Differential Gene Expression in Jatropha curcas In Vitro Cultures Exposed to Microgravity

Jatropha (Jatropha curcas L.) has been identified as a suitable species for biofuel production.  However, the species is undomesticated and breeding and genetic improvement programs are necessary. Microgravity offers a unique environment for assessment of genetic variation that can be used in genetic improvement programs.  The objective of this study was to evaluate the differential gene expression of in vitro jatropha cultures exposed to microgravity.  In vitro cultures of two jatropha accessions (Brazil, India) were initiated from cotyledon (CO), leaf (L) and stem (ST) sections. Different RNA isolation protocols were evaluated to assure sufficient RNA of good quality for subsequent microarray analysis. Groups of 10 petri dishes containing treatments (accession x explant tissue) were arranged in Group Activation Pack flight hardware (petriGAPs), previously validated for spaceflight experiments, and exposed to microgravity for periods varying from 14 to 125 days under different space shuttle missions (STS-133 and STS-135).  Once returned, cultures were processed for RNA isolation and subsequent microarray analysis.  Comparisons were performed between ground and orbit samples for the effects of medium and microgravity exposure time. The type of tissue influenced the efficiency of the RNA isolation and the Plant Reagent and Trizol protocols returned the best RNA quality and quantity.  Microarray analysis revealed differential patterns of gene expression between ground and orbit samples and expression patterns were affected by accession and explant tissue.  For all comparisons performed, between 9 and 522 genes were differentially expressed.  Over 20% of those genes were expressed at higher levels by over 2-fold.  Higher levels of differential gene expression were observed in orbit.  Gene expression was also affected by exposure time to microgravity, with periods of 111 days showing higher expression levels. Specific groups of genes showing differential gene expression were grouped using a cluster analysis.  The roles of different gene groups and the observed genetic changes are discussed for their potential impact on new cultivar development. Differential gene expression induced by microgravity may assist in future genetic improvement programs.