The quest for osmotic stress markers in Musa: From protein to gene and back in a non-model crop

Abstract

Bananas and plantains are a major staple food and export product in more than 120 countries with a worldwide production of over 135 million tonnes per year. The Laboratory of Tropical Crop Improvement hosts the Bioversity International Musa Germplasm Transit Centre which contains the world s largest banana collection with over 1400 accessions kept as in vitro plants.Water is one of the most limiting abiotic stress factors in banana production. We therefore designed a long term experimental set-up to screen the available Musa biodiversity for drought tolerance in which osmotic stress research is a first step. This research was executed at three levels: cell cultures, heterotrophic in vitro plants and autotrophic plants.Research on banana cell cultures identified more than fifty potential osmotic stress markers via proteomics and transcriptomics. To evaluate the suitability of these stress markers for future use in high-throughput screening of banana varieties, we assessed the four most promising via qPCR. We showed that all four candidates reacted to the stress treatment. One (phosphoglycerate kinase) was validated as an osmotic stress marker.Then our focus shifted from the model on cell cultures towards the plant level. We developed a heterotrophic in vitro growth model to screen five varieties representing different genome constitutions present in Musa. The proteome of the variety with the smallest growth reduction was analyzed by two-dimensional gel electrophoresis. We successfully identified 24 proteins as potential osmotic stress markers of which five (PR10, isoflavone reductase, glutathione-S-transferase, S-adenosyl methionine synthase and phosphoglucomutase) had already been identified in cell cultures and we showed that proteins belonging to the defense and reactive oxygen species metabolism and to the energy metabolism contributed to the new homeostasis in the stressed in vitro plants.Further proteomic research on autotrophic plants again revealed 35 potential stress markers of which six (HSP20, HSP70, glutathione-S-transferase, S-adenosyl methionine synthase, sucrose synthase and phosphoglycerate kinase) had already been identified in cell cultures and/or in vitro plants. Finally we focused our research on one interesting osmotic stress marker protein family, HSP70. It is not uncommon to identify several spots on a gel from two-dimensional gel electrophoresis with the same general identification of the gene family. Gene families in banana consist of paralogs, genes related by duplication within a genome, and allelic variants, genes at the same locus of homologous chromosomes. HSP70 was identified in a trail of six spots. With the availability of the Musa A and B genomes and the combinatorial use of gel-based and gel-free proteomics techniques, we were able to pinpoint in an ABB variety which paralogs and/or allelic variants were expressed and were present in the spots. We also identified an osmotic stress-responsive HSP70 encoded by the paralog located on chromosome 2.The nine osmotic stress markers (HSP20, HSP70, PR10, isoflavone reductase, glutathione-S-transferase, S-adenosyl methionine synthase, sucrose synthase, phosphoglucomutase and phosphoglycerate kinase) identified in this research should now be screened in several varieties and validated under real drought conditions. Combining the validated stress marker genes with phenotyping approaches will help in the future to diagnose the severity of stress and finally drought stress tolerance marker will aid us in the identification of drought tolerant varieties and facilitate banana breeding for drought tolerance.status: publishe