MOLECULAR MECHANISM OF NONCELL AUTONOMOUS RNA TRAFFICKING
In plants, most of the cell divisions only occur at specialized zones known as meristem. However, the perception of the biotic and abiotic stimulates most rely on other part of the tissues such as leaves. In order to incorporate these environmental challenges into their developmental program, plants take advantage of vascular system to traffic information molecules. It has been well established that micromolecules, such as hormones, sugars, and small peptides, can transport through phloem and regulate plant development or defensive response. Recently, accumulated evidences showed that the macromolecules, including proteins and RNA, might also function as cell-to-cell or long-distance information signals.
Although RNA has been identified from phloem exudates over several decades, it has been thought to be reflected the contamination during phloem sap collection. The first compelling evidence demonstrates that RNA is bona fide phloem sap component is provided by potato SUT1 (SUCROSE TRANSPORTER 1). Using in situ hybridization, the SUT1 RNA has been shown to be localized in companion cells (CC) and sieve elements (SE), especially in plasmodesmata (PD) that connected CC and SE. The lack of nucleus in SE suggests the SUT1 RNA detected in the SE might be transcribed within the CC and then transported into the SE via PD. Using pumpkin (Cucurbita maxima) as a model system, a number of phloem sap RNA (pRNA) have been identified. Results obtained from the characterization of two pRNA, CmPP16 and CmNACP, are consistent with the notion that pRNA have the ability to traffic over long-distance to the scion apex. The first evidence indicates that pRNA might function as a long-distance signaling molecule was provided by analysis of tomato mