Gene Discovery and Characterization from Non-sequenced Medicinal Plants

Plant-derived secondary metabolites still constitute important resources for currently prescribed drugs and for the discovery of active principles which are effective in new indication areas. Promising compounds are of-

Pichia pastoris酵母中表达人源中和性抗甲型肝炎病毒scFv-Fc融合抗体的研究

为了探讨人源抗甲型肝炎(甲肝)病毒scFv-Fc融合抗体在酵母中的表达特性,将获得的人源抗甲肝病毒中和性单链可变区抗体(scFv抗体)基因克隆入含信号肽及人IgG1Fc抗体基因的酵母细胞表达载体中,获得了一株中和性人源抗甲肝病毒pPiscFv-FcHA16融合抗体的分泌表达,并对表达产物进行了纯化。同时对表达产物的生物学特性进行了一系列鉴定。表达的pPiscFv-FcHA16融合抗体为具有不同糖基化形式的同源二聚体,与相应的CHO细胞表达的IgG抗体相比,pPiscFv-FcHA16融合抗体仍保持很好的抗原结合活性,以及与中和性鼠抗甲肝病毒单克隆抗体的竞争抑制能力。同时也保持了对甲肝病毒的体外中和活性。这些结果表明,在酵母中表达的单链可变区(scFv)与IgG1Fc区的融合抗体具有很好的生物学活性,有希望用做体外诊断,用纯化相应的抗原,或者可能用于体内预防与治疗。

Role of Arabidopsis UV RESISTANCE LOCUS 8 in Plant Growth Reduction under Osmotic Stress and Low Levels of UV-B

In high-light environments, plants are exposed to different types of stresses, such as an excess of UV-B, but also drought stress which triggers a common morphogenic adaptive response resulting in a general reduction of plant growth. Here, we report that the Arabidopsis thaliana UVRESISTANCE LOCUS 8 （UVR8） gene, a known regulator of the UV-B morphogenic response, was able to complement a Saccharomyces cerevisiae osmo-sensitive mutant and its expression was induced after osmotic or salt stress in Arabidopsis plants. Under low levels of UV-B, plants overexpressing UVR8 are dwarfed with a reduced root development and accumulate more flavonoids compared to control plants. The growth defects are mainly due to the inhibition of cell expansion. The growth inhibition triggered by UVR8 overexpression in plants under low levels of UV-B was exacerbated by mannitol-induced osmotic stress, but it was not significantly affected by ionic stress. In contrast, uvr8-6 mutant plants do not differ from wild-type plants under standard conditions, but they show an increased shoot growth under high-salt stress. Our data suggest that UVR8-mediated accumulation of flavonoid and possibly changes in auxin homeostasis are the underlying mechanism of the observed growth phenotypes and that UVR8 might have an important role for integrating plant growth and stress signals.

The Anaphase-Promoting Complex/Cyclosome in Control of Plant Development

Temporal controlled degradation of key cell division proteins ensures a correct onset of the different cell cycle phases and exit from the cell division program. In light of the cell cycle, the Anaphase-Promoting Complex/Cyclosome （APC/C） is an important conserved multi-subunit ubiquitin ligase, marking targets for degradation by the 26S protea- some. However, whereas the APC/C has been studied extensively in yeast and mammals, only in the last decade has the plant APC/C started to unveil its secrets. Research results have shown the importance of the APC/C core complex and its activators during gametogenesis, growth, hormone signaling, symbiotic interactions, and endoreduplication onset. In addition, recently, the first plant APC/C inhibitors have been reported, allowing a fine-tuning of APC/C activity during the cell cycle. Together with the identification of the first APC/C targets, a picture emerges of APC/C activity being essential for many different developmental processes.

Higher Plant Proteins of Cyanobacterial Origin:Are They or Are They Not Preferentially Targeted to Chloroplasts?

Dear Editor, What does the evolutionary origin of a plant protein tell about its subcellular localization？ Naively thinking, one would assume that plant proteins that were originally encoded in the endosymbiont genome are targeted to the chloroplast. However, published data seem to support only a loose link between evolutionary origin and subcel- lular localization. About half of the Arabidopsis proteins with a detectable cyanobacterial ortholog are targeted to subcellular compartments other than the chloroplast （Martin et al., 2002）. H

The Formation of Anthocyanic Vacuolar Inclusions in Arabidopsis thaliana and Implications for the Sequestration of Anthocyanin Pigments

Anthocyanins are flavonoid pigments that accumulate in the large central vacuole of most plants. Inside the vacuole, anthocyanins can be found uniformly distributed or as part of sub-vacuolar pigment bodies, the Anthocyanic Vacuolar Inclusions （AVIs）. Using Arabidopsis seedlings grown under anthocyanin-inductive conditions as a model to un- derstand how AVIs are formed, we show here that the accumulation of AVIs strongly correlates with the formation of cyanidin 3-glucoside （C3G） and derivatives. Arabidopsis mutants that fail to glycosylate anthocyanidins at the 5-0 position （Sgt mutant） accumulate AVIs in almost every epidermal cell of the cotyledons, as compared to wild-type seedlings, where only a small fraction of the cells show AVIs. A similar phenomenon is observed when seedlings are treated with vanadate. Highlighting a role for autophagy in the formation of the AVIs, we show that various mutants that interfere with the autophagic process （atg mutants） display lower numbers of AVIs, in addition to a reduced accumulation of anthocyanins. Interestingly, vanadate increases the numbers of AVIs in the atg mutants, suggesting that several pathways might participate in AVl formation. Taken together, our results suggest novel mechanisms for the formation of sub-vacuolar compartments capable of accumulating anthocyanin pigments.

From Squalene to Brassinolide： The Steroid Metabolic and Signaling Pathways across the Plant Kingdom

The plant steroid hormones, brassinosteroids （BRs）, and their precursors, phytosterols, play major roles in plant growth, development, and stress tolerance. Here, we review the impressive progress made during recent years in elucidating the components of the sterol and BR metabolic and signaling pathways, and in understanding their mecha- nism of action in both model plants and crops, such as Arabidopsis and rice. We also discuss emerging insights into the regulations of these pathways, their interactions with other hormonal pathways and multiple environmental signals, and the putative nature of sterols as signaling molecules.

Molecular and Physiological Analysis of Growth-Limiting Drought Stress in Brachypodium distachyon Leaves

The drought-tolerant grass Brachypodium distachyon is an emerging model species for temperate grasses and cereal crops. To explore the usefulness of this species for drought studies, a reproducible in vivo drought assay was developed. Spontaneous soil drying led to a 45% reduction in leaf size, and this was mostly due to a decrease in cell expansion, whereas cell division remained largely unaffected by drought. To investigate the molecular basis of the observed leaf growth reduction, the third Brachypodium leaf was dissected in three zones, namely proliferation, expan- sion, and mature zones, and subjected to transcriptome analysis, based on a whole-genome tiling array. This approach allowed us to highlight that transcriptome profiles of different developmental leaf zones respond differently to drought. Several genes and functional processes involved in drought tolerance were identified. The transcriptome data suggest an increased energy availability in the proliferation zones, along with an up-regulation of sterol synthesis that may influ-ence membrane fluidity. This information may be used to improve the tolerance of temperate cereals to drought, which is undoubtedly one of the major environmental challenges faced by agriculture today and in the near future.

Anterograde and Retrograde Regulation of Nuclear Genes Encoding Mitochondrial Proteins during Growth, Development, and Stress

Mitochondrial biogenesis and function in plants require the expression of over 1000 nuclear genes encoding mitochondrial proteins （NGEMPs）. The expression of these genes is regulated by tissue-specific, developmental, internal, and external stimuli that result in a dynamic organelle involved in both metabolic and a variety of signaling processes. Although the metabolic and biosynthetic machinery of mitochondria is relatively well understood, the factors that regu- late these processes and the various signaling pathways involved are only beginning to be identified at a molecular level. The molecular components of anterograde （nuclear to mitochondrial） and retrograde （mitochondrial to nuclear） signaling pathways that regulate the expression of NGEMPs interact with chloroplast-, growth-, and stress-signaling pathways in the cell at a variety of levels, with common components involved in transmission and execution of these signals. This positions mitochondria as important hubs for signaling in the cell, not only in direct signaling of mitochondrial function per se, but also in sensing and/or integrating a variety of other internal and external signals. This integrates and optimizes growth with energy metabolism and stress responses, which is required in both photosynthetic and non-photosynthetic cells.

Cellular and Molecular Requirements for Polar PIN Targeting and Transcytosis in Plants

The polar, sub-cellular localization of PIN auxin efflux carriers determines the direction of intercellular auxin flow, thus defining the spatial aspect of auxin signalling. Dynamic, transcytosis-like relocalizations of PIN proteins occur in response to external and internal signals, integrating these signals into changes in auxin distribution. Here, we examine the cellular and molecular mechanisms of polar PIN delivery and transcytosis. The mechanisms of the ARF-GEF-dependent polar targeting and transcytosis are well conserved and show little variations among diverse Arabidopsis ecotypes consistent with their fundamental importance in regulating plant development. At the cellular level, we refine previous findings on the role of the actin cytoskeleton in apical and basal PIN targeting, and identify a previously unknown role for microtubules, specifically in basal targeting. PIN protein delivery to different sides of the cell is mediated by ARFdependent trafficking with a previously unknown complex level of distinct ARF-GEF vesicle trafficking regulators. Our data suggest that alternative recruitment of PIN proteins by these distinct pathways can account for cell type- and cargo-specific aspects of polar targeting, as well as for polarity changes in response to different signals. The resulting dynamic PIN positioning to different sides of cells defines a three-dimensional pattern of auxin fluxes within plant tissues.

Spatial H202 Signaling Specificity： H202 from Chloroplasts and Peroxisomes Modulates the Plant Transcriptome Differentially

Hydrogen peroxide （H2O2） operates as a signaling molecule in eukaryotes, but the specificity of its signal- ing capacities remains largely unrevealed. Here, we analyzed whether a moderate production of H2O2 from two different plant cellular compartments has divergent effects on the plant transcriptome. Arabidopsis thaliana overexpressing glycolate oxidase in the chloroplast （Fahnenstich et al., 2008; Balazadeh et al., 2012） and plants deficient in peroxisomal catalase （Queval et al., 2007; Inze et al., 2012） were grown under non-photorespiratory conditions and then transferred to photorespiratory conditions to foster the production of H202 in both organelles. We show that H202 originating in a specific organelle induces two types of responses： one that integrates signals independently from the subcellular site of H202 production and another that is dependent on the H2O2 production site. H2O2 produced in peroxisomes induces transcripts involved in protein repair responses, while H2O2 produced in chloroplasts induces early signaling responses, including transcription factors and biosynthetic genes involved in production of secondary signaling messengers. There is a significant bias towards the induction of genes involved in responses to wounding and pathogen attack by chloroplas- tic-produced H202, including indolic glucosinolates-, camalexin-, and stigmasterol-biosynthetic genes. These transcriptional responses were accompanied by the accumulation of 4-methoxy-indol-3-ylmethyl glucosinolate and stigmasterol.

New Strigolactone Analogs as Plant Hormones with Low Activities in the Rhizosphere

Strigolactones （SLs） are known not only as plant hormones, but also as rhizosphere signals for establishing symbiotic and parasitic interactions. The design of new specific SL analogs is a challenging goal in understanding the basic plant biology and is also useful to control plant architectures without favoring the development of parasitic plants. Two different molecules （23 （3＇-methyI-GR24）, 31 （thia-3＇-methyl-debranone-like molecule）） already described, and a new one （AR36）, for which the synthesis is presented, are biologically compared with the well-known GR24 and the recently identified CISA-1. These different structures emphasize the wide range of parts attached to the D-ring for the bioactivity as a plant hormone. These new compounds possess a common dimethylbutenolide motif but their structure varies in the ABC part of the molecules： 23 has the same ABC part as GR24, while 31 and AR36 carry, respectively, an aromatic ring and an acyclic carbon chain. Detailed information is given for the bioactivity of such derivatives in strigolactone synthesis or in perception mutant plants （pea rmsl and rms4, Arabidopsis max2 and, max4） for different hormonal functions along with their action in the rhizosphere on arbuscular mycorrhizal hyphal growth and parasitic weed germination.

