Molecular evolution and diversification of proteins involved in miRNA maturation pathway

Small RNAs (smRNA, 19–25 nucleotides long), which are transcribed by RNA polymerase II, regulate the expression of genes involved in a multitude of processes in eukaryotes. miRNA biogenesis and the proteins involved in the biogenesis pathway differ across plant and animal lineages. The major proteins constituting the biogenesis pathway, namely, the Dicers (DCL/DCR) and Argonautes (AGOs), have been extensively studied. However, the accessory proteins (DAWDLE (DDL), SERRATE (SE), and TOUGH (TGH)) of the pathway that differs across the two lineages remain largely uncharacterized. We present the first detailed report on the molecular evolution and divergence of these proteins across eukaryotes. Although DDL is present in eukaryotes and prokaryotes, SE and TGH appear to be specific to eukaryotes. The addition/deletion of specific domains and/or domain-specific sequence divergence in the three proteins points to the observed functional divergence of these proteins across the two lineages, which correlates with the differences in miRNA length across the two lineages. Our data enhance the current understanding of the structure–function relationship of these proteins and reveals previous unexplored crucial residues in the three proteins that can be used as a basis for further functional characterization. The data presented here on the number of miRNAs in crown eukaryotic lineages are consistent with the notion of the expansion of the number of miRNA-coding genes in animal and plant lineages correlating with organismal complexity. Whether this difference in functionally correlates with the diversification (or presence/absence) of the three proteins studied here or the miRNA signaling in the plant and animal lineages is unclear. Based on our results of the three proteins studied here and previously available data concerning the evolution of miRNA genes in the plant and animal lineages, we believe that miRNAs probably evolved once in the ancestor to crown eukaryotes and have diversified independently in the eukaryotes

NHX antiporters regulate the pH of endoplasmic reticulum and auxin-mediated development

AtNHX5 and AtNHX6 are endosomal Na+,K+/H+ antiporters that are critical for growth and development in Arabidopsis, but the mechanism behind their action remains unknown. Here, we report that AtNHX5 and AtNHX6, functioning as H+ leak, control auxin homeostasis and auxin-mediated development. We found that nhx5 nhx6 exhibited growth variations of auxin-related defects. We further showed that nhx5 nhx6 was affected in auxin homeostasis. Genetic analysis showed that AtNHX5 and AtNHX6 were required for the function of the ER-localized auxin transporter PIN5. Although AtNHX5 and AtNHX6 were co-localized with PIN5 at ER, they did not interact directly. Instead, the conserved acidic residues in AtNHX5 and AtNHX6, which are essential for exchange activity, were required for PIN5 function. AtNHX5 and AtNHX6 regulated the pH in ER. Overall, AtNHX5 and AtNHX6 may regulate auxin transport across the ER via the pH gradient created by their transport activity. H+-leak pathway provides a fine-tuning mechanism that controls cellular auxin fluxes

Mutually opposing activity of PIN7 splicing isoforms is required for auxin-mediated tropic responses in Arabidopsis thaliana

Summary Advanced transcriptome sequencing has uncovered that the majority of eukaryotic genes undergo alternative splicing (AS). Nonetheless, little effort has been dedicated to investigating the functional relevance of particular splicing events, even those in the key developmental and hormonal regulators. Combining approaches of genetics, biochemistry and advanced confocal microscopy, we describe the impact of alternative splicing on the PIN7 gene in the plant model Arabidopsis thaliana. PIN7 encodes a polarly localized transporter for the phytohormone auxin and produces two evolutionary-conserved transcripts PIN7a and PIN7b. PIN7a and PIN7b, differing in a 4-amino acid motif, exhibit almost identical expression pattern and subcellular localization. We reveal that they closely associate and mutually influence their mobility within the plasma membrane. Phenotypic complementation tests indicate that the functional contribution of PIN7b per se is minor, but it markedly reduces the prominent PIN7a activity, which is required for correct seedling apical hook formation and auxin-mediated tropic responses. Our results establish alternative splicing of the PIN family as a conserved, functionally relevant mechanism, unveiling an additional regulatory level of auxin-mediated plant development.Advanced transcriptome sequencing has revealed that the majority of eukaryotic genes undergo alternative splicing (AS). Nonetheless, little effort has been dedicated to investigating the functional relevance of particular splicing events, even those in the key developmental and hormonal regulators. Combining approaches of genetics, biochemistry and advanced confocal microscopy, we describe the impact of alternative splicing on the PIN7 gene in the model plant Arabidopsis thaliana. PIN7 encodes a polarly localized transporter for the phytohormone auxin and produces two evolutionarily conserved transcripts, PIN7a and PIN7b. PIN7a and PIN7b, differing in a four amino acid stretch, exhibit almost identical expression patterns and subcellular localization. We reveal that they are closely associated and mutually influence each other's mobility within the plasma membrane. Phenotypic complementation tests indicate that the functional contribution of PIN7b per se is minor, but it markedly reduces the prominent PIN7a activity, which is required for correct seedling apical hook formation and auxin-mediated tropic responses. Our results establish alternative splicing of the PIN family as a conserved, functionally relevant mechanism, revealing an additional regulatory level of auxin-mediated plant development.Peer reviewe

