Perfect and imperfect views of ultraconserved sequences.

Across the human genome, there are nearly 500 'ultraconserved' elements: regions of at least 200 contiguous nucleotides that are perfectly conserved in both the mouse and rat genomes. Remarkably, the majority of these sequences are non-coding, and many can function as enhancers that activate tissue-specific gene expression during embryonic development. From their first description more than 15 years ago, their extreme conservation has both fascinated and perplexed researchers in genomics and evolutionary biology. The intrigue around ultraconserved elements only grew with the observation that they are dispensable for viability. Here, we review recent progress towards understanding the general importance and the specific functions of ultraconserved sequences in mammalian development and human disease and discuss possible explanations for their extreme conservation.

Intrinsic toxicity of the cellular prion protein is regulated by its conserved central region.

The conserved central region (CR) of PrPC has been hypothesized to serve as a passive linker connecting the protein's toxic N-terminal and globular C-terminal domains. Yet, deletion of the CR causes neonatal fatality in mice, implying the CR possesses a protective function. The CR encompasses the regulatory α-cleavage locus, and additionally facilitates a regulatory metal ion-promoted interaction between the PrPC N- and C-terminal domains. To elucidate the role of the CR and determine why CR deletion generates toxicity, we designed PrPC constructs wherein either the cis-interaction or α-cleavage are selectively prevented. These constructs were interrogated using nuclear magnetic resonance, electrophysiology, and cell viability assays. Our results demonstrate the CR is not a passive linker and the native sequence is crucial for its protective role over the toxic N-terminus, irrespective of α-cleavage or the cis-interaction. Additionally, we find that the CR facilitates homodimerization of PrPC , attenuating the toxicity of the N-terminus.

Plant organellar DNA polymerases bypass thymine glycol using two conserved lysine residues.

Plant organelles cope with endogenous DNA damaging agents, byproducts of respiration and photosynthesis, and exogenous agents like ultraviolet light. Plant organellar DNA polymerases (DNAPs) are not phylogenetically related to yeast and metazoan DNAPs and they harbor three insertions not present in any other DNAPs. Plant organellar DNAPs from Arabidopsis thaliana (AtPolIA and AtPolIB) are translesion synthesis (TLS) DNAPs able to bypass abasic sites, a lesion that poses a strong block to replicative polymerases. Besides abasic sites, reactive oxidative species and ionizing radiation react with thymine resulting in thymine glycol (Tg), a DNA adduct that is also a strong block to replication. Here, we report that AtPolIA and AtPolIB bypass Tg by inserting an adenine opposite the lesion and efficiently extend from a Tg-A base pair. The TLS ability of AtPolIB is mapped to two conserved lysine residues: K593 and K866. Residue K593 is situated in insertion 1 and K866 is in insertion 3. With basis on the location of both insertions on a structural model of AtPolIIB, we hypothesize that the two positively charged residues interact to form a clamp around the primer-template. In contrast with nuclear and bacterial replication, where lesion bypass involves an interplay between TLS and replicative DNA polymerases, we postulate that plant organellar DNAPs evolved to exert replicative and TLS activities.

Conservation and innovation: Plastome evolution during rapid radiation of Rhodiola on the Qinghai-Tibetan Plateau.

