Timothy W. Conner

Identification of mRNAs with enhanced expression in ripening strawberry fruit using polymerase chain reaction differential display.

Fruit ripening is a complex developmental process that involves specific changes in gene expression and cellular metabolism. In climateric fruits these events are coordinated by the gaseous hormone ethylene, which is synthesized autocatalytically in the early stages of ripening. Nonclimacteric fruits do not synthesize or respond to ethylene in this manner, yet undergo many of the same physiological and biochemical changes associated with the production of a ripe fruit. To gain insight into the molecular determinants associated with nonclimacteric fruit ripening, we examined mRNA populations in ripening strawberry fruit using polymerase chain reaction (PCR) differential display. Five mRNAs with ripening-enhanced expression were identified using this approach. Three of the mRNAs appear to be fruit-specific, with little or no expression detected in vegetative tissues. Sequence analysis of cDNA clones revealed positive identities for three of the five mRNAs based on homology to known proteins. These results indicate that the differential display technique can be a useful tool to study fruit ripening and other developmental processes in plants at the RNA level.

Developmental and transgenic analysis of two tomato fruit enhanced genes

Tomato fruit development is characterized by distinct developmental stages: fruit set, periods of rapid cell division and cell expansion, and the period where processes associated with ripening are dominant. During each of these stages, different aspects of cellular metabolism are favored. Accompanying these developmental changes are dramatic differences in gene expression, with a subset of genes being expressed early and a subset being expressed later in development. We have isolated and characterized several sequences from tomato that are expressed primarily in immature green fruit. Two of these genes (Tfm7 and Tfm5) have been characterized more extensively and their sequence indicates that they encode proteins corresponding to a proline-rich protein (PRP) and a glycine-rich protein (GRP). RNA blot analysis indicates that the transcripts from these genes are present at the earliest stages of fruit development, and continue to be expressed throughout the growth period of the fruit. Expression analysis during development indicates that the gene encoding the PRP may be down-regulated by ethylene. As a means to understanding the functional significance and the transcriptional contribution of these tissue-limited proteins during development, we constructed promoter-reporter gene fusions to identify which cell types express each of these sequences. GUS protein produced in transgenic plants by both promoter-reporter gene constructs was detected in most tissues of the fruit including the pericarp, columella, and placental tissues of young immature fruit through the mature green stage. However, only one of the promoter sequences conferred expression in the fruit locular tissue.

RNA-binding protein-mediated translational repression of transgene expression in plants

We have demonstrated that RNA-binding proteins from coliphages and yeast can function as translational repressors in plants. RNA sequences called translational operators were inserted at a cap-proximal position in the 5′-UTR of mRNAs of two reporter genes, gusor aroA:CP4. Translation of the reporter mRNAs was efficiently repressed when the RNA binding protein that specifically binds to its cognate operator was co-expressed. The efficiency of translational repression by RNA-binding protein positively correlated with the amount of binding protein in transformed plant cells. Detailed studies on coliphage MS2 coat protein-mediated translational repression also suggested that the efficiency of translational repression was position-dependent. A translational operator situated at the cap-proximal position was more efficient in conferring repression than one that was placed cap-distal. Translational repression can be an efficient means for regulation of transgene expression, thereby broadening current approaches for transgene regulation in plants.