Transcriptome analysis of nodes and buds from high and low tillering switchgrass inbred lines.

In the last two decades switchgrass has received increasing attention as a promising bioenergy feedstock. Biomass is the principal trait for improvement in switchgrass breeding programs and tillering is an important component of biomass yield. Switchgrass inbred lines derived from a single parent showing vast variation in tiller number trait was used in this study. Axillary buds, which can develop into tillers, and node tissues, which give rise to axillary buds, were collected from high and low tillering inbred lines growing in field conditions. RNA from buds and nodes from the contrasting inbred lines were used for transcriptome profiling with switchgrass Affymetrix genechips. Nearly 7% of the probesets on the genechip exhibited significant differential expression in these lines. Real-time PCR analysis of 30 genes confirmed the differential expression patterns observed with genechips. Cluster analysis aided in identifying probesets unique to high or low tillering lines as well as those specific to buds or nodes of high tillering lines. Rice orthologs of the switchgrass genes were used for gene ontology (GO) analysis with AgriGO. Enrichment of genes associated with amino acid biosynthesis, lipid transport and vesicular transport were observed in low tillering lines. Enrichment of GOs for translation, RNA binding and gene expression in high tillering lines were indicative of active metabolism associated with rapid growth and development. Identification of different classes of transcription factor genes suggests that regulation of many genes determines the complex process of axillary bud initiation and development. Genes identified in this study will complement the current ongoing efforts in quantitative trait loci mapping of tillering in switchgrass.

Phylogenetic and expression analysis of RNA-binding proteins with triple RNA recognition motifs in plants.

The superfamily of RNA binding proteins (RBPs) is vastly expanded in plants compared to other eukaryotes. A subfamily of RBPs that contain three RNA recognition motifs (RRMs) from the Arabidopsis (24), rice (19) and poplar (37) genomes was analyzed in this study. Phylogenetic analysis with full-length protein sequences of 80 RBPs identified nine clades. The largest clade, comprising 23 members, showed high homology to human RBPs involved in oxidative signaling. Digital northern analysis revealed that Arabidopsis RBPs are transcriptionally responsive to biotic, abiotic and hormonal treatments. Northern blot analysis of eight Arabidopsis RBPs belonging to the tobacco RBP45/47 family showed that these genes respond to ozone stress. AtRBP45b, which shows closest homology to the yeast oxidative stress regulatory protein, CSX1, was expressed in multiple tissues. Two novel splice variant forms of AtRBP45b were identified by 3'RACE analysis. Based on RT-PCR, splice variant AtRBP45b-SV1 was observed only in response to mechanical wounding caused by pathogen or chemical infiltrations and was not detectable in response to salt or temperature stress. Electrophoretic mobility shift assay demonstrated that recombinant full-length and splice variant forms of AtRBP45b bound synthetic RNA. Identifying in vivo RNA targets of AtRBP45b will aid in determining the precise functional role of these proteins during oxidative signaling.

Identification of stress-responsive genes in plants using suppression subtraction hybridization: ozone stress as an example.

Among the open-ended techniques for identifying differentially expressed genes in response to stress, the PCR-based suppression subtraction hybridization (SSH) is widely used. The popularity of this technique stems from the ease of conducting this procedure in any laboratory set up for basic molecular biology research. Further, the availability of a comprehensive kit for conducting suppression subtractions from BD Biosciences has made this technique easy to adapt and adopt to any biological system. In this chapter we describe in detail the SSH procedure and explain the subtle changes that have been incorporated to make this technique adaptable for identifying stress-responsive genes in plants.

Ozone responsive genes in Medicago truncatula: analysis by suppression subtraction hybridization.

Acute ozone is a model abiotic elicitor of oxidative stress in plants. In order to identify genes that are important for conferring ozone resistance or sensitivity we used two accessions of Medicago truncatula with contrasting responses to this oxidant. We used suppression subtraction hybridization (SSH) to identify genes differentially expressed in ozone-sensitive Jemalong and ozone-resistant JE154 following exposure to 300 nLL(-1) of ozone for 6h. Following differential screening of more than 2500 clones from four subtraction libraries, more than 800 clones were selected for sequencing. Sequence analysis of these clones identified 239 unique contigs. Fifteen novel genes of unknown functions were identified. A majority of the ozone responsive genes identified in this study were present in the Medicago truncatula EST collections. Genes induced in JE154 were associated with adaptive responses to stress, while in Jemalong, the gene ontologies for oxidative stress, cell growth, and translation were enriched. A meta-analysis of ozone responsive genes using the Genvestigator program indicated enrichment of ABA and auxin responsive genes in JE154, while cytokinin response genes were induced in Jemalong. In resistant JE154, down regulation of photosynthesis-related genes and up regulation of genes responding to low nitrate leads us to speculate that lowering carbon-nitrogen balance may be an important resource allocation strategy for overcoming oxidative stress. Temporal profiles of select genes using real-time PCR analysis showed that most of the genes in Jemalong were induced at the later time points and is consistent with our earlier microarray studies. Inability to mount an early active transcriptional reprogramming in Jemalong may be the cause for an inefficient defense response that in turn leads to severe oxidative stress and culminates in cell death.