Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR

<p>Abstract</p> <p>Background</p> <p>The wild grass species <it>Brachypodium distachyon </it>(Brachypodium hereafter) is emerging as a new model system for grass crop genomics research and biofuel grass biology. A draft nuclear genome sequence is expected to be publicly available in the near future; an explosion of gene expression studies will undoubtedly follow. Therefore, stable reference genes are necessary to normalize the gene expression data.</p> <p>Results</p> <p>A systematic exploration of suitable reference genes in Brachypodium is presented here. Nine reference gene candidates were chosen, and their gene sequences were obtained from the Brachypodium expressed sequence tag (EST) databases. Their expression levels were examined by quantitative real-time PCR (qRT-PCR) using 21 different Brachypodium plant samples, including those from different plant tissues and grown under various growth conditions. Effects of plant growth hormones were also visualized in the assays. The expression stability of the candidate genes was evaluated using two analysis software packages, geNorm and NormFinder. In conclusion, the ubiquitin-conjugating enzyme 18 gene (<it>UBC18</it>) was validated as a suitable reference gene across all the plant samples examined. While the expression of the polyubiquitin genes (<it>Ubi4 </it>and <it>Ubi10</it>) was most stable in different plant tissues and growth hormone-treated plant samples, the expression of the S-adenosylmethionine decarboxylase gene (<it>SamDC</it>) ranked was most stable in plants grown under various environmental stresses.</p> <p>Conclusion</p> <p>This study identified the reference genes that are most suitable for normalizing the gene expression data in Brachypodium. These reference genes will be particularly useful when stress-responsive genes are analyzed in order to produce transgenic plants that exhibit enhanced stress resistance.</p

The chromosome content and genotype of two wheat cell lines and of their somatic fusion product with oat

Somatic hybridization seeks to genetically combine phylogenetically distant parents. An effective system has been established in bread wheat (Triticum aestivum L.) involving protoplasts from a non-totipotent cell line adapted to in vitro culture (T1) in combination with totipotent protoplasts harvested from embryogenic calli (T2). Here, we report the karyotype and genotype of T1 and T2. Line T1 carries nine A (A-genome of wheat), seven B (B-genome of wheat) and eight D (D-genome of wheat) genome chromosomes, while T2 cells have 12 A, 10 B and 12 D genome chromosomes. Rates of chromosome aberration in the B- and D-genomes were more than 25%, higher than in the A-genome. DNA deletion rates were 55.6% in T1 and 19.4% in T2, and DNA variation rates were 8.3% in T1 and 13.9% in T2. Rate of DNA elimination was B- > D- > A-genome in both T1 and T2. The same set of cytological and genetic assays was applied to a derivative of the somatic fusion between protoplasts of T1, T2 and oat (Avena sativa L.). The regenerant plants were near euploid with respect to their wheat complement. Six wheat chromosome arms—4AL, 3BS, 4BL, 3DS, 6DL and 7DL—carried small introgressed segments of oat chromatin. A genotypic analysis of the hybrid largely confirmed this cytologically-based diagnosis

The dynamic changes of DNA methylation and histone modifications of salt responsive transcription factor genes in soybean.

Epigenetic modification contributes to the regulation of gene expression and plant development under salinity stress. Here we describe the identification of 49 soybean transcription factors by microarray analysis as being inducible by salinity stress. A semi-quantitative RT-PCR-based expression assay confirmed the salinity stress inducibility of 45 of these 49 transcription factors, and showed that ten of them were up-regulated when seedlings were exposed to the demethylation agent 5-aza-2-deoxycytidine. Salinity stress was shown to affect the methylation status of four of these ten transcription factors (one MYB, one b-ZIP and two AP2/DREB family members) using a combination of bisulfite sequencing and DNA methylation-sensitive DNA gel blot analysis. ChIP analysis indicated that the activation of three of the four DNA methylated transcription factors was correlated with an increased level of histone H3K4 trimethylation and H3K9 acetylation, and/or a reduced level of H3K9 demethylation in various parts of the promoter or coding regions. Our results suggest a critical role for some transcription factors' activation/repression by DNA methylation and/or histone modifications in soybean tolerance to salinity stress

Molecular and functional profiling of Arabidopsis pathogenesis-related genes: Insights into their roles in salt response of seed germination

