Phloem long-distance trafficking of GIBBERELLIC ACID-INSENSITIVE RNA regulates leaf development.

The phloem translocation stream contains a population of RNA molecules, suggesting plants use RNA to integrate developmental processes, at the whole-plant level. In the present study, we analyzed the role of long-distance trafficking in the delivery of transcripts from two members of the GRAS family, namely CmGAIP and GAI. These two homologs were chosen because of their involvement as transcriptional regulators in GA signaling. A combination of pumpkin, tomato and Arabidopsis was employed to examine the processes involved in long-distance delivery, to sink tissues, of RNA for engineered dominant gain-of-function pumpkin (Cmgaip) and Arabidopsis (DeltaDELLA-gai) genes. Our studies demonstrate that gai RNA entry into functional sieve elements occurs via a selective process. Both engineered mutant gai transcripts were able to exit the scion phloem and traffic cell to cell into the shoot apex. Delivery of Cmgaip and DeltaDELLA-gai RNA mediated highly reproducible changes in leaf phenotype in transgenic tomato lines grown under greenhouse conditions. Phenotypic analysis indicated that tomato leaflet morphology was influenced quite late in development. In addition, tissue sink strength did not appear to dictate gai RNA delivery, suggesting complexity in the process underlying macromolecular trafficking. These results establish that the molecular properties of the Cmgaip and DeltaDELLA-gai transcripts are compatible with the tomato cell-to-cell and long-distance macromolecular trafficking systems. An important conclusion, based on our work, is that control over GAI RNA delivery, via the phloem, may be regulated by sequence motifs conserved between plant families. We propose that RNA delivery via the phloem allows for flexibility in fine tuning of developmental programs to ensure newly developing leaves are optimized for performance under the prevailing environmental conditions.

A systemic small RNA signaling system in plants.

Systemic translocation of RNA exerts non-cell-autonomous control over plant development and defense. Long-distance delivery of mRNA has been proven, but transport of small interfering RNA and microRNA remains to be demonstrated. Analyses performed on phloem sap collected from a range of plants identified populations of small RNA species. The dynamic nature of this population was reflected in its response to growth conditions and viral infection. The authenticity of these phloem small RNA molecules was confirmed by bioinformatic analysis; potential targets for a set of phloem small RNA species were identified. Heterografting studies, using spontaneously silencing coat protein (CP) plant lines, also established that transgene-derived siRNA move in the long-distance phloem and initiate CP gene silencing in the scion. Biochemical analysis of pumpkin (Cucurbita maxima) phloem sap led to the characterization of C. maxima Phloem SMALL RNA BINDING PROTEIN1 (CmPSRP1), a unique component of the protein machinery probably involved in small RNA trafficking. Equivalently sized small RNA binding proteins were detected in phloem sap from cucumber (Cucumis sativus) and lupin (Lupinus albus). PSRP1 binds selectively to 25-nucleotide single-stranded RNA species. Microinjection studies provided direct evidence that PSRP1 could mediate the cell-to-cell trafficking of 25-nucleotide single-stranded, but not double-stranded, RNA molecules. The potential role played by PSRP1 in long-distance transmission of silencing signals is discussed with respect to the pathways and mechanisms used by plants to exert systemic control over developmental and physiological processes.