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  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 06 August 2013, Vol.110(32), pp.13198-203
    Description: Inflorescence architecture of barley (Hordeum vulgare L.) is common among the Triticeae species, which bear one to three single-flowered spikelets at each rachis internode. Triple spikelet meristem is one of the unique features of barley spikes, in which three spikelets (one central and two lateral spikelets) are produced at each rachis internode. Fertility of the lateral spikelets at triple spikelet meristem gives row-type identity to barley spikes. Six-rowed spikes show fertile lateral spikelets and produce increased grain yield per spike, compared with two-rowed spikes with sterile lateral spikelets. Thus, far, two loci governing the row-type phenotype were isolated in barley that include Six-rowed spike1 (Vrs1) and Intermedium-C. In the present study, we isolated Six-rowed spike4 (Vrs4), a barley ortholog of the maize (Zea mays L.) inflorescence architecture gene RAMOSA2 (RA2). Eighteen coding mutations in barley RA2 (HvRA2) were specifically associated with lateral spikelet fertility and loss of spikelet determinacy. Expression analyses through mRNA in situ hybridization and microarray showed that Vrs4 (HvRA2) controls the row-type pathway through Vrs1 (HvHox1), a negative regulator of lateral spikelet fertility in barley. Moreover, Vrs4 may also regulate transcripts of barley SISTER OF RAMOSA3 (HvSRA), a putative trehalose-6-phosphate phosphatase involved in trehalose-6-phosphate homeostasis implicated to control spikelet determinacy. Our expression data illustrated that, although RA2 is conserved among different grass species, its down-stream target genes appear to be modified in barley and possibly other species of tribe Triticeae.
    Keywords: Egg Apparatus1 ; Cytokinin ; Grain Number ; Yield Potential ; Gene Expression Regulation, Plant ; Hordeum -- Genetics ; Inflorescence -- Genetics ; Plant Proteins -- Genetics
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 2
    Language: English
    In: 2012, Vol.7(7), p.e41867
    Description: Cell specification and differentiation in the endosperm of cereals starts at the maternal-filial boundary and generates the endosperm transfer cells (ETCs). Besides the importance in assimilate transfer, ETCs are proposed to play an essential role in the regulation of endosperm differentiation by affecting development of proximate endosperm tissues. We attempted to identify signalling elements involved in early endosperm differentiation by using a combination of laser-assisted microdissection and 454 transcriptome sequencing. ; 454 sequencing of the differentiating ETC region from the syncytial state until functionality in transfer processes captured a high proportion of novel transcripts which are not available in existing barley EST databases. Intriguingly, the ETC-transcriptome showed a high abundance of elements of the two-component signalling (TCS) system suggesting an outstanding role in ETC differentiation. All components and subfamilies of the TCS, including distinct kinds of membrane-bound receptors, have been identified to be expressed in ETCs. The TCS system represents an ancient signal transduction system firstly discovered in bacteria and has previously been shown to be co-opted by eukaryotes, like fungi and plants, whereas in animals and humans this signalling route does not exist. Transcript profiling of TCS elements by qRT-PCR suggested pivotal roles for specific phosphorelays activated in a coordinated time flow during ETC cellularization and differentiation. ETC-specificity of transcriptionally activated TCS phosphorelays was assessed for early differentiation and cellularization contrasting to an extension of expression to other grain tissues at the beginning of ETC maturation. Features of candidate genes of distinct phosphorelays and transcriptional activation of genes putatively implicated in hormone signalling pathways hint at a crosstalk of hormonal influences, putatively ABA and ethylene, and TCS signalling. ; Our findings suggest an integral function for the TCS in ETC differentiation possibly coupled to sequent hormonal regulation by ABA and ethylene.