Molecular evolution and diversification of the SMXL gene family

Strigolactones (SLs) are a relatively recent addition to the list of plant hormones that control different aspects of plant development. SL signalling is perceived by an alpha/beta hydrolase, DWARF 14 (D14). A close homolog of D14, KARRIKIN INSENSTIVE2 (KAI2), is involved in perception of an uncharacterized molecule called karrikin (KAR). Recent studies in Arabidopsis identified the SUPPRESSOR OF MAX2 1 (SMAX1) and SMAX1-LIKE 7 (SMXL7) to be potential SCF-MAX2 complex-mediated proteasome targets of KAI2 and D14, respectively. Genetic studies on SMXL7 and SMAX1 demonstrated distinct developmental roles for each, but very little is known about these repressors in terms of their sequence features. In this study, we performed an extensive comparative analysis of SMXLs and determined their phylogenetic and evolutionary history in the plant lineage. Our results show that SMXL family members can be subdivided into four distinct phylogenetic clades/classes, with an ancient SMAX1. Further, we identified the Glade-specific motifs that have evolved and that might act as determinants of SL-KAR signalling specificity. These specificities resulted from functional diversities among the clades. Our results suggest that a gradual co-evolution of SMXL members with their upstream receptors D14/KAI2 provided an increased specificity to both the SL perception and response in land plants

Role of the Arabidopsis PIN6 auxin transporter in auxin homeostasis and auxin-mediated development

Plant-specific PIN-formed (PIN) efflux transporters for the plant hormone auxin are required for tissue-specific directional auxin transport and cellular auxin homeostasis. The Arabidopsis PIN protein family has been shown to play important roles in developmental processes such as embryogenesis, organogenesis, vascular tissue differentiation, root meristem patterning and tropic growth. Here we analyzed roles of the less characterised Arabidopsis PIN6 auxin transporter. PIN6 is auxin-inducible and is expressed during multiple auxin–regulated developmental processes. Loss of pin6 function interfered with primary root growth and lateral root development. Misexpression of PIN6 affected auxin transport and interfered with auxin homeostasis in other growth processes such as shoot apical dominance, lateral root primordia development, adventitious root formation, root hair outgrowth and root waving. These changes in auxin-regulated growth correlated with a reduction in total auxin transport as well as with an altered activity of DR5-GUS auxin response reporter. Overall, the data indicate that PIN6 regulates auxin homeostasis during plant development.Christopher I. Cazzonelli, Marleen Vanstraelen, Sibu Simon, Kuide Yin, Ashley Carron-Arthur, Nazia Nisar, Gauri Tarle, Abby J. Cuttriss¤, Iain R. Searle, Eva Benkova, Ulrike Mathesius, Josette Masle, Jiří Friml, Barry J. Pogso

