitochondria and chloroplasts, probably via binding to organellar transcripts. These results confirm, but massively extend, the very sparse observations previously obtained from detailed characterization of individual mutants in other organisms.

The gene encoding Arabidopsis thaliana mitochondrial ribosomal protein S13 is a recent duplication of the gene encoding plastid S13.

A gene encoding mitochondrial S13 is generally present in the mitochondrial genome of higher plants, but is lacking from the Arabidopsis thaliana mitochondrial genome. Previous research has failed to identify a nuclear gene capable of encoding a mitochondrial S13 protein or the protein itself. Doubts have even been raised as to whether a mitochondrial S13 exists in Arabidopsis. Here, we show that the nuclear gene encoding the plastid S13 has been partially duplicated in A. thaliana, such that the copy has lost the exon encoding the plastid transit peptide and acquired a sequence capable of encoding a mitochondrial targeting sequence. The two S13 sequences were fused to green fluorescent protein and shown to be targeted to plastids and mitochondria respectively.