Location: Plant Genetics Research
Title: A Combined Subtractive Suppression Hybridization and Expression Profiling Strategy to Identify Novel Desiccation Response Transcripts From Tortula ruralis Gametophytes Authors
Submitted to: Physiologia Plantarum
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 21, 2009
Publication Date: July 31, 2009
Citation: Oliver, M.J., Hudgeons, J., Dowd, S.E., Payton, P.R. 2009. A Combined Subtractive Suppression Hybridization and Expression Profiling Strategy to Identify Novel Desiccation Response Transcripts From Tortula ruralis Gametophytes. Physiologia Plantarum. 136:437-460. Interpretive Summary: An understanding of cellular survival and recovery from extremes of water deficits has important implications in both agriculture and plant biology in general and could lead to novel strategies for crop improvement for drought tolerance. In this manuscript we describe our work to understand the genes that are necessary for the survival of plants that are naturally adapted to live in extreme environments, especially those that can survive complete loss of water from their vegetative tissues. We have chosen a simple plant, a bryophyte, as a model for these studies and taken a genomics approach to develop a catalogue of those genes that respond to both the loss of water during dehydration and the reestablishment of normal metabolism following hydration of the dried tissues. We used an innovative approach to narrow the search for genes of interest by only looking at those active genes that differ in the treated plants from those that are active in control plants. We have managed to isolate over two hundred genes that respond specifically to dehydration and rehydration only 37% of which have been described in other plant responses. With this new list of genes we can start to determine which ones are critical for dehydration tolerance and develop the new strategies we will require for crop improvement that are required to meet the challenges of a changing climate.
Technical Abstract: Tortula ruralis is truly desiccation tolerant, capable of surviving - 540 MPa of water deficit, and as such it offers a comparative model for gene expression studies related to the response of crop species (that only survive water deficits of, in general, -2.5 MPa) to dehydration stress. This vegetative desiccation tolerance phenotype of T. ruralis is critical in elucidating the evolutionary, and perhaps gene functionality, connections between desiccation tolerance and water stress responses in sensitive species. We have constructed two Subtractive Suppression Hybridization (SSH) libraries that are designed to enrich for differentially expressed low-abundance transcripts contained within gametophytic cells either in the slow-dried state (mRNP sequestrated rehydrin transcripts) or cells that have been rapidly dried, rehydrated and sampled at 2h of hydration (rehydrin and recovery transcripts) when the translational change in gene expression is at its peak. From these libraries we isolated a random sample of 768 clones, 384 from each library, sequenced them to establish a collection of 614 unique EST contigs, and arrayed them in a cDNA array for expression profiling. However, only 31% of these unique contigs could be annotated by virtue of similarity to the sequences of known genes archived in the public databases. Utilizing a hybridization strategy involving both total and polysomal RNA extracts we were able to establish a limited assessment of the sequestered transcript pool in the dried gametophytes (mRNP fraction) and the pool of transcripts allotted for the recovery response following rehydration. Of note was the involvement of Lea proteins in the rehydration response of the moss following desiccation and the wide range of biological processes that are involved in the survival of the gametophytes. Of the genes that are up-regulated and that could be annotated, almost half have been previously associated with plant environmental stress responses, of the remainder many are associated with either the protein synthetic machinery or its activity. The analysis of the expression profiles and transcript abundances during both dehydration and rehydration offers new insights into the processes involved in vegetative desiccation tolerance and may lead to novel mechanisms for crop improvement with regards to drought tolerance.