    Keywords: Research Article ; Agriculture ; Biology ; Genetics And Genomics ; Plant Biology ; Computational Biology
    E-ISSN: 1932-6203
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  • 3
    Language: English
    In: Molecular Phylogenetics and Evolution, March 2013, Vol.66(3), pp.617-627
    Description: ► We provide the first exhaustive phylogeny of crocuses based on nuclear sequences. ► We reconstruct chromosome number evolution and polyploidization in . ► 8 out of 15 series of are monophyletic, as well as probably both sections. ► Chromosome numbers increased multiple times independently by polyploidization. ► Supernumerary B chromosomes evolved at least five times independently within consists of about 100 species distributed from western Europe and northern Africa to western China, with the center of diversity on the Balkan Peninsula and in Asia Minor. Our study focuses on clarifying phylogenetic relationships and chromosome number evolution within the genus using sequences of the chloroplast L-F region, the nuclear ribosomal DNA internal transcribed spacer (ITS) region, and a part of the nuclear single-copy gene . In a combined dataset of ITS and L-F sequences, 115 individuals representing 110 taxa from both subgenera and all sections and series of were analyzed with Bayesian phylogenetic inference. For 79 individuals representing 74 taxa were included, and for the majority of them PCR amplicons were cloned and up to eight clones per individual were sequenced to detect allopolyploidization events. species were included as outgroup in both analyses. Characteristics of seed surface structures were evaluated by scanning electron microscopy. Phylogenetic analysis of ITS/ L-F data resulted in a monophyletic genus , probably monophyletic sections and , and inferred monophyly for eight of the 15 series of the genus. The aggregate, thought to be consisting of closely related subspecies, was found to be polyphyletic, the taxa occurring within three major clades in the phylogenetic tree. Cloning of resulted in the detection of homoeologous copies in about one third of the taxa of section , indicating an allotetraploid origin of this section. Reconstruction of chromosome number evolution along the phylogenetic tree using a probabilistic and a parsimony approach arrived at partly contradictory results. Both analyses agreed however on the occurrence of multiple polyploidization and dysploidy events. B chromosomes evolved at least five times independently within the genus, preferentially in clades characterized by karyotype changes.
    Keywords: B Chromosome ; Chromosome Number ; Crocus ; Evolution ; Internal Transcribed Spacer (Its) ; Pcosat103 ; Phylogeny ; Trnl-F ; Seed Testa ; Whole Genome Duplication ; Biology
    ISSN: 1055-7903
    E-ISSN: 1095-9513
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  • 4
    Language: English
    In: PLoS ONE, 01 January 2015, Vol.10(10), p.e0140476
    Description: Barley is an attractive vehicle for producing recombinant protein, since it is a readily transformable diploid crop species in which doubled haploids can be routinely generated. High amounts of protein are naturally accumulated in the grain, but optimal endosperm-specific promoters have yet to be perfected. Here, the oat GLOBULIN1 promoter was combined with the legumin B4 (LeB4) signal peptide and the endoplasmic reticulum (ER) retention signal (SE)KDEL. Transgenic barley grain accumulated up to 1.2 g/kg dry weight of recombinant protein (GFP), deposited in small roundish compartments assumed to be ER-derived protein bodies. The molecular farming potential of the system was tested by generating doubled haploid transgenic lines engineered to synthesize the anti-HIV-1 monoclonal antibody 2G12 with up to 160 μg recombinant protein per g grain. The recombinant protein was deposited at the periphery of protein bodies in the form of a mixture of various N-glycans (notably those lacking terminal N-acetylglucosamine residues), consistent with their vacuolar localization. Inspection of protein-A purified antibodies using surface plasmon resonance spectroscopy showed that their equilibrium and kinetic rate constants were comparable to those associated with recombinant 2G12 synthesized in Chinese hamster ovary cells.
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 5
    In: Plant Journal, October 2011, Vol.68(1), pp.40-50
    Description: The histone H3 variant (CENH3) of centromeric nucleosomes is essential for kinetochore assembly and thus for chromosome segregation in eukaryotes. The mechanism(s) that determine centromere identity, assembly and maintenance of kinetochores are still poorly understood. Although the role of CENH3 during mitosis has been studied in several organisms, little is known about its meiotic function. We show that RNAi‐mediated CENH3 knockdown in caused dwarfism as the result of a reduced number of mitotic divisions. The remaining mitotic divisions appeared to be error‐free. CENH3 RNAi transformants had reduced fertility because of frequently disturbed meiotic chromosome segregation. N‐terminally truncated EYFP–CENH3(C) is deposited to and functional within Arabidopsis centromeres of mitotic chromosomes, but cannot be loaded onto centromeres of meiotic nuclei. Thus the N‐terminal part is apparently required for CENH3 loading during meiosis. EYFP–CENH3(C) expression reduces the amount of endogenous CENH3, thus mimicking the effect of RNAi. The consequences of reduced endogenous CENH3 and lack of meiotic incorporation of EYFP–CENH3(C) are reduced fertility caused by insufficient CENH3 loading to the centromeres of meiotic chromosomes, subsequent lagging of chromosomes and formation of micronuclei.
