Isolation of plant transcription factors using a modified yeast one-hybrid system

BACKGROUND: The preparation of expressional cDNA libraries for use in the yeast two-hybrid system is quick and efficient when using the dedicated Clontech™ product, the MATCHMAKER Library Construction and Screening Kit 3. This kit employs SMART technology for the amplification of full-length cDNAs, in combination with cloning using homologous recombination. Unfortunately, such cDNA libraries prepared directly in yeast can not be used for the efficient recovery of purified plasmids and thus are incompatible with existing yeast one-hybrid systems, which use yeast transformation for the library screen. RESULTS: Here we propose an adaptation of the yeast one-hybrid system for identification and cloning of transcription factors using a MATCHMAKER cDNA library. The procedure is demonstrated using a cDNA library prepared from the liquid part of the multinucleate coenocyte of wheat endosperm. The method is a modification of a standard one-hybrid screening protocol, utilising a mating step to introduce the library construct and reporter construct into the same cell. Several novel full length transcription factors from the homeodomain, AP2 domain and E2F families of transcription factors were identified and isolated. CONCLUSION: In this paper we propose a method to extend the compatibility of MATCHMAKER cDNA libraries from yeast two-hybrid screens to one-hybrid screens. The utility of the new yeast one-hybrid technology is demonstrated by the successful cloning from wheat of full-length cDNAs encoding several transcription factors from three different families

Evolutionary conservation and regulation of particular alternative splicing events in plant SR proteins

Alternative splicing is an important mechanism for fine tuning of gene expression at the post-transcriptional level. SR proteins govern splice site selection and spliceosome assembly. The Arabidopsis genome encodes 19 SR proteins, several of which have no orthologues in metazoan. Three of the plant specific subfamilies are characterized by the presence of a relatively long alternatively spliced intron located in their first RNA recognition motif, which potentially results in an extremely truncated protein. In atRSZ33, a member of the RS2Z subfamily, this alternative splicing event was shown to be autoregulated. Here we show that atRSp31, a member of the RS subfamily, does not autoregulate alternative splicing of its similarily positioned intron. Interestingly, this alternative splicing event is regulated by atRSZ33. We demonstrate that the positions of these long introns and their capability for alternative splicing are conserved from green algae to flowering plants. Moreover, in particular alternative splicing events the splicing signals are embedded into highly conserved sequences. In different taxa, these conserved sequences occur in at least one gene within a subfamily. The evolutionary preservation of alternative splice forms together with highly conserved intron features argues for additional functions hidden in the genes of these plant-specific SR proteins

Assessment of the sewage flotation process use

Import 05/08/2014Bakalářská práce je zaměřena do oblasti čistění odpadních vod. Je zde řešena možnost využití flotace k odstraňování sraženiny fosforu vznikající při terciárním čištění odpadní vody. Úvodní část je teoretická a popisuje čištění odpadních vod, flotaci jako separační technologie a srážení fosforu. Druhá část je praktická, která popisuje experimentální zařízení.The thesis is focused on the area of wastewater treatment. There addressed the possibility of using flotation to remove the precipitate formed during the phosphorus tertiary wastewater treatment. The first part is theoretical and describes wastewater treatment, flotation separation technology as a precipitation of phosphorus. The second part is practical, which describes the experimental facility.Prezenční546 - Institut environmentálního inženýrstvívelmi dobř

Expression Level of the DREB2-Type Gene, Identified with Amplifluor SNP Markers, Correlates with Performance, and Tolerance to Dehydration in Bread Wheat Cultivars from Northern Kazakhstan

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.A panel of 89 local commercial cultivars of bread wheat was tested in field trials in the dry conditions of Northern Kazakhstan. Two distinct groups of cultivars (six cultivars in each group), which had the highest and the lowest grain yield under drought were selected for further experiments. A dehydration test conducted on detached leaves indicated a strong association between rates of water loss in plants from the first group with highest grain yield production in the dry environment relative to the second group. Modern high-throughput Amplifluor Single Nucleotide Polymorphism (SNP) technology was applied to study allelic variations in a series of drought-responsive genes using 19 SNP markers. Genotyping of an SNP in the TaDREB5 (DREB2-type) gene using the Amplifluor SNP marker KATU48 revealed clear allele distribution across the entire panel of wheat accessions, and distinguished between the two groups of cultivars with high and low yield under drought. Significant differences in expression levels of TaDREB5 were revealed by qRT-PCR. Most wheat plants from the first group of cultivars with high grain yield showed slight up-regulation in the TaDREB5 transcript in dehydrated leaves. In contrast, expression of TaDREB5 in plants from the second group of cultivars with low grain yield was significantly down-regulated. It was found that SNPs did not alter the amino acid sequence of TaDREB5 protein. Thus, a possible explanation is that alternative splicing and up-stream regulation of TaDREB5 may be affected by SNP, but these hypotheses require additional analysis (and will be the focus of future studies)

The General Transcription Repressor TaDr1 Is Co-expressed With TaVrn1 and TaFT1 in Bread Wheat Under Drought