The amount of plastome sequence data available has soared in the last decade, but the nature of plastome evolution during rapid radiations is largely unknown. Moreover, although there is increasing evidence showing that plastomes may have undergone adaptive evolution in order to allow adaptation to various environments, few studies have systematically investigated the role of the plastome in alpine adaptation. To address these questions, we sequenced and analyzed 12 representative species of Rhodiola, a genus which includes ca. 70 perennial herbs mainly growing in alpine habitats in the Qinghai-Tibet Plateau and the Hengduan Mountains. Rapid radiation in this genus was triggered by the uplift of the Qinghai-Tibet Plateau. We also included nine species of Crassulaceae as the outgroups. All plastomes were conserved with respect to size, structure, and gene content and order, with few variations: each contained 134 genes, including 85 protein-coding genes, 37 tRNAs, 8 rRNAs, and 4 potential pseudogenes. Four types of repeat sequence were detected. Slight contraction and expansion of the inverted repeats were also revealed. Both the genome-wide alignment and sequence polymorphism analyses showed that the inverted repeats and coding regions were more conserved than the single-copy regions and the non-coding regions. Positive selection analyses identified three genes containing sites of positive selection (rpl16, ndhA, ndhH), and one gene with a faster than average rate of evolution (psaA). The products of these genes may be involved in the adaptation of Rhodiola to alpine environments such as low CO2 concentration and high-intensity light.

The HARP-like domain-containing protein AH2/ZRANB3 binds to PCNA and participates in cellular response to replication stress.

Proteins with annealing activity are newly identified ATP-dependent motors that can rewind RPA-coated complementary single-stranded DNA bubbles. AH2 (annealing helicase 2, also named as ZRANB3) is the second protein with annealing activity, the function of which is still unknown. Here, we report that AH2 is recruited to stalled replication forks and that cells depleted of AH2 are hypersensitive to replication stresses. Furthermore, AH2 binds to PCNA, which is crucial for its function at stalled replication forks. Interestingly, we identified a HARP-like (HPL) domain in AH2 that is indispensible for its annealing activity in vitro and its function in vivo. Moreover, searching of HPL domain in SNF2 family of proteins led to the identification of SMARCA1 and RAD54L, both of which possess annealing activity. Thus, this study not only demonstrates the in vivo functions of AH2, but also reveals a common feature of this new subfamily of proteins with annealing activity.

Intracellular Binding Site for a Positive Allosteric Modulator of the Dopamine D1 Receptor.

The binding site for DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one], a positive allosteric modulator (PAM) of the dopamine D1 receptor, was identified and compared with the binding site for CID 2886111 [N-(6-tert-butyl-3-carbamoyl-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)pyridine-4-carboxamide], a reference D1 PAM. From D1/D5 chimeras, the site responsible for potentiation by DETQ of the increase in cAMP in response to dopamine was narrowed down to the N-terminal intracellular quadrant of the receptor; arginine-130 in intracellular loop 2 (IC2) was then identified as a critical amino acid based on a human/rat species difference. Confirming the importance of IC2, a ß2-adrenergic receptor construct in which the IC2 region was replaced with its D1 counterpart gained the ability to respond to DETQ. A homology model was built from the agonist-state ß2-receptor structure, and DETQ was found to dock to a cleft created by IC2 and adjacent portions of transmembrane helices 3 and 4 (TM3 and TM4). When residues modeled as pointing into the cleft were mutated to alanine, large reductions in the potency of DETQ were found for Val119 and Trp123 (flanking the conserved DRY sequence in TM3), Arg130 (located in IC2), and Leu143 (TM4). The D1/D5 difference was found to reside in Ala139; changing this residue to methionine as in the D5 receptor reduced the potency of DETQ by approximately 1000-fold. None of these mutations affected the activity of CID 2886111, indicating that it binds to a different allosteric site. When combined, DETQ and CID 2886111 elicited a supra-additive response in the absence of dopamine, implying that both PAMs can bind to the D1 receptor simultaneously.

TM8 represses developmental timing in Nicotiana benthamiana and has functionally diversified in angiosperms.