Addressing the Role of microRNAs in Reprogramming Leaf Growth during Drought Stress in Brachypodium distachyon

Plant responses to drought are regulated by complex genetic and epigenetic networks leading to rapid reprogramming of plant growth, miRNAs have been widely indicated as key players in the regulation of growth and development. The role of miRNAs in drought response was investigated in young leaves of Brachypodium dis- tachyon, a drought-tolerant monocot model species. Adopting an in vivo drought assay, shown to cause a dramatic reduction in leaf size, mostly due to reduced cell expansion, small RNA libraries were produced from proliferating and expanding leaf cells. Next-generation sequencing data were analyzed using an in-house bioinformatics pipeline allowing the identification of 66 annotated miRNA genes and 122 new high confidence predictions greatly expand- ing the number of known Brachypodium miRNAs. In addition, we identified four TAS3 loci and a large number of siRNA-producing loci that show characteristics suggesting that they may represent young miRNA genes. Most miR- NAs showed a high expression level, consistent with their involvement in early leaf development and cell identity. Proliferating and expanding leaf cells respond differently to drought treatment and differential expression analyses suggest novel evidence for an miRNA regulatory network controlling cell division in both normal and stressed condi-tions and demonstrate that drought trigger a genetic reprogramming of leaf growth in which miRNAs are deeply involved.

Enquiry into the Topology of Plasma Membrane- Localized PIN Auxin Transport Components

Auxin directs plant ontogenesis via differential accumulation within tissues depending largely on the activity of PIN proteins that mediate auxin efflux from cells and its directional cell-to-cell transport. Regard- less of the developmental importance of PINs, the structure of these transporters is poorly characterized. Here, we present experimental data concerning protein topology of plasma membrane-localized PINs. Utilizing approaches based on pH-dependent quenching of fluorescent reporters combined with immuno- localization techniques, we mapped the membrane topology of PINs and further cross-validated our results using available topology modeling software. We delineated the topology of PIN1 with two transmembrane （TM） bundles of five m-helices linked by a large intracellular loop and a C-terminus positioned outside the cytoplasm. Using constraints derived from our experimental data, we also provide an updated position of helical regions generating a verisimilitude model of PIN1. Since the canonical long PINs show a high degree of conservation in TM domains and auxin transport capacity has been demonstrated for Arabidopsis representatives of this group, this empirically enhanced topological model of PIN1 will be an important starting point for further studies on PIN structure-function relationships. In addition, we have established protocols that can be used to probe the topology of other plasma membrane proteins in plants.

Cellular Auxin Homeostasis： Gatekeeping Is Housekeeping

The phytohormone auxin is essential for plant development and contributes to nearly every aspect of the plant life cycle. The spatio-temporal distribution of auxin depends on a complex interplay between auxin metabolism and cell-to-cell auxin transport. Auxin metabolism and transport are both crucial for plant development; however, it largely remains to be seen how these processes are integrated to ensure defined cellular auxin levels or even gradients within tissues or organs. In this review, we provide a glance at very diverse topics of auxin biology, such as biosynthesis, conjugation, oxidation, and transport of auxin. This broad, but certainly superficial, overview highlights the mutual importance of auxin metabolism and transport. Moreover, it allows pinpointing how auxin metabolism and transport get integrated to jointly regulate cellular auxin homeostasis. Even though these processes have been so far only separately studied, we assume that the phytohormonal crosstalk integrates and coordinates auxin metabolism and transport. Besides the integrative power of the global hormone signaling, we additionally introduce the hypothetical concept considering auxin transport components as gatekeepers for auxin responses.

