A simple and high-sensitivity method for analysis of ubiquitination and polyubiquitination based on wheat cell-free protein synthesis

<p>Abstract</p> <p>Background</p> <p>Ubiquitination is mediated by the sequential action of at least three enzymes: the E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) proteins. Polyubiquitination of target proteins is also implicated in several critical cellular processes. Although Arabidopsis genome research has estimated more than 1,300 proteins involved in ubiquitination, little is known about the biochemical functions of these proteins. Here we demonstrate a novel, simple and high-sensitive method for <it>in vitro </it>analysis of ubiquitination and polyubiquitination based on wheat cell-free protein synthesis and luminescent detection.</p> <p>Results</p> <p>Using wheat cell-free synthesis, 11 E3 proteins from Arabidopsis full-length cDNA templates were produced. These proteins were analyzed either in the translation mixture or purified recombinant protein from the translation mixture. In our luminescent method using FLAG- or His-tagged and biotinylated ubiquitins, the polyubiquitin chain on AtUBC22, UPL5 and UPL7 (HECT) and CIP8 (RING) was detected. Also, binding of ubiquitin to these proteins was detected using biotinylated ubiquitin and FLAG-tagged recombinant protein. Furthermore, screening of the RING 6 subgroup demonstrated that At1g55530 was capable of polyubiquitin chain formation like CIP8. Interestingly, these ubiquitinations were carried out without the addition of exogenous E1 and/or E2 proteins, indicating that these enzymes were endogenous to the wheat cell-free system. The amount of polyubiquitinated proteins in the crude translation reaction mixture was unaffected by treatment with MG132, suggesting that our system does not contain 26S proteasome-dependent protein degradation activity.</p> <p>Conclusion</p> <p>In this study, we developed a simple wheat cell-free based luminescence method that could be a powerful tool for comprehensive ubiquitination analysis.</p

Recent advances in the characterization of plant transcriptomes in response to drought, salinity, heat, and cold stress [version 1; peer review: 2 approved]

Despite recent advancements in plant molecular biology and biotechnology, providing food security for an increasing world population remains a challenge. Drought (water scarcity), salinity, heat, and cold stress are considered major limiting factors that affect crop production both qualitatively and quantitatively. Therefore, the development of cost-effective and environmentally friendly strategies will be needed to resolve these agricultural problems. This will require a comprehensive understanding of transcriptomic alterations that occur in plants in response to varying levels of environmental stresses, singly and in combination. Here, we briefly discuss the current status and future challenges in plant research related to understanding transcriptional changes that occur in response to drought, salinity, heat, and cold stress

Acetic Acid Treatment Enhances Drought Avoidance in Cassava (Manihot esculenta Crantz)

The external application of acetic acid has recently been reported to enhance survival of drought in plants such as Arabidopsis, rapeseed, maize, rice, and wheat, but the effects of acetic acid application on increased drought tolerance in woody plants such as a tropical crop “cassava” remain elusive. A molecular understanding of acetic acid-induced drought avoidance in cassava will contribute to the development of technology that can be used to enhance drought tolerance, without resorting to transgenic technology or advancements in cassava cultivation. In the present study, morphological, physiological, and molecular responses to drought were analyzed in cassava after treatment with acetic acid. Results indicated that the acetic acid-treated cassava plants had a higher level of drought avoidance than water-treated, control plants. Specifically, higher leaf relative water content, and chlorophyll and carotenoid levels were observed as soils dried out during the drought treatment. Leaf temperatures in acetic acid-treated cassava plants were higher relative to leaves on plants pretreated with water and an increase of ABA content was observed in leaves of acetic acid-treated plants, suggesting that stomatal conductance and the transpiration rate in leaves of acetic acid-treated plants decreased to maintain relative water contents and to avoid drought. Transcriptome analysis revealed that acetic acid treatment increased the expression of ABA signaling-related genes, such as OPEN STOMATA 1 (OST1) and protein phosphatase 2C; as well as the drought response and tolerance-related genes, such as the outer membrane tryptophan-rich sensory protein (TSPO), and the heat shock proteins. Collectively, the external application of acetic acid enhances drought avoidance in cassava through the upregulation of ABA signaling pathway genes and several stress responses- and tolerance-related genes. These data support the idea that adjustments of the acetic acid application to plants is useful to enhance drought tolerance, to minimize the growth inhibition in the agricultural field

Construction and EST sequencing of full-length, drought stress cDNA libraries for common beans (Phaseolus vulgaris L.)

