Genome-wide transcriptomic analysis of the effects of sub-ambient atmospheric oxygen and elevated atmospheric carbon dioxide levels on gametophytes of the moss, Physcomitrella patens.

It is widely accepted that atmospheric O2 has played a key role in the development of life on Earth, as evident from the coincidence between the rise of atmospheric O2 concentrations in the Precambrian and biological evolution. Additionally, it has also been suggested that low atmospheric O2 is one of the major drivers for at least two of the five mass-extinction events in the Phanerozoic. At the molecular level, our understanding of the responses of plants to sub-ambient O2 concentrations is largely confined to studies of the responses of underground organs, e.g. roots to hypoxic conditions. Oxygen deprivation often results in elevated CO2 levels, particularly under waterlogged conditions, due to slower gas diffusion in water compared to air. In this study, changes in the transcriptome of gametophytes of the moss Physcomitrella patens arising from exposure to sub-ambient O2 of 13% (oxygen deprivation) and elevated CO2 (1500 ppmV) were examined to further our understanding of the responses of lower plants to changes in atmospheric gaseous composition. Microarray analyses revealed that the expression of a large number of genes was affected under elevated CO2 (814 genes) and sub-ambient O2 conditions (576 genes). Intriguingly, the expression of comparatively fewer numbers of genes (411 genes) was affected under a combination of both sub-ambient O2 and elevated CO2 condition (low O2-high CO2). Overall, the results point towards the effects of atmospheric changes in CO2 and O2 on transcriptional reprogramming, photosynthetic regulation, carbon metabolism, and stress responses.

In silico identification of miRNAs and their target genes and analysis of gene co-expression network in saffron (Crocus sativus L.) stigma.

As an aromatic and colorful plant of substantive taste, saffron (Crocus sativus L.) owes such properties of matter to growing class of the secondary metabolites derived from the carotenoids, apocarotenoids. Regarding the critical role of microRNAs in secondary metabolic synthesis and the limited number of identified miRNAs in C. sativus, on the other hand, one may see the point how the characterization of miRNAs along with the corresponding target genes in C. sativus might expand our perspectives on the roles of miRNAs in carotenoid/apocarotenoid biosynthetic pathway. A computational analysis was used to identify miRNAs and their targets using EST (Expressed Sequence Tag) library from mature saffron stigmas. Then, a gene co- expression network was constructed to identify genes which are potentially involved in carotenoid/apocarotenoid biosynthetic pathways. EST analysis led to the identification of two putative miRNAs (miR414 and miR837-5p) along with the corresponding stem- looped precursors. To our knowledge, this is the first report on miR414 and miR837-5p in C. sativus. Co-expression network analysis indicated that miR414 and miR837-5p may play roles in C. sativus metabolic pathways and led to identification of candidate genes including six transcription factors and one protein kinase probably involved in carotenoid/apocarotenoid biosynthetic pathway. Presence of transcription factors, miRNAs and protein kinase in the network indicated multiple layers of regulation in saffron stigma. The candidate genes from this study may help unraveling regulatory networks underlying the carotenoid/apocarotenoid biosynthesis in saffron and designing metabolic engineering for enhanced secondary metabolites.

Evaluation of the use of the polyubiquitin genes, Ubi4 and Ubi10 as reference genes for expression studies in Brachypodium distachyon.

BACKGROUND: Brachypodium distachyon is emerging as the model plant for temperate grass research and the genome of the community line Bd21 has been sequenced. Additionally, techniques have been developed for Agrobacterium-mediated transformation for the generation of T-DNA insertional lines. Recently, it was reported that expression of the polyubiquitin genes, Ubi4 and Ubi10 are stable in different tissues and growth hormone-treated plant samples, leading to the conclusion that both Ubi4 and Ubi10 are good reference genes for normalization of gene expression data using real-time, quantitative PCR (qPCR). PRINCIPAL FINDINGS: Mining of the Joint Genome Institute (JGI) 8X Brachypodium distachyon genome assembly showed that Ubi4 and Ubi10 share a high level of sequence identity (89%), and in silico analyses of the sequences of Ubi4 (Bradi3g04730) and Ubi10 (Bradi1g32860) showed that the primers used previously exhibit multiple binding sites within the coding sequences arising from the presence of tandem repeats of the coding regions. This can potentially result in over-estimation of steady-state levels of Ubi4 and Ubi10. Additionally, due to the high level of sequence identity between both genes, primers used previously for amplification of Ubi4 can bind to Ubi10 and vice versa, resulting in the formation of non-specific amplification products. CONCLUSIONS: The results from this study indicate that the primers used previously were not sufficiently robust and specific. Additionally, their use would result in over-estimation of the steady-state expression levels of Ubi4. Our results question the validity of using the previously proposed primer sets for qPCR amplification of Ubi4 and Ubi10. We demonstrate that primers designed to target the 3'-UTRs of Ubi4 and Ubi10 are better suited for real-time normalization of steady-state expression levels in Brachypodium distachyon.