Rip-Chip for global analysis of cell-specific gene expression in Arabidopsis.

MARÍA EUGENIA ZANETTI, ANGELIKA MUSTROPH Y JULIA BAILEYSERRES

Mar del Plata, Buenos Aires,. Argentina

Congreso; XLII Reunión anual de SAIB; 2007

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

In higher eukaryotic organisms, gene expression is regulated at different levels. Two significant deficiencies of routine mRNA expression profiling studies are, (1) the abundance of an individual mRNA may not reflect the level of its translation, and (2) the abundance of an mRNA in specific cell-types cannot be gleaned from organ extracts. To circumvent these challenges and augment the elucidation of developmental processes and environmental responses, we have developed a Rip-Chip (“RNA immunopurification and microarray chip hybridization”) technology to isolate polyribosomes for global characterization of cell-specific gene expression in Arabidopsis thaliana (Zanetti et al 2005, Plant Physiol 138: 624). The basis of this technology is the use of cell-type specific promoters to drive the expression of an epitope-tagged ribosomal protein (r-protein). Polyribosomes are affinity purified and the associated mRNAs are extracted, amplified and hybridized to DNA microarrays to analyze mRNA expression. In plants with a cauliflower mosaic virus 35S:HF-RPL18 transgene immunopurification with anti-FLAG agarose beads yielded 60-Svedberg ribosomal subunits, intact 80-Svedberg monosomes and polysomes. The immunopurified complexes included putative cytosolic RPs of Arabidopsis and ribosome-associated proteins, as well as full-length transcripts of high and low abundance. Whole-genome profiling using long DNA oligonucleotide-based microarrays provided a high level of reproducibility between polysomal mRNA samples immunopurified from two independent biological replicates (r approximately 0.90). Comparison of immunopurified and total cellular RNA samples revealed that for most of the genes, the mRNAs were associated with the epitope-tagged polysomal complexes, with an average relative level of association of 62.06% ± 4.39%. The results demonstrate that the immunopurification of polysomes can be a valuable tool for the quantification of mRNAs present in translation complexes in plant cells. This technology has been extended to evaluation of mRNA populations at the cell- or tissue-specific level by regulation of the tagged RP with distinct promoters: SCR, scarecrow; SHR, shortroot, WOL, wooden leg, GL2, glabra, etc.  Recent Rip-Chip experiments were conducted to evaluate global gene expression in specific cell-types of Arabidopsis roots (cortex, endodermis, vasculature, etc.) kept in the air (normal) or subjected to hypoxia during 2 hours.  The results suggest that both qualitative and quantitative differences exist in the polysomal mRNA levels under hypoxia conditions in each root cell-type.