<p>Abstract</p> <p>Background</p> <p>Common bean is an important legume crop with only a moderate number of short expressed sequence tags (ESTs) made with traditional methods. The goal of this research was to use full-length cDNA technology to develop ESTs that would overlap with the beginning of open reading frames and therefore be useful for gene annotation of genomic sequences. The library was also constructed to represent genes expressed under drought, low soil phosphorus and high soil aluminum toxicity. We also undertook comparisons of the full-length cDNA library to two previous non-full clone EST sets for common bean.</p> <p>Results</p> <p>Two full-length cDNA libraries were constructed: one for the drought tolerant Mesoamerican genotype BAT477 and the other one for the acid-soil tolerant Andean genotype G19833 which has been selected for genome sequencing. Plants were grown in three soil types using deep rooting cylinders subjected to drought and non-drought stress and tissues were collected from both roots and above ground parts. A total of 20,000 clones were selected robotically, half from each library. Then, nearly 10,000 clones from the G19833 library were sequenced with an average read length of 850 nucleotides. A total of 4,219 unigenes were identified consisting of 2,981 contigs and 1,238 singletons. These were functionally annotated with gene ontology terms and placed into KEGG pathways. Compared to other EST sequencing efforts in common bean, about half of the sequences were novel or represented the 5' ends of known genes.</p> <p>Conclusions</p> <p>The present full-length cDNA libraries add to the technological toolbox available for common bean and our sequencing of these clones substantially increases the number of unique EST sequences available for the common bean genome. All of this should be useful for both functional gene annotation, analysis of splice site variants and intron/exon boundary determination by comparison to soybean genes or with common bean whole-genome sequences. In addition the library has a large number of transcription factors and will be interesting for discovery and validation of drought or abiotic stress related genes in common bean.</p

Characterization of expressed sequence tags from a full-length enriched cDNA library of Cryptomeria japonica male strobili

<p>Abstract</p> <p>Background</p> <p><it>Cryptomeria japonica </it>D. Don is one of the most commercially important conifers in Japan. However, the allergic disease caused by its pollen is a severe public health problem in Japan. Since large-scale analysis of expressed sequence tags (ESTs) in the male strobili of <it>C. japonica </it>should help us to clarify the overall expression of genes during the process of pollen development, we constructed a full-length enriched cDNA library that was derived from male strobili at various developmental stages.</p> <p>Results</p> <p>We obtained 36,011 expressed sequence tags (ESTs) from either one or both ends of 19,437 clones derived from the cDNA library of <it>C. japonica </it>male strobili at various developmental stages. The 19,437 cDNA clones corresponded to 10,463 transcripts. Approximately 80% of the transcripts resembled ESTs from <it>Pinus </it>and <it>Picea</it>, while approximately 75% had homologs in <it>Arabidopsis</it>. An analysis of homologies between ESTs from <it>C. japonica </it>male strobili and known pollen allergens in the Allergome Database revealed that products of 180 transcripts exhibited significant homology. Approximately 2% of the transcripts appeared to encode transcription factors. We identified twelve genes for MADS-box proteins among these transcription factors. The twelve MADS-box genes were classified as <it>DEF/GLO/GGM13-, AG-, AGL6-, TM3- </it>and <it>TM8</it>-like MIKC^Cgenes and type I MADS-box genes.</p> <p>Conclusion</p> <p>Our full-length enriched cDNA library derived from <it>C. japonica </it>male strobili provides information on expression of genes during the development of male reproductive organs. We provided potential allergens in <it>C. japonica</it>. We also provided new information about transcription factors including MADS-box genes expressed in male strobili of <it>C. japonica</it>. Large-scale gene discovery using full-length cDNAs is a valuable tool for studies of gymnosperm species.</p