The general transcription repressor, TaDr1 gene, was identified during screening of a wheat SNP database using the Amplifluor-like SNP marker KATU-W62. Together with two genes described earlier, TaDr1A and TaDr1B, they represent a set of three homeologous genes in the wheat genome. Under drought, the total expression profiles of all three genes varied between different bread wheat cultivars. Plants of four high-yielding cultivars exposed to drought showed a 2.0–2.4-fold increase in TaDr1 expression compared to controls. Less strong, but significant 1.3–1.8-fold up-regulation of the TaDr1 transcript levels was observed in four low-yielding cultivars. TaVrn1 and TaFT1, which controls the transition to flowering, revealed similar profiles of expression as TaDr1. Expression levels of all three genes were in good correlation with grain yields of evaluated cultivars growing in the field under water-limited conditions. The results could indicate the involvement of all three genes in the same regulatory pathway, where the general transcription repressor TaDr1 may control expression of TaVrn1 and TaFT1 and, consequently, flowering time. The strength of these genes expression can lead to phenological changes that affect plant productivity and hence explain differences in the adaptation of the examined wheat cultivars to the dry environment of Northern and Central Kazakhstan. The Amplifluor-like SNP marker KATU-W62 used in this work can be applied to the identification of wheat cultivars differing in alleles at the TaDr1 locus and in screening hybrids

Role of homeodomain leucine zipper (HD-Zip) iv transcription factors in plant development and plant protection from deleterious environmental factors

Homeobox genes comprise an important group of genes that are responsible for regulation of developmental processes. These genes determine cell differentiation and cell fate in all eukaryotic organisms, starting from the early stages of embryo development. Homeodomain leucine zipper (HD-Zip) transcription factors are unique to the plant kingdom. Members of the HD-Zip IV subfamily have a complex domain topology and can bind several cis-elements with overlapping sequences. Many of the reported HD-Zip IV genes were shown to be specifically or preferentially expressed in plant epidermal or sub-epidermal cells. HD-Zip IV TFs were found to be associated with differentiation and maintenance of outer cell layers, and regulation of lipid biosynthesis and transport. Insights about the role of these proteins in plant cuticle formation, and hence their possible involvement in plant protection from pathogens and abiotic stresses has just started to emerge. These roles make HD-Zip IV proteins an attractive tool for genetic engineering of crop plants. To this end, there is a need for in-depth studies to further clarify the function of each HD-Zip IV subfamily member in commercially important plant species.William Chew, Maria Hrmova and Sergiy Lopat

Ectopic Expression of atRSZ33 Reveals Its Function in Splicing and Causes Pleiotropic Changes in Development

Splicing provides an additional level in the regulation of gene expression and contributes to proteome diversity. Herein, we report the functional characterization of a recently described plant-specific protein, atRSZ33, which has characteristic features of a serine/arginine-rich protein and the ability to interact with other splicing factors, implying that this protein might be involved in constitutive and/or alternative splicing. Overexpression of atRSZ33 leads to alteration of splicing patterns of atSRp30 and atSRp34/SR1, indicating that atRSZ33 is indeed a splicing factor. Moreover, atRSZ33 is a regulator of its own expression, as splicing of its pre-mRNA is changed in transgenic plants. Investigations by promoter-β-glucuronidase (GUS) fusion and in situ hybridization revealed that atRSZ33 is expressed during embryogenesis and early stages of seedling formation, as well as in flower and root development. Ectopic expression of atRSZ33 caused pleiotropic changes in plant development resulting in increased cell expansion and changed polarization of cell elongation and division. In addition, changes in activity of an auxin-responsive promoter suggest that auxin signaling is disturbed in these transgenic plants

The impact of drought on wheat leaf cuticle properties

Abstract Background The plant cuticle is the outermost layer covering aerial tissues and is composed of cutin and waxes. The cuticle plays an important role in protection from environmental stresses and glaucousness, the bluish-white colouration of plant surfaces associated with cuticular waxes, has been suggested as a contributing factor in crop drought tolerance. However, the cuticle structure and composition is complex and it is not clear which aspects are important in determining a role in drought tolerance. Therefore, we analysed residual transpiration rates, cuticle structure and epicuticular wax composition under well-watered conditions and drought in five Australian bread wheat genotypes, Kukri, Excalibur, Drysdale, RAC875 and Gladius, with contrasting glaucousness and drought tolerance. Results Significant differences were detected in residual transpiration rates between non-glaucous and drought-sensitive Kukri and four glaucous and drought-tolerant lines. No simple correlation was found between residual transpiration rates and the level of glaucousness among glaucous lines. Modest differences in the thickness of cuticle existed between the examined genotypes, while drought significantly increased thickness in Drysdale and RAC875. Wax composition analyses showed various amounts of C31 β-diketone among genotypes and increases in the content of alkanes under drought in all examined wheat lines. Conclusions The results provide new insights into the relationship between drought stress and the properties and structure of the wheat leaf cuticle. In particular, the data highlight the importance of the cuticle’s biochemical makeup, rather than a simple correlation with glaucousness or stomatal density, for water loss under limited water conditions