Drought and tropospheric ozone are escalating climate change problems that can co-occur. In this study, we observed Medicago truncatula cultivar Jemalong that is sensitive to ozone and drought stress when applied singly, showed tolerance when subjected to a combined application of these stresses. Lowered stomatal conductance may be a vital tolerance mechanism to overcome combined ozone and drought. Sustained increases in both reduced ascorbate and glutathione in response to combined stress may play a role in lowering reactive oxygen species and nitric oxide toxicity. Transcriptome analysis indicated that genes associated with glucan metabolism, responses to temperature and light signalling may play a role in dampening ozone responses due to drought-induced stomatal closure during combined occurrence of these two stresses. Gene ontologies for jasmonic acid signalling and innate immunity were enriched among the 300 differentially expressed genes unique to combined stress. Differential expression of transcription factors associated with redox, defence signalling, jasmonate responses and chromatin modifications may be important for evoking novel gene networks during combined occurrence of drought and ozone. The alterations in redox milieu and distinct transcriptome changes in response to combined stress could aid in tweaking the metabolome and proteome to annul the detrimental effects of ozone and drought in Jemalong.

BackgroundSwitchgrass (Panicum virgatum L.) is a C4 perennial grass and widely popular as an important bioenergy crop. To accelerate the pace of developing high yielding switchgrass cultivars adapted to diverse environmental niches, the generation of genomic resources for this plant is necessary. The large genome size and polyploid nature of switchgrass makes whole genome sequencing a daunting task even with current technologies. Exploring the transcriptional landscape using next generation sequencing technologies provides a viable alternative to whole genome sequencing in switchgrass.Principal FindingsSwitchgrass cDNA libraries from germinating seedlings, emerging tillers, flowers, and dormant seeds were sequenced using Roche 454 GS-FLX Titanium technology, generating 980,000 reads with an average read length of 367 bp. De novo assembly generated 243,600 contigs with an average length of 535 bp. Using the foxtail millet genome as a reference greatly improved the assembly and annotation of switchgrass ESTs. Comparative analysis of the 454-derived switchgrass EST reads with other sequenced monocots including Brachypodium, sorghum, rice and maize indicated a 70–80% overlap. RPKM analysis demonstrated unique transcriptional signatures of the four tissues analyzed in this study. More than 24,000 ESTs were identified in the dormant seed library. In silico analysis indicated that there are more than 2000 EST-SSRs in this collection. Expression of several orphan ESTs was confirmed by RT-PCR.SignificanceWe estimate that about 90% of the switchgrass gene space has been covered in this analysis. This study nearly doubles the amount of EST information for switchgrass currently in the public domain. The celerity and economical nature of second-generation sequencing technologies provide an in-depth view of the gene space of complex genomes like switchgrass. Sequence analysis of closely related members of the NAD+-malic enzyme type C4 grasses such as the model system Setaria viridis can serve as a viable proxy for the switchgrass genome.

Ozone is a model abiotic elicitor of reactive oxygen species (ROS). ROS are important oxidative signaling molecules coordinating plant development and responses to biotic and abiotic stresses. Recently, microRNAs have been described as important players in regulating stress responses in plants. In this research we examined the miRNAs that are differentially expressed early in response to ozone in the Arabidopsis thaliana ecotype Col-0 that is tolerant to this oxidant. We used a plant miRNA array to identify 22 miRNA families that are differentially expressed within one hour of ozone fumigation. Majority of these miRNAs were also reported in response to UV-B stress. Analysis of the miRNA target genes showed a strong negative correlation to the miRNA expression. In silico promoter analysis of miRNA genes identified several stress responsive cis-elements that were enriched in the promoters of ozone responsive genes. Majority of the target genes of ozone responsive miRNAs were associated with developmental processes. Based on these results we suggest that post-transcriptional gene regulation via miRNAs may aid in resource allocation by downregulating developmental processes to cater to the oxidative stress demands on plants.

Arabidopsis nudix hydrolase 7 (AtNudt7) plays an important role in regulating redox homeostasis during stress/defense signaling and seed germination. The early responsiveness of AtNudt7 provides a useful marker especially during oxidative cell death in plants. Nuclear run-on assays demonstrate that AtNudt7 is transcriptionally regulated. AtNUDT7 promoter-GUS transgenic plants show rapid inducibility in response to ozone and pathogens. A 16-bp insertion containing a GCC-box motif was identified in the promoter of a Ws-2 ecotype and was absent in Col-0. The 16-bp sequence was identified in 5% of the Arabidopsis ecotypes used in the 1001 genome sequencing project. The kinetics of expression of Ethylene Response Factor 1 (ERF1), a GCC-box binding factor is in synchrony with expression of AtNudt7 in response to ozone stress. ERF1 protein binds to the GCC-box motif in the AtNUDT7 promoter. In silico analysis of erf1 mutant and overexpressor lines supports a role for this protein in regulating AtNUDT7 expression.