Comparative genomic in situ hybridization (cGISH) analysis of the genomic relationships among Sinapis arvensis, Brassica rapa and Brassica nigra

To further understand the relationships between the SS genome of Sinapis arvensis and the AA, BB genomes in Brassica, genomic DNA of Sinapis arvensis was hybridized to the metaphase chromosomes of Brassica nigra (BB genome), and the metaphase chromosomes and interphase nucleus of Brassica rapa (AA genome) by comparative genomic in situ hybridization (cGISH). As a result, every chromosome of B. nigra had signals along the whole chromosomal length. However, only half of the condensed heterochromatic areas in the interphase nucleus and the chromosomes showed rich signals in Brassica rapa. Interphase nucleus and the metaphase chromosomes of S. arvensis were simultaneously hybridized with digoxigenin-labeled genomic DNA of B. nigra and biotin-labeled genomic DNA of B. rapa. Signals of genomic DNA of B. nigra hybridized throughout the length of all chromosomes and all the condensed heterochromatic areas in the interphase nucleus, except chromosome 4, of which signals were weak in centromeric regions. Signals of the genomic DNA of B. rapa patterned the most areas of ten chromosomes and ten condensed heterochromatic areas, others had less signals. The results showed that the SS genome had homology with AA and BB genomes, but the homology between SS genome and AA genome was clearly lower than that between the SS genome and BB genome.     

The Arabidopsis LARP1s are Involved in Regulation of Seed Germination

Members of La-related protein (LARP) 1 family spread widely in various species, and they are involved in regulating many important biological processes in mammal, yeast, and fruit fly. However, functional research of LARP1s in plants is limited so far. In Arabidopsis, there are three members in LARP1 family, LARP1a, 1b, and 1c. Here, we found that the mutation of LARP1 genes delayed seed germination, implying that LARP1 proteins might be positive factors of seed germination in Arabidopsis. Moreover, the larp1 mutants showed more sensitive to abscisic acid (ABA) and paclobutrazol (PAC), as larp1 mutants displayed low rate of germination in medium contained ABA or PAC. Temporal and spatial expression analyses revealed that LARP1s were more abundant in seeds, especially in imbibed seeds. Subcellular localization analysis revealed that all LARP1 proteins could localize to the P-bodies, suggesting that LARP1s might play a role in RNA processes. Taken together, our results unravel new conserved functions of LARP1s in the regulation of Arabidopsis seed germination.

     

Kv Channel S1-S2 Linker Working as a Binding Site of Human β-Defensin 2 for Channel Activation Modulation

Among the three extracellular domains of the tetrameric voltage-gated K⁺ (Kv) channels consisting of six membrane-spanning helical segments named S1–S6, the functional role of the S1-S2 linker still remains unclear because of the lack of a peptide ligand. In this study, the Kv1.3 channel S1-S2 linker was reported as a novel receptor site for human β-defensin 2 (hBD2). hBD2 shifts the conductance-voltage relationship curve of the human Kv1.3 channel in a positive direction by nearly 10.5 mV and increases the activation time constant for the channel. Unlike classical gating modifiers of toxin peptides from animal venoms, which generally bind to the Kv channel S3-S4 linker, hBD2 only targets residues in both the N and C termini of the S1-S2 linker to influence channel gating and inhibit channel currents. The increment and decrement of the basic residue number in a positively charged S4 sensor of Kv1.3 channel yields conductance-voltage relationship curves in the positive direction by ∼31.2 mV and 2–4 mV, which suggests that positively charged hBD2 is anchored in the channel S1-S2 linker and is modulating channel activation through electrostatic repulsion with an adjacent S4 helix. Together, these findings reveal a novel peptide ligand that binds with the Kv channel S1-S2 linker to modulate channel activation. These findings also highlight the functional importance of the Kv channel S1-S2 linker in ligand recognition and modification of channel activation.     

SjAPI,the First Functionally Characterized Ascaris-Type Protease Inhibitor from Animal Venoms

Background

Serine protease inhibitors act as modulators of serine proteases, playing important roles in protecting animal toxin peptides from degradation. However, all known serine protease inhibitors discovered thus far from animal venom belong to the Kunitz-type subfamily, and whether there are other novel types of protease inhibitors in animal venom remains unclear.

Principal Findings

Here, by screening scorpion venom gland cDNA libraries, we identified the first Ascaris-type animal toxin family, which contains four members: Scorpiops jendeki Ascaris-type protease inhibitor (SjAPI), Scorpiops jendeki Ascaris-type protease inhibitor 2 (SjAPI-2), Chaerilus tricostatus Ascaris-type protease inhibitor (CtAPI), and Buthus martensii Ascaris-type protease inhibitor (BmAPI). The detailed characterization of Ascaris-type peptide SjAPI from the venom gland of scorpion Scorpiops jendeki was carried out. The mature peptide of SjAPI contains 64 residues and possesses a classical Ascaris-type cysteine framework reticulated by five disulfide bridges, different from all known protease inhibitors from venomous animals. Enzyme and inhibitor reaction kinetics experiments showed that recombinant SjAPI was a dual function peptide with α-chymotrypsin- and elastase-inhibiting properties. Recombinant SjAPI inhibited α-chymotrypsin with a Ki of 97.1 nM and elastase with a Ki of 3.7 μM, respectively. Bioinformatics analyses and chimera experiments indicated that SjAPI contained the unique short side chain functional residues “AAV” and might be a useful template to produce new serine protease inhibitors.

Conclusions/Significance

To our knowledge, SjAPI is the first functionally characterized animal toxin peptide with an Ascaris-type fold. The structural and functional diversity of animal toxins with protease-inhibiting properties suggested that bioactive peptides from animal venom glands might be a new source of protease inhibitors, which will accelerate the development of diagnostic and therapeutic agents for human diseases that target diverse proteases.     

A Single Nucleotide Deletion in Gibberellin20-oxidase1 Causes Alpine Dwarfism in Arabidopsis

Alpine dwarfism is widely observed in alpine plant populations and often considered a high-altitude adaptation, yet its molecular basis and ecological relevance remain unclear. In this study, we used map-based cloning and field transplant experiments to investigate dwarfism in natural Arabidopsis (Arabidopsis thaliana) accessions collected from the Swiss Alps. A loss-of-function mutation due to a single nucleotide deletion in gibberellin20-oxidase1 (GA5) was identified as the cause of dwarfism in an alpine accession. The mutated allele, ga5-184, was found in two natural Arabidopsis populations collected from one geographic region at high altitude, but was different from all other reported ga5 null alleles, suggesting that this allele has evolved locally. In field transplant experiments, the dwarf accession with ga5-184 exhibited a fitness pattern consistent with adaptation to high altitude. Across a wider array of accessions from the Swiss Alps, plant height decreased with altitude of origin, but fitness patterns in the transplant experiments were variable and general altitudinal adaptation was not evident. In general, our study provides new insights into molecular basis and possible ecological roles of alpine dwarfism, and demonstrates the importance of the GA-signaling pathway for the generation of ecologically relevant variation in higher plants.Adaptation to environmental conditions is one of the evolutionary processes that can lead to lineage divergence and the generation of biodiversity (Savolainen et al., 2013). Local adaptation within species may evolve in spatially heterogeneous environments if the strength of divergent selection among populations overrides other evolutionary forces (e.g. genetic drift, gene flow), and is manifested in an increased fitness of local genotypes in the local habitat when compared with foreign genotypes in the local habitat and when compared with fitness of the local genotypes in foreign habitats (Kawecki and Ebert, 2004; Savolainen et al., 2013). It is often a challenge for classic studies of local adaptation to identify traits mediating local adaptation and the underlying genetic architectures. To date, our knowledge on the molecular basis of local adaptation remains remarkably limited, even though this topic has been extensively studied in recent decades (Bergelson and Roux, 2010; Barrett and Hoekstra, 2011). A particular difficulty in studying the molecular basis of local adaptation comes from the need to combine meaningful field experiments together with relevant molecular studies to provide solid evidence on the adaptive value of phenotypes and associated genotypes. Therefore, a complete study of molecular mechanisms of adaptation ideally requires (1) identifying candidate traits under natural selection or relevant for adaptation, (2) isolating genes/loci that influence candidate traits, and (3) quantifying the fitness (adaptive value) conferred by different alleles under natural conditions (Bergelson and Roux, 2010). In the model plant Arabidopsis (Arabidopsis thaliana) and its relatives, as well as in several animal systems, great advances in detecting the molecular basis of adaptation have been made (e.g. Linnen et al., 2009; Fournier-Level et al., 2011; Hancock et al., 2011). Nevertheless, discussions on adaptionist storytelling or of producing molecular spandrels have been recurrent (for review, see Barrett and Hoekstra, 2011).Alpine habitats are characterized by a fine mosaic of heterogeneous and often extreme environmental conditions (Körner, 2003; Byars et al., 2007; Pico, 2012). Along altitudinal gradients, changes in environmental conditions are often steep, and plants growing in alpine areas have developed a variety of morphological and physiological adaptations that allow them to cope with extreme conditions. Alpine plant dwarfism, that is, reduced plant stature with increasing altitude, is one of the most common characteristics observed in plant populations originating from high altitudes (Clausen et al., 1948; Körner, 2003). Alpine dwarfism is thought to help alpine plants take advantage of the higher ambient temperature close to the soil surface, allocate more resources to reproduction, decrease damage from strong wind, and reduce evaporation (Turesson, 1922; Körner, 2003). Therefore, alpine dwarfism is widely considered as adaptive in plants (Turesson, 1922; Clausen et al., 1948; Körner, 2003; Byars et al., 2007; Gonzalo-Turpin and Hazard, 2009). However, whether alpine dwarfism is indeed adaptive and how it is genetically controlled remain unknown in many species. This may be partially attributable to the limited genomic resources that are available for alpine plant species, which are usually nonmodel organisms. Hence, studying the molecular basis of this ecologically important trait in model organisms such as Arabidopsis can give much-needed insights (Bergelson and Roux, 2010).Dwarfism and semidwarfism are important and well-studied traits in agriculture because they help overcome lodging and thus substantially contributed to the green revolution in the last century, the unprecedented increase in crop yields due to the adoption of genetically improved crop varieties (Peng et al., 1999; Khush, 2001). It has repeatedly been found in a diversity of plants that dwarfism and semidwarfism were due to deficiencies in either signaling or biosynthesis of GA (e.g. Spray et al., 1996; Peng et al., 1999; Monna et al., 2002). Dwarfism and dwarfing alleles have also been reported in natural Arabidopsis accessions (Barboza et al., 2013), and other recent studies have shown evidence of population differentiation and climatic adaptation along altitudinal gradients in this model plant (Montesinos et al., 2009; Méndez-Vigo et al., 2011, 2013; Montesinos-Navarro et al., 2011; Pico, 2012; Suter et al., 2014; Luo et al., 2015). In this study, we have investigated plant dwarfism in natural Arabidopsis accessions collected in the Swiss Alps (Supplemental Table S1) and identified a loss-of-function mutation in gibberellin20-oxidase1 (GA5, also called GA20ox1) as the cause of dwarfism in an Arabidopsis accession collected from high altitude (2,012 m above sea level [a.s.l.]). In field transplant experiments, this accession displayed fitness patterns consistent with altitudinal adaptation. Across a larger set of regional accessions, including 10 further accessions without the dwarfing mutation, plant height decreased with altitude of origin; however, this pattern could not be tied to adaptive differentiation along altitude.     

Cloning and Genomic Characterization of a Natural Insecticidal Peptide LaIT1 with Unique DDH Structural Fold

Two native peptides with disulfide‐directed hairpin (DDH) fold, LaIT1 and LITX, were recently isolated from scorpion venom, a development that offered insights into exploring the evolutionary linkage between DDH and inhibitor cystine knot (ICK) peptides. In this work, we isolated and identified the full‐length cDNAs of LaTI1, a representative member with DDH fold, and further determined its complete gene structure. The precursor organization of LaIT1 is similar to that of ICK peptides. The LaIT1 gene contains four exons interrupted by three unique introns and differed from ICK peptides, suggesting divergent genomic organizations of DDH peptides and ICK peptides. Phylogenetic analysis further showed that the “simple” DDH peptide originates from the “complex” ICK peptide, rather than the reverse. To the best of our knowledge, this is the first report on the genomic organization of DDH‐fold peptides, and it presents new evidence of an evolutionary linkage between ICK and DDH peptides.     

Characterization of a new Kv1.3 channel-specific blocker, J123, from the scorpion Buthus martensii Karsch

The potassium channel Kv1.3 is an attractive pharmacological target for T-cell-mediated autoimmune diseases, and specific and selective peptidic blockers of Kv1.3 channels have served as valuable therapeutic leads for treating these diseases. Here, we found a new peptide toxin, J123, with 43 amino acids including six cysteine residues by screening the venomous cDNA library of scorpion Buthus martensii Karsch, which has been used as traditional medicine in China for more than 2000 years. The sequence analysis suggested that peptide J123 constituted a new member of the alpha-KTx toxins. The electrophysiological experiments further indicated that peptide J123 has a novel pharmacological profiles: it blocked Kv1.3 channel with high potency (IC(50)=0.79nM), and exhibited good selectivity on Kv1.3 over Kv1.1 (>1000-fold) and Kv1.2 (about 30-fold), respectively. Furthermore, peptide J123 had no activity on SKCa2 and SKCa3 channels at micromolar concentration level. Based on the pharmacological activities, the possible channel-interacting surface of peptide J123 was also predicted by molecular modeling and docking. All these data not only enrich the knowledge of the structure-function relationship of the new Kv1.3-speicific peptide but also present a potential drug candidate for selectively targeting Kv1.3 channels.     

<Emphasis Type="Italic">Arabidopsis</Emphasis> KHZ1 and KHZ2, two novel non-tandem CCCH zinc-finger and K-homolog domain proteins,have redundant roles in the regulation of flowering and senescence

Key message

Progression of the infection canal that conducts rhizobia to the nodule primordium requires a functional Rab GTPase located in Golgi/trans-Golgi that also participate in root hair polar growth.

Abstract

Common bean (Phaseolus vulgaris) symbiotically associates with its partner Rhizobium etli, resulting in the formation of root nitrogen-fixing nodules. Compatible bacteria can reach cortical cells in a tightly regulated infection process, in which the specific recognition of signal molecules is a key step to select the symbiotic partner. In this work, we show that RabA2, a monomeric GTPase from common bean, is required for the progression of the infection canal, referred to as the infection thread (IT), toward the cortical cells. Expression of miss-regulated mutant variants of RabA2 resulted in an increased number of abortive infection events, including bursting of ITs and a reduction in the number of nodules. Nodules formed in these plants were small and contained infected cells with disrupted symbiosome membranes, indicating either early senescence of these cells or defects in the formation of the symbiosome membrane during bacterial release. RabA2 localized to mobile vesicles around the IT, but mutations that affect GTP hydrolysis or GTP/GDP exchange modified this localization. Colocalization of RabA2 with ArfA1 and a Golgi marker indicates that RabA2 localizes in Golgi stacks and the trans-Golgi network. Our results suggest that RabA2 is part of the vesicle transport events required to maintain the integrity of the membrane during IT progression.