BACKGROUND: MADS-box genes are key regulators of plant reproductive development and members of most lineages of this gene family have been extensively studied. However, the function and diversification of the ancient TM8 lineage remains elusive to date. The available data suggest a possible function in flower development in tomato and fast evolution through numerous gene loss events in flowering plants. RESULTS: We show the broad conservation of TM8 within angiosperms and find that in contrast to other MADS-box gene lineages, no gene duplicates have been retained after major whole genome duplication events. Through knock-down of NbTM8 by virus induced gene silencing in Nicotiana benthamiana, we show that NbTM8 represses miR172 together with another MADS-box gene, SHORT VEGETATIVE PHASE (NbSVP). In the closely related species Petunia hybrida, PhTM8 is not expressed under the conditions we investigated and consistent with this, a knock-out mutant did not show a phenotype. Finally, we generated transgenic tomato plants in which TM8 was silenced or ectopically expressed, but these plants did not display a clear phenotype. Therefore, no clear function could be confirmed for Solanum lycopersium. CONCLUSIONS: While the presence of TM8 is generally conserved, it remains difficult to propose a general function in angiosperms. Based on all the available data to date, supplemented with our own results, TM8 function seems to have diversified quickly throughout angiosperms and acts as repressor of miR172 in Nicotiana benthamiana, together with NbSVP.

Dynamic Changes in ANGUSTIFOLIA3 Complex Composition Reveal a Growth Regulatory Mechanism in the Maize Leaf.

Most molecular processes during plant development occur with a particular spatio-temporal specificity. Thus far, it has remained technically challenging to capture dynamic protein-protein interactions within a growing organ, where the interplay between cell division and cell expansion is instrumental. Here, we combined high-resolution sampling of the growing maize (Zea mays) leaf with tandem affinity purification followed by mass spectrometry. Our results indicate that the growth-regulating SWI/SNF chromatin remodeling complex associated with ANGUSTIFOLIA3 (AN3) was conserved within growing organs and between dicots and monocots. Moreover, we were able to demonstrate the dynamics of the AN3-interacting proteins within the growing leaf, since copurified GROWTH-REGULATING FACTORs (GRFs) varied throughout the growing leaf. Indeed, GRF1, GRF6, GRF7, GRF12, GRF15, and GRF17 were significantly enriched in the division zone of the growing leaf, while GRF4 and GRF10 levels were comparable between division zone and expansion zone in the growing leaf. These dynamics were also reflected at the mRNA and protein levels, indicating tight developmental regulation of the AN3-associated chromatin remodeling complex. In addition, the phenotypes of maize plants overexpressing miRNA396a-resistant GRF1 support a model proposing that distinct associations of the chromatin remodeling complex with specific GRFs tightly regulate the transition between cell division and cell expansion. Together, our data demonstrate that advancing from static to dynamic protein-protein interaction analysis in a growing organ adds insights in how developmental switches are regulated.

Genome-wide identification of conserved and novel microRNAs in one bud and two tender leaves of tea plant (Camellia sinensis) by small RNA sequencing, microarray-based hybridization and genome survey scaffold sequences.

BACKGROUND: MicroRNAs (miRNAs) are important for plant growth and responses to environmental stresses via post-transcriptional regulation of gene expression. Tea, which is primarily produced from one bud and two tender leaves of the tea plant (Camellia sinensis), is one of the most popular non-alcoholic beverages worldwide owing to its abundance of secondary metabolites. A large number of miRNAs have been identified in various plants, including non-model species. However, due to the lack of reference genome sequences and/or information of tea plant genome survey scaffold sequences, discovery of miRNAs has been limited in C. sinensis. RESULTS: Using small RNA sequencing, combined with our recently obtained genome survey data, we have identified and analyzed 175 conserved and 83 novel miRNAs mainly in one bud and two tender leaves of the tea plant. Among these, 93 conserved and 18 novel miRNAs were validated using miRNA microarray hybridization. In addition, the expression pattern of 11 conserved and 8 novel miRNAs were validated by stem-loop-qRT-PCR. A total of 716 potential target genes of identified miRNAs were predicted. Further, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that most of the target genes were primarily involved in stress response and enzymes related to phenylpropanoid biosynthesis. The predicted targets of 4 conserved miRNAs were further validated by 5'RLM-RACE. A negative correlation between expression profiles of 3 out of 4 conserved miRNAs (csn-miR160a-5p, csn-miR164a, csn-miR828 and csn-miR858a) and their targets (ARF17, NAC100, WER and MYB12 transcription factor) were observed. CONCLUSION: In summary, the present study is one of few such studies on miRNA detection and identification in the tea plant. The predicted target genes of majority of miRNAs encoded enzymes, transcription factors, and functional proteins. The miRNA-target transcription factor gene interactions may provide important clues about the regulatory mechanism of these miRNAs in the tea plant. The data reported in this study will make a huge contribution to knowledge on the potential miRNA regulators of the secondary metabolism pathway and other important biological processes in C. sinensis.