Analýza komplexity procesů souvisejících s auxinem a jejich regulace

Souhrn Rostlinný hormon (fytohormon) auxin je důležitým regulátorem růstu a vývoje rostlin. Koncentrační maxima auxinu, nutná pro regulaci vývoje rostlin, se vytvářejí na základě spolupůsobení metabolických procesů, pasivní difuse molekul auxinu přes buněčnou membránu a aktivního transportu auxinu prostřednictvím přenašečů. V naší práci jsme použili skupinu sloučenin strukturně příbuzných hlavnímu endogennímu auxinu kyselině indol-3-octové a syntetickým auxinům kyselinám 2,4-dichlor- fenoxyoctové a naftalen-1-octové. Cílem bylo charakterizovat specifitu vůči auxinům u vývojově významných procesů, jako je aktivní transport auxinu přes buněčnou membránu a "genomický (transkripční)" a "negenomický (netranskripční)" přenos auxinem neseného signálu. Kromě vlastní charakterizace fyziologické aktivity daných látek jsme chtěli přispět k pochopení komplexní regulace fyziologických procesů závislých na auxinu a případně nalézt takové látky, jejichž chování se bude u jednotlivých procesů výrazně lišit. S využitím vybraných látek spolu s dalšími molekulárními nástroji jsme analyzovali negenomický mechanismus přenosu auxinového signálu, způsob účinku styrylových FM (Fei-Mao)-barviv na dynamiku membránově lokalizovaných auxinových transportérů, a účast ostatních fytohormonů - jmenovitě cytokininů - při regulaci hladin...Phytohormone auxin plays an important role in various aspects of plant growth and development. The necessary concentration maxima at the region of its action are achieved by auxin metabolism, passive diffusion of auxin molecules across plasma membrane and by the carrier-mediated auxin transport, as well as by modulation of these processes. In our study we used a group of compounds structurally related to major endogenous auxin indole-3-acetic acid, as well as synthetic auxins 2,4-dichlorophenoxy acetic acid (2,4- D) and naphthalene-1-acetic acid (NAA). We aimed to characterize the auxin specificity of developmentally important processes such as carrier-mediated auxin transport, and 'genomic' (transcriptional) and 'non-genomic' (transcriptional) auxin signaling. In addition to the characterization of these compounds we also hoped to get an insight into the complex regulatory mechanism of auxin-related processes and to possibly find a particular compound with distinct behavior towards particular processes. By making use of such compounds and other molecular tools we aimed to analyze the mechanism of 'non-genomic' auxin signaling, to understand the mode of action of FM (Fei Mao) styryl dyes on the dynamics of membrane- localized auxin transporters, and to study the involvement of other phytohormones...Katedra biochemieDepartment of BiochemistryPřírodovědecká fakultaFaculty of Scienc

Analyzing the complexity of auxin-related processes and their regulation

Phytohormone auxin plays an important role in various aspects of plant growth and development. The necessary concentration maxima at the region of its action are achieved by auxin metabolism, passive diffusion of auxin molecules across plasma membrane and by the carrier-mediated auxin transport, as well as by modulation of these processes. In our study we used a group of compounds structurally related to major endogenous auxin indole-3-acetic acid, as well as synthetic auxins 2,4-dichlorophenoxy acetic acid (2,4- D) and naphthalene-1-acetic acid (NAA). We aimed to characterize the auxin specificity of developmentally important processes such as carrier-mediated auxin transport, and 'genomic' (transcriptional) and 'non-genomic' (transcriptional) auxin signaling. In addition to the characterization of these compounds we also hoped to get an insight into the complex regulatory mechanism of auxin-related processes and to possibly find a particular compound with distinct behavior towards particular processes. By making use of such compounds and other molecular tools we aimed to analyze the mechanism of 'non-genomic' auxin signaling, to understand the mode of action of FM (Fei Mao) styryl dyes on the dynamics of membrane- localized auxin transporters, and to study the involvement of other phytohormones..

Why plants need more than one type of auxin

The versatile functionality and physiological importance of the phytohormone auxin is a major focus of attention in contemporary plant science. Recent studies have substantially contributed to our understanding of the molecular mechanisms underlying the physiological role of auxin in plant development. The mechanism of auxin action includes both fast responses not involving gene expression, possibly mediated by Auxin Binding Protein 1 (ABP1), and slower responses requiring auxin-regulated gene expression mediated by F-box proteins. These two mechanisms of action have been described to varying degrees for the major endogenous auxin indole-3-acetic acid (IAA) and for the synthetic auxins 2,4- dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (NAA). However, in addition to IAA, plants synthesize three other compounds that are commonly regarded as "endogenous auxins", namely, 4-chloroindole-3-acetic acid (4-Cl-IAA), indole-3-butyric acid (IBA) and phenylacetic acid (PAA). Although a spectrum of auxinic effects has been identified for all these as well as several other endogenous compounds, we remain largely ignorant of many aspects of their mechanisms of action and the extent to which they contribute to auxin-regulated plant development. Here, we briefly summarize the action of IBA, 4-Cl-IAA and PAA, and discuss the extent to which their action overlaps with that of IAA or results from their metabolic conversions to IAA. Other possible pathways for their action are considered. We present a scheme for homeostatic regulation of IAA levels that embraces other endogenous auxins in terms of the described mechanism of auxin action including its receptor and downstream signal transduction events

Trismus as a first presenting complaint in a case of myasthenia gravis

The initial presentation of myasthenia gravis as trismus is very rare and no previous reports have been found in the literature. A 35-year-old male presented to the outpatient unit of our department with inability to clench well and to open his mouth. Physical examination revealed that he had clinical findings consistent with the signs and symptoms of myasthenia gravis. He was immediately referred to a neurologist, who confirmed that he was in an advanced stage of myasthenia gravis with severe deficit to his respiratory muscles and he was promptly treated. He is presently on a maintenance drug therapy. To our knowledge, this is the first reported case of myasthenia gravis whose initial presentation was trismus. This case presents a rare but important diagnosis that should be added to the differential diagnosis of trismus