RARGE: a large-scale database of RIKEN Arabidopsis resources ranging from transcriptome to phenome

The RIKEN Arabidopsis Genome Encyclopedia (RARGE) database houses information on biological resources ranging from transcriptome to phenome, including RIKEN Arabidopsis full-length (RAFL) complementary DNAs (cDNAs), their promoter regions, Dissociation (Ds) transposon-tagged lines and expression data from microarray experiments. RARGE provides tools for searching by resource code, sequence homology or keyword, and rapid access to detailed information on the resources. We have isolated 245 946 RAFL cDNA clones and collected 11 933 transposon-tagged lines, which are available from the RIKEN Bioresource Center and are stored in RARGE. The RARGE web interface can be accessed at http://rarge.gsc.riken.jp/. Additionally, we report 90 000 new RAFL cDNA clones here

Comparative genomic analysis of 1047 completely sequenced cDNAs from an Arabidopsis-related model halophyte, Thellungiella halophila

<p>Abstract</p> <p>Background</p> <p><it>Thellungiella halophila </it>(also known as <it>T. salsuginea</it>) is a model halophyte with a small size, short life cycle, and small genome. <it>Thellungiella </it>genes exhibit a high degree of sequence identity with Arabidopsis genes (90% at the cDNA level). We previously generated a full-length enriched cDNA library of <it>T. halophila </it>from various tissues and from whole plants treated with salinity, chilling, freezing stress, or ABA. We determined the DNA sequences of 20 000 cDNAs at both the 5'- and 3' ends, and identified 9569 distinct genes.</p> <p>Results</p> <p>Here, we completely sequenced 1047 <it>Thellungiella </it>full-length cDNAs representing abiotic-stress-related genes, transcription factor genes, and protein phosphatase 2C genes. The predicted coding sequences, 5'-UTRs, and 3'-UTRs were compared with those of orthologous genes from Arabidopsis for length, sequence similarity, and structure. The 5'-UTR sequences of <it>Thellungiella </it>and Arabidopsis orthologs shared a significant level of similarity, although the motifs were rearranged. While examining the stress-related <it>Thellungiella </it>coding sequences, we found a short splicing variant of <it>T. halophila </it><it>salt overly sensitive 1 </it>(<it>ThSOS1</it>), designated <it>ThSOS1S</it>. ThSOS1S contains the transmembrane domain of ThSOS1 but lacks the C-terminal hydrophilic region. The expression level of <it>ThSOS1S </it>under normal growth conditions was higher than that of <it>ThSOS1</it>. We also compared the expression levels of Na⁺-transport-system genes between <it>Thellungiella </it>and Arabidopsis by using full-length cDNAs from each species as probes. Several genes that play essential roles in Na⁺excretion, compartmentation, and diffusion (<it>SOS1</it>, <it>SOS2</it>, <it>NHX1</it>, and <it>HKT1</it>) were expressed at higher levels in <it>Thellungiella </it>than in Arabidopsis.</p> <p>Conclusions</p> <p>The full-length cDNA sequences obtained in this study will be essential for the ongoing annotation of the <it>Thellungiella </it>genome, especially for further improvement of gene prediction. Moreover, they will enable us to find splicing variants such as <it>ThSOS1S </it>(AB562331).</p

An Arabidopsis SBP-domain fragment with a disrupted C-terminal zinc-binding site retains its tertiary structure

AbstractSQUAMOSA promoter-binding proteins (SBPs) form a major family of plant-specific transcription factors, mainly related to flower development. SBPs share a highly conserved DNA-binding domain of ∼80 amino acids (SBP domain), which contains two non-interleaved zinc-binding sites formed by eight conserved Cys or His residues. In the present study, an Arabidopsis SPL12 SBP-domain fragment that lacks a Cys residue involved in the C-terminal zinc-binding pocket was found to retain a folded structure, even though only a single Zn2+ ion binds to the fragment. Solution structure of this fragment determined by NMR is very similar to the previously determined structures of the full SBP domains of Arabidopsis SPL4 and SPL7. Considering the previous observations that chelating all the Zn2+ ions of SBPs resulted in the complete unfolding of the structure and that a mutation of the Cys residue equivalent to that described above impaired the DNA-binding activity, we propose that the Zn2+ ion at the N-terminal site is necessary to maintain the overall tertiary structure, while the Zn2+ ion at the C-terminal site is necessary for the DNA binding, mainly by guiding the basic C-terminal loop to correctly fit into the DNA groove