Three conserved C-terminal residues of influenza fusion peptide alter its behavior at the membrane interface.

The N-terminal fragment of the viral hemagglutinin HA2 subunit is termed a fusion peptide (HAfp). The 23-amino acid peptide (HAfp1-23) contains three C-terminal W21-Y22-G23 residues which are highly conserved among serotypes of influenza A and has been shown to form a tight helical hairpin very distinct from the boomerang structure of HAfp1-20. We studied the effect of peptide length on fusion properties, structural dynamics, and binding to the membrane interface. We developed a novel fusion visualization assay based on FLIM microscopy on giant unilamellar vesicles (GUV). By means of molecular dynamics simulations and spectroscopic measurements, we show that the presence of the three C-terminal W21-Y22-G23 residues promotes the hairpin formation, which orients perpendicularly to the membrane plane and induces more disorder in the surrounding lipids than the less structured HAfp1-20. Moreover, we report cholesterol-enriched domain formation induced exclusively by the longer fusion peptide.

Identification of Conserved Moieties in Metabolic Networks by Graph Theoretical Analysis of Atom Transition Networks.

Conserved moieties are groups of atoms that remain intact in all reactions of a metabolic network. Identification of conserved moieties gives insight into the structure and function of metabolic networks and facilitates metabolic modelling. All moiety conservation relations can be represented as nonnegative integer vectors in the left null space of the stoichiometric matrix corresponding to a biochemical network. Algorithms exist to compute such vectors based only on reaction stoichiometry but their computational complexity has limited their application to relatively small metabolic networks. Moreover, the vectors returned by existing algorithms do not, in general, represent conservation of a specific moiety with a defined atomic structure. Here, we show that identification of conserved moieties requires data on reaction atom mappings in addition to stoichiometry. We present a novel method to identify conserved moieties in metabolic networks by graph theoretical analysis of their underlying atom transition networks. Our method returns the exact group of atoms belonging to each conserved moiety as well as the corresponding vector in the left null space of the stoichiometric matrix. It can be implemented as a pipeline of polynomial time algorithms. Our implementation completes in under five minutes on a metabolic network with more than 4,000 mass balanced reactions. The scalability of the method enables extension of existing applications for moiety conservation relations to genome-scale metabolic networks. We also give examples of new applications made possible by elucidating the atomic structure of conserved moieties.

The Conserved Tetratricopeptide Repeat-Containing C-Terminal Domain of Pseudomonas aeruginosa FimV Is Required for Its Cyclic AMP-Dependent and -Independent Functions.

UNLABELLED: FimV is a Pseudomonas aeruginosa inner membrane protein that regulates intracellular cyclic AMP (cAMP) levels-and thus type IV pilus (T4P)-mediated twitching motility and type II secretion (T2S)-by activating the adenylate cyclase CyaB. Its cytoplasmic domain contains three predicted tetratricopeptide repeat (TPR) motifs separated by an unstructured region: two proximal to the inner membrane and one within the "FimV C-terminal domain," which is highly conserved across diverse homologs. Here, we present the crystal structure of the FimV C terminus, FimV861-919, containing a TPR motif decorated with solvent-exposed, charged side chains, plus a C-terminal capping helix. FimV689, a truncated form lacking this C-terminal motif, did not restore wild-type levels of twitching or surface piliation compared to the full-length protein. FimV689 failed to restore wild-type levels of the T4P motor ATPase PilU or T2S, suggesting that it was unable to activate cAMP synthesis. Bacterial two-hybrid analysis showed that TPR3 interacts directly with the CyaB activator, FimL. However, FimV689 failed to restore wild-type motility in a fimV mutant expressing a constitutively active CyaB (fimV cyaB-R456L), suggesting that the C-terminal motif is also involved in cAMP-independent functions of FimV. The data show that the highly conserved TPR-containing C-terminal domain of FimV is critical for its cAMP-dependent and -independent functions. IMPORTANCE: FimV is important for twitching motility and cAMP-dependent virulence gene expression in P. aeruginosa FimV homologs have been identified in several human pathogens, and their functions are not limited to T4P expression. The C terminus of FimV is remarkably conserved among otherwise very diverse family members, but its role is unknown. We provide here biological evidence for the importance of the C-terminal domain in both cAMP-dependent (through FimL) and -independent functions of FimV. We present X-ray crystal structures of the conserved C-terminal domain and identify a consensus sequence for the C-terminal TPR within the conserved domain. Our data extend our knowledge of FimV's functionally important domains, and the structures and consensus sequences provide a foundation for studies of FimV and its homologs.

Conserved non-coding elements and cis regulation: actions speak louder than words.

It is a truth (almost) universally acknowledged that conserved non-coding genomic sequences function in the cis regulation of neighbouring genes. But is this a misconception? The literature is strewn with examples of conserved non-coding sequences being able to drive reporter expression, but the extent to which such sequences are actually used endogenously in vivo is only now being rigorously explored using unbiased genome-scale approaches. Here, we review the emerging picture, examining the extent to which conserved non-coding sequences equivalently regulate gene expression in different species, or at different developmental stages, and how genomics approaches are revealing the relationship between sequence conservation and functional use of cis-regulatory elements.

Conserved and divergent functions of Nfix in skeletal muscle development during vertebrate evolution.

During mouse skeletal muscle development, the Nfix gene has a pivotal role in regulating fetal-specific transcription. Zebrafish and mice share related programs for muscle development, although zebrafish develops at a much faster rate. In fact, although mouse fetal muscle fibers form after 15 days of development, in fish secondary muscle fibers form by 48 hours post-fertilization in a process that until now has been poorly characterized mechanically. In this work, we studied the zebrafish ortholog Nfix (nfixa) and its role in the proper switch to the secondary myogenic wave. This allowed us to highlight evolutionarily conserved and divergent functions of Nfix. In fact, the knock down of nfixa in zebrafish blocks secondary myogenesis, as in mouse, but also alters primary slow muscle fiber formation. Moreover, whereas Nfix mutant mice are motile, nfixa knockdown zebrafish display impaired motility that probably depends upon disruption of the sarcoplasmic reticulum. We conclude that, during vertebrate evolution, the transcription factor Nfix lost some specific functions, probably as a consequence of the different environment in which teleosts and mammals develop.

Phylogenetic tree-informed microRNAome analysis uncovers conserved and lineage-specific miRNAs in Camellia during floral organ development.

In plants, miRNAs are endogenous small RNAs derived from single-stranded precursors with hairpin structures. The evolution of miRNAs and their targets represents one of the most dynamic circuits directing gene expression, which may play fundamental roles in shaping the development of distinct plant organs. Here we performed high-throughput small RNA sequencing in five organ types of Camellia azalea to capture the spatial profile of small non-coding RNA. In total we obtained >227 million high-quality reads and identified 175 miRNAs with mature and precursor sequences. We aligned the miRNAs to known miRNA databases and revealed some conserved as well as 'newly evolved' miRNA genes. Twelve miRNAs were identified to be specific in the genus Camellia, supporting the lineage-specific manner of expansion of 'young' miRNAs. Through differential expression analysis, we showed that many miRNAs were preferentially abundant in certain organ types. Moreover, hierarchical clustering analysis revealed distinctive expression patterns of tissue-specific miRNAs. Gene Ontology enrichment analysis of targets of stamen- and carpel-specific miRNA subclusters showed that miRNA-target regulatory circuits were involved in many important biological processes, enabling their proper specification and organogenesis, such as 'DNA integration' and 'fruit development'. Further, quantitative PCR of key miRNAs and their target genes revealed anti-correlated patterns, and uncovered the functions of key miRNA-target pairs in different floral organs. Taken together, this work yielded valuable information on miRNA-target regulation in the control of floral organ development and sheds light on the evolution of lineage-specific miRNAs in Camellia.

Essential role of conserved DUF177A protein in plastid 23S rRNA accumulation and plant embryogenesis.

DUF177 proteins are nearly universally conserved in bacteria and plants except the Chlorophyceae algae. Thus far, duf177 mutants in bacteria have not established a function. In contrast, duf177a mutants have embryo lethal phenotypes in maize and Arabidopsis. In maize inbred W22, duf177a mutant embryos arrest at an early transition stage, whereas the block is suppressed in the B73 inbred background, conditioning an albino seedling phenotype. Background-dependent embryo lethal phenotypes are characteristic of maize plastid gene expression mutants. Consistent with the plastid gene expression hypothesis, quantitative real-time PCR revealed a significant reduction of 23S rRNA in an Escherichia coli duf177 knockout. Plastid 23S rRNA contents of duf177a mutant tissues were also markedly reduced compared with the wild-type, whereas plastid 16S, 5S, and 4.5S rRNA contents were less affected, indicating that DUF177 is specifically required for accumulation of prokaryote-type 23S rRNA. An AtDUF177A-green fluorescent protein (GFP) transgene controlled by the native AtDUF177A promoter fully complemented the Arabidopsis atduf177a mutant. Transient expression of AtDUF177A-GFP in Nicotiana benthamiana leaves showed that the protein was localized in chloroplasts. The essential role of DUF177A in chloroplast-ribosome formation is reminiscent of IOJAP, another highly conserved ribosome-associated protein, suggesting that key mechanisms controlling ribosome formation in plastids evolved from non-essential pathways for regulation of the prokaryotic ribosome.

A highly conserved sequence associated with the HIV gp41 loop region is an immunomodulator of antigen-specific T cells in mice.

Modulation of T-cell responses by HIV occurs via distinct mechanisms, 1 of which involves inactivation of T cells already at the stage of virus-cell fusion. Hydrophobic portions of the gp41 protein of the viral envelope that contributes to membrane fusion may modulate T-cell responsiveness. Here we found a highly conserved sequence (termed "ISLAD") that is associated with the membranotropic gp41 loop region. We showed that ISLAD has the ability to bind the T-cell membrane and to interact with the T-cell receptor (TCR) complex. Furthermore, ISLAD inhibited T-cell proliferation and interferon-γ secretion that resulted from TCR engagement through antigen-presenting cells. Moreover, administering ISLAD (10 µg per mouse) to an experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis reduced the severity of the disease. This was related to the inhibition of pathogenic T-cell proliferation and to reduced pro-inflammatory cytokine secretion in the lymph nodes of ISLAD-treated EAE mice. The data suggest that T-cell inactivation by HIV during membrane fusion may lie in part in this conserved sequence associated with the gp41 loop region.

Discovery of insect and human dengue virus host factors.