    Keywords: Centromere ; Kinetochore ; Cenh3 ; Arabidopsis Thaliana ; Rnai ; Meiosis
    ISSN: 0960-7412
    E-ISSN: 1365-313X
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  • 6
    In: Plant Journal, August 2012, Vol.71(4), pp.639-655
    Description: Barley endosperm cells differentiate into transfer cells (ETCs) opposite the nucellar projection. To comprehensively analyse ETC differentiation, laser microdissection‐based transcript and metabolite profiles were obtained from laser microdissected tissues and cell morphology was analysed. Flange‐like secondary‐wall ingrowths appeared between 5 and 7 days after pollination within the three outermost cell layers. Gene expression analysis indicated that ethylene‐signalling pathways initiate ETC morphology. This is accompanied by gene activity related to cell shape control and vesicle transport, with abundant mitochondria and endomembrane structures. Gene expression analyses indicate predominant formation of hemicelluloses, glucuronoxylans and arabinoxylans, and transient formation of callose, together with proline and 4‐hydroxyproline biosynthesis. Activation of the methylation cycle is probably required for biosynthesis of phospholipids, pectins and ethylene. Membrane microdomains involving sterols/sphingolipids and remorins are potentially involved in ETC development. The transcriptional activity of assimilate and micronutrient transporters suggests ETCs as the main uptake organs of solutes into the endosperm. Accordingly, the endosperm grows maximally after ETCs are fully developed. Up‐regulated gene expression related to amino acid catabolism, C:N balances, carbohydrate oxidation, mitochondrial activity and starch degradation meets high demands for respiratory energy and carbohydrates, required for cell proliferation and wall synthesis. At 10 days after pollination, ETCs undergo further differentiation, potentially initiated by abscisic acid, and metabolism is reprogrammed as shown by activated storage and stress‐related processes. Overall, the data provide a comprehensive view of barley ETC differentiation and development, and identify candidate genes and associated pathways.
    Keywords: Barley Endosperm ; Differentiation ; Endosperm Transfer Cells ; Transcript Profiling ; Metabolite Profiling ; Assimilate Transport
    ISSN: 0960-7412
    E-ISSN: 1365-313X
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  • 7
    In: New Phytologist, March 2017, Vol.213(4), pp.1909-1924
    Description: The formation of gametes is a prerequisite for any sexually reproducing organism in order to complete its life cycle. In plants, female gametes are formed in a multicellular tissue, the female gametophyte or embryo sac. Although the events leading to the formation of the female gametophyte have been morphologically characterized, the molecular control of embryo sac development remains elusive. We used single and double mutants as well as cell‐specific marker lines to characterize a novel class of gene regulators in Arabidopsis thaliana, the RWP‐RK domain‐containing (RKD) transcription factors. Morphological and histological analyses were conducted using confocal laser scanning and differential interference contrast microscopy. Gene expression and transcriptome analyses were performed using quantitative reverse transcription−PCR and RNA sequencing, respectively. Our results showed that RKD genes are expressed during distinct stages of embryo sac development. Morphological analysis of the mutants revealed severe distortions in gametophyte polarity and cell differentiation. Transcriptome analysis revealed changes in the expression of several gametophyte‐specific gene families (RKD2 and RKD3) and ovule development‐specific genes (RKD3), and identified pleiotropic effects on phytohormone pathways (RKD5). Our data provide novel insight into the regulatory control of female gametophyte development. RKDs are involved in the control of cell differentiation and are required for normal gametophytic development.