Pathogenesis-related (PR) proteins are a group of heterogeneous proteins encoded by genes that are rapidly induced by pathogenic infections and by salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). They are widely used as molecular markers for resistance response to pathogens and systemic acquired response (SAR). However, recent studies have shown that the PR genes are also regulated by environmental factors, including light and abiotic stresses, and by developmental cues, suggesting that they also play a role in certain stress responses and developmental processes. In this work, we systematically examined the expression patterns of Arabidopsis PR genes. We also investigated the effects of environmental stresses and growth hormones on the expression of PR genes. We found that individual PR genes are temporally and spatially regulated in distinct patterns. In addition, they are differentially regulated by plant growth hormones, including SA, ABA, JA, ET and brassinosteroid (BR), and by diverse abiotic stresses, supporting the contention that the PR proteins play a role in plant developmental processes other than disease resistance response. Interestingly, PR-3 was induced significantly by high salt in an ABA-dependent manner. Consistent with this, a T-DNA insertional knockout plant with disruption of the PR-3 gene showed a significantly reduced rate of seed germination in the presence of high salt. It is thus proposed that PR-3 mediates ABA-dependent salt stress signals that affect seed germination in Arabidopsis. PR-4 and PR-5 also contributed to salt regulation of seed germination, although their effects were not as evident as those of PR-3

WRKY71 acts antagonistically against salt-delayed flowering in Arabidopsis thaliana

Soil salinity affects various aspects of plant growth and development including flowering. Usually, plants show a delayed flowering phenotype under high salinity conditions, whereas some plants will risk their life to continue to grow, thereby escaping serious salt stress to achieve reproductive success. However, the molecular mechanisms of the escape strategies are not clear yet. In this work, we report that the transcription factor WRKY71 helps escape salt stress in Arabidopsis. The expression of the WRKY71 wild-type (WT) allele was salinity inducible. Compared with Col-0, high salt stress caused only a marginal delay in the flowering time of the activation-tagged mutant WRKY71-1D. However, flowering in the RNA interference (RNAi)-based multiple WRKY knock-out mutant (w71w8 + 28RNAi) was dramatically later than in the WT under high salinity conditions. Meanwhile, expression of FLOWERING LOCUS T (FT) and LEAFY (LFY) was greater in WRKY71-1D than in the WT, and lower in w71w8 + 28RNAi under salinity-stressed conditions. The suggestion is that WRKY71 activity hastens flowering, thereby providing a means for the plant to complete its life cycle in the presence of salt stress.

GUS activity for miR165a/166b, REV, and WUS/CLV3 in in vitro direct Arabidopsis thaliana shoot regeneration

In Arabidopsis thaliana, the process of shoot regeneration in vitro requires the presence of specific miRNAs. We describe here the beta-glucuronidase (GUS) expression domains for miR165a/166b, REV, and WUS/CLV3 during direct shoot regeneration. Increased GUS activity of miR166b and REV were first detected within the shoot apical meristem of explants, while no pmiR165a::GUS activity appeared there. The zone of pWUS::GUS activity covered the inner sides of developing protuberances, while that of pCLV3::GUS was more restricted. Once the primary shoot had emerged from the protuberance, pREV::GUS activity was turned on throughout the protuberance. pmiR165a::GUS activity was detected in a small number of protuberance surface cells, while pWUS::GUS activity was restricted to within a few cells beneath the protuberance surface. After the differentiation of leaf-like structures, GUS activity for miR165a and miR166b appeared largely on their abaxial surface, while pWUS::GUS activity was concentrated at the apex of the primary shoot, and no pCLV3::GUS activity was detectable. Following the formation of secondary shoots, pmiR165a::GUS activity was detected on their abaxial surface. GUS activity for miR166b, REV, and WUS/CLV3 were concentrated in the stem apical meristem. The observations suggested that each member of this set of genes might play a distinct role in both primary and secondary shoot regeneration

Cloning and Characterization of a <i>Phragmites australis</i> Phytochelatin Synthase (<i>PaPCS</i>) and Achieving Cd Tolerance in Tall Fescue

<div><p>The production of phytochelatins (PCs) provides an important means for plants to achieve tolerance to cadmium (Cd) toxicity. A reed gene encoding PC synthase (<i>PaPCS</i>) was isolated and its function tested through its heterologous expression in a strain of yeast sensitive to Cd. Subsequently, the Cd sensitive and high biomass accumulating species tall fescue was transformed either with <i>PaPCS</i> or <i>PaGCS</i> (a glutamyl cysteine synthetase gene of reed) on their own (single transformants), or with both genes together in the same transgene cassette (double transformant). The single and double transformants showed greater Cd tolerance and accumulated more Cd and PC than wild type plants, and their Cd leaf/root ratio content was higher. The ranking in terms of Cd and PC content for the various transgenic lines was double transformants><i>PaGCS</i> single transformants><i>PaPCS</i> single transformants>wild type. Thus <i>PaGCS</i> appears to exert a greater influence than <i>PaPCS</i> over PC synthesis and Cd tolerance/accumulation. The double transformant has interesting potential for phytoremediation.</p></div