Osmotic Stress Modulates the Balance between Exocytosis and Clathrin-Mediated Endocytosis in Arabidopsis thaliana

The sessile life style of plants creates the need to deal with an often adverse environment, in which water availability can change on a daily basis, challenging the cellular physiology and integrity. Changes in os- motic conditions disrupt the equilibrium of the plasma membrane： hypoosmotic conditions increase and hyperosmotic environment decrease the cell volume. Here, we show that short-term extracellular osmotic treatments are closely followed by a shift in the balance between endocytosis and exocytosis in root mer- istem cells. Acute hyperosmotic treatments （ionic and nonionic） enhance clathrin-mediated endocytosis simultaneously attenuating exocytosis, whereas hypoosmotic treatments have the opposite effects. In addition to clathrin recruitment to the plasma membrane, components of early endocytic trafficking are essential during hyperosmotic stress responses. Consequently, growth of seedlings defective in elements of clathrin or early endocytic machinery is more sensitive to hyperosmotic treatments. We also found that the endocytotic response to a change of osmotic status in the environment is dominant over the presum- ably evolutionary more recent regulatory effect of plant hormones, such as auxin. These results imply that osmotic perturbation influences the balance between endocytosis and exocytosis acting through clathrin- mediated endocytosis. We propose that tension on the plasma membrane determines the addition or removal of membranes at the cell surface, thus preserving cell integrity.

A Fluorescent Alternative to the Synthetic Strigolactone GR24

Strigolactones have recently been implicated in both above- and below-ground developmental pathways in higher plants. To facilitate the molecular and chemical properties of strigolactones in vitro and in vivo, we have devel- oped a fluorescent strigolactone molecule, CISA-1, synthesized via a novel method which was robust, high-yielding, and used simple starting materials. We demonstrate that CISA-1 has a broad range of known strigolactone activities and further report on an adventitious rooting assay in Arabidopsis which is a highly sensitive and rapid method for testing biological activity of strigolactone analogs. In this rooting assay and the widely used Orobanche germination assay, CISA-1 showed stronger biological activity than the commonly tested GR24. CISA-1 and GR24 were equally effective at inhibiting branching in Arabidopsis inflorescence stems. In both the branching and adventitious rooting assay, we also demonstrated that CISA-1 activity is dependent on the max strigolactone signaling pathway. In water methanol solu- tions, CISA-1 was about threefold more stable than GR24, which may contribute to the increased activity observed in the various biological tests.

PEP7 acts as a peptide ligand for the receptor kinase SIRK1 to regulate aquaporin-mediated water influx and lateral root growth

Plant receptors constitute a large protein family that regulates various aspects of development and responses to external cues.Functional characterization of this protein family and the identification of their ligands remain major challenges in plant biology.Previously,we identified plasma membrane-intrinsic sucrose-induced receptor kinase 1(SIRK1)and Qian Shou kinase 1(QSK1)as receptor/co-receptor pair involved in the regulation of aquaporins in response to osmotic conditions induced by sucrose.In this study,we identified a member of the elicitor peptide(PEP)family,namely PEP7,as the specific ligand of th receptor kinase SIRK1.PEP7 binds to the extracellular domain of SIRK1 with a binding constant of 1.44±0.79μM and is secreted to the apoplasm specifically in response to sucrose treatment.Stabilization of a signaling complex involving SIRK1,QSK1,and aquaporins as substrates is mediated by alterations in the external sucrose concentration or by PEP7 application.Moreover,the presence of PEP7 induces the phosphorylation of aquaporins in vivo and enhances water influx into protoplasts.Disturbed water influx,in turn,led to delayed lateral root development in the pep7 mutant.The loss-of-function mutant of SIRK1 is not responsive to external PEP7 treatment regarding kinase activity,aquaporin phosphorylation,water influx activity,and lateral root development.Taken together,our data indicate that the PEP7/SIRK1/QSK1 complex represents a crucial perception and response module that mediates sucrose-controlled water flux in plants and lateral root development.

The Sensor Histidine Kinases AHK2 and AHK3 Proceed into Multiple Serine/Threonine/Tyrosine Phosphorylation Pathways in Arabidopsis thaliana

Cytokinins are N^6-substituted adenine derivatives that are involved in the regulation of numerous aspects of plant growth and development. These include the control of cell division, leaf senescence, apical dominance, sink^source relationship, vascular and embryonic development, and apical meristem activity （Kieber and Schaller, 2014）. In addition, there is increasing evidence for the function of cytokinins in abiotic stress responses （Zwack and Rashotte, 2015）.