Sequencing analysis of 20,000 full-length cDNA clones from cassava reveals lineage specific expansions in gene families related to stress response

<p>Abstract</p> <p>Background</p> <p>Cassava, an allotetraploid known for its remarkable tolerance to abiotic stresses is an important source of energy for humans and animals and a raw material for many industrial processes. A full-length cDNA library of cassava plants under normal, heat, drought, aluminum and post harvest physiological deterioration conditions was built; 19968 clones were sequence-characterized using expressed sequence tags (ESTs).</p> <p>Results</p> <p>The ESTs were assembled into 6355 contigs and 9026 singletons that were further grouped into 10577 scaffolds; we found 4621 new cassava sequences and 1521 sequences with no significant similarity to plant protein databases. Transcripts of 7796 distinct genes were captured and we were able to assign a functional classification to 78% of them while finding more than half of the enzymes annotated in metabolic pathways in Arabidopsis. The annotation of sequences that were not paired to transcripts of other species included many stress-related functional categories showing that our library is enriched with stress-induced genes. Finally, we detected 230 putative gene duplications that include key enzymes in reactive oxygen species signaling pathways and could play a role in cassava stress response features.</p> <p>Conclusion</p> <p>The cassava full-length cDNA library here presented contains transcripts of genes involved in stress response as well as genes important for different areas of cassava research. This library will be an important resource for gene discovery, characterization and cloning; in the near future it will aid the annotation of the cassava genome.</p

Transcriptome Analyses of a Salt-Tolerant Cytokinin-Deficient Mutant Reveal Differential Regulation of Salt Stress Response by Cytokinin Deficiency

Soil destruction by abiotic environmental conditions, such as high salinity, has resulted in dramatic losses of arable land, giving rise to the need of studying mechanisms of plant adaptation to salt stress aimed at creating salt-tolerant plants. Recently, it has been reported that cytokinins (CKs) regulate plant environmental stress responses through two-component systems. A decrease in endogenous CK levels could enhance salt and drought stress tolerance. Here, we have investigated the global transcriptional change caused by a reduction in endogenous CK content under both normal and salt stress conditions. Ten-day-old Arabidopsis thaliana wild-type (WT) and CK-deficient ipt1,3,5,7 plants were transferred to agar plates containing either 0 mM (control) or 200 mM NaCl and maintained at normal growth conditions for 24 h. Our experimental design allowed us to compare transcriptome changes under four conditions: WT-200 mM vs. WT-0 mM, ipt1,3,5,7-0 mM vs. WT-0 mM, ipt1,3,5,7-200 mM vs. ipt1,3,5,7-0 mM and ipt1,3,5,7-200 mM vs. WT-200 mM NaCl. Our results indicated that the expression of more than 10% of all of the annotated Arabidopsis genes was altered by CK deficiency under either normal or salt stress conditions when compared to WT. We found that upregulated expression of many genes encoding either regulatory proteins, such as NAC, DREB and ZFHD transcription factors and the calcium sensor SOS3, or functional proteins, such as late embryogenesis-abundant proteins, xyloglucan endo-transglycosylases, glycosyltransferases, glycoside hydrolases, defensins and glyoxalase I family proteins, may contribute to improved salt tolerance of CK-deficient plants. We also demonstrated that the downregulation of photosynthesis-related genes and the upregulation of several NAC genes may cause the altered morphological phenotype of CK-deficient plants. This study highlights the impact of CK regulation on the well-known stress-responsive signaling pathways, which regulate plant adaptation to high salinity as well as other environmental stresses