Dengue fever is the most frequent arthropod-borne viral disease of humans, with almost half of the world's population at risk of infection. The high prevalence, lack of an effective vaccine, and absence of specific treatment conspire to make dengue fever a global public health threat. Given their compact genomes, dengue viruses (DENV-1-4) and other flaviviruses probably require an extensive number of host factors; however, only a limited number of human, and an even smaller number of insect host factors, have been identified. Here we identify insect host factors required for DENV-2 propagation, by carrying out a genome-wide RNA interference screen in Drosophila melanogaster cells using a well-established 22,632 double-stranded RNA library. This screen identified 116 candidate dengue virus host factors (DVHFs). Although some were previously associated with flaviviruses (for example, V-ATPases and alpha-glucosidases), most of the DVHFs were newly implicated in dengue virus propagation. The dipteran DVHFs had 82 readily recognizable human homologues and, using a targeted short-interfering-RNA screen, we showed that 42 of these are human DVHFs. This indicates notable conservation of required factors between dipteran and human hosts. This work suggests new approaches to control infection in the insect vector and the mammalian host.

Molecular cloning and potential function prediction of homologous SOC1 genes in tree peony.

KEY MESSAGE: The central flower integrator PsSOC1 was isolated and its expression profiles were analyzed; then the potential function of PsSOC1 in tree peony was postulated. The six flowering genes PrSOC1, PdSOC1, PsSOC1, PsSOC1-1, PsSOC1-2, and PsSOC1-3 were isolated from Paeonia rockii, Paeonia delavayi, and Paeonia suffruticosa, respectively. Sequence comparison analysis showed that the six genes were highly conserved and shared 99.41% nucleotide identity. Further investigation suggested PsSOC1 was highly homologous to the floral integrators, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), from Arabidopsis. Phylogenetic analysis showed that the SOC1 protein clustering has family specificity and PsSOC1 has a close relationship with homologous SOC1 from Asteraceae species. The studies of PsSOC1's expression patterns in different buds and flower buds, and vegetative organs indicated that PsSOC1 could express in both vegetative and reproductive organs. While the expression of PsSOC1 in different developmental stages of buds was different; high expression levels of PsSOC1 occurred in the bud at the bud sprouting stage and the type I aborted the flower bud. PsSOC1 expression was also shown to be affected by gibberellins (GA), low temperature, and photoperiod. One of the pathways that regulates tree peony flowering may be the GA-inductive pathway. Ectopic expression of PsSOC1 in tobacco demonstrated that greater PsSOC1 expression in the transgenic tobacco plants not only promoted plant growth, but also advanced the flowering time. Finally, the potential function of PsSOC1 in tree peony was postulated.

Functional role of evolutionarily highly conserved residues, N-glycosylation level and domains of the Leishmania miltefosine transporter-Cdc50 subunit.

Cdc50 (cell-cycle control protein 50) is a family of conserved eukaryotic proteins that interact with P4-ATPases (phospholipid translocases). Cdc50 association is essential for the endoplasmic reticulum export of P4-ATPases and proper translocase activity. In the present study, we analysed the role of Leishmania infantum LiRos3, the Cdc50 subunit of the P4-ATPase MLF (miltefosine) transporter [LiMT (L. infantum MLF transporter)], on trafficking and complex functionality using site-directed mutagenesis and domain substitution. We identified 22 invariant residues in the Cdc50 proteins from L. infantum, human and yeast. Seven of these residues are found in the extracellular domain of LiRos3, the conservation of which is critical for ensuring that LiMT arrives at the plasma membrane. The substitution of other invariant residues affects complex trafficking to a lesser extent. Furthermore, invariant residues located in the N-terminal cytosolic domain play a role in the transport activity. Partial N-glycosylation of LiRos3 reduces MLF transport and total N-deglycosylation completely inhibits LiMT trafficking to the plasma membrane. One of the N-glycosylation residues is invariant along the Cdc50 family. The transmembrane and exoplasmic domains are not interchangeable with the other two L. infantum Cdc50 proteins to maintain LiMT interaction. Taken together, these findings indicate that both invariant and N-glycosylated residues of LiRos3 are implicated in LiMT trafficking and transport activity.