    Keywords: Arabidopsis Thaliana ; Cell Differentiation ; Female Gametophyte ; Rkd ; Transcription Factors
    ISSN: 0028-646X
    E-ISSN: 1469-8137
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  • 8
    In: PLoS ONE, 2013, Vol.8(4)
    Description: The steady state level of integral membrane proteins is dependent on a strictly controlled delivery and removal. Here we show that Dendra2, a green-to-red photoconvertible fluorescent protein, is a suitable tool to study protein turnover in plants. We characterized the fluorescence properties of Dendra2 expressed either as a free protein or as a tag in Arabidopsis thaliana roots and optimized photoconversion settings to study protein turnover. Dendra2 was fused to the PIN2 protein, an auxin transporter in the root tip, and by time-lapse imaging and assessment of red and green signal intensities in the membrane after photoconversion we quantified directly and simultaneously the rate of PIN2 delivery of the newly synthesized protein into the plasma membrane as well as the disappearance of the protein from the plasma membrane due to degradation. Additionally we have verified several factors which are expected to affect PIN2 protein turnover and therefore potentially regulate root growth.
    Keywords: Research Article ; Biology ; Chemistry
    E-ISSN: 1932-6203
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  • 9
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 14 July 2015, Vol.112(28), pp.8656-60
    Description: Centromeres play a pivotal role in maintaining genome integrity by facilitating the recruitment of kinetochore and sister-chromatid cohesion proteins, both required for correct chromosome segregation. Centromeres are epigenetically specified by the presence of the histone H3 variant (CENH3). In this study, we investigate the role of the highly conserved γ-tubulin complex protein 3-interacting proteins (GIPs) in Arabidopsis centromere regulation. We show that GIPs form a complex with CENH3 in cycling cells. GIP depletion in the gip1gip2 knockdown mutant leads to a decreased CENH3 level at centromeres, despite a higher level of Mis18BP1/KNL2 present at both centromeric and ectopic sites. We thus postulate that GIPs are required to ensure CENH3 deposition and/or maintenance at centromeres. In addition, the recruitment at the centromere of other proteins such as the CENP-C kinetochore component and the cohesin subunit SMC3 is impaired in gip1gip2. These defects in centromere architecture result in aneuploidy due to severely altered centromeric cohesion. Altogether, we ascribe a central function to GIPs for the proper recruitment and/or stabilization of centromeric proteins essential in the specification of the centromere identity, as well as for centromeric cohesion in somatic cells.
    Keywords: Arabidopsis ; Mzt1 ; Centromere Assembly ; Centromeric Cohesion ; Ploidy Stability ; Centromere ; Arabidopsis -- Genetics ; Arabidopsis Proteins -- Physiology ; Carrier Proteins -- Physiology
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 10
    In: New Phytologist, April 2012, Vol.194(1), pp.142-157
    Description: • Owing to its evolutional plasticity and adaptability, barley (Hordeum vulgare) is one of the most widespread crops in the world. Despite this evolutionary success, sexual reproduction of small grain cereals is poorly investigated, making discovery of novel genes and functions a challenging priority. Barley gene Jekyll appears to be a key player in grain development; however, its role in flowers has remained unknown. • Here, we studied RNAi lines of barley, where Jekyll expression was repressed to different extents. The impact of Jekyll on flower development was evaluated based on differential gene expression analysis applied to anthers and gynoecia of wildtype and transgenic plants, as well as using isotope labeling experiments, hormone analysis, immunogold‐ and TUNEL‐assays and in situ hybridization. • Jekyll is expressed in nurse tissues mediating gametophyte–sporophyte interaction in anthers and gynoecia, where JEKYLL was found within the intracellular membranes. The repression of Jekyll impaired pollen maturation, anther dehiscence and induced a significant loss of fertility. The presence of JEKYLL on the pollen surface also hints at possible involvement in the fertilization process. • We conclude that the role of Jekyll in cereal sexual reproduction is clearly much broader than has been hitherto realized.
    Keywords: Cereals ; Fertility ; Flower ; Gametophyte–Sporophyte Interactions ; Gene Expression ; Sexual Reproduction
    ISSN: 0028-646X
    E-ISSN: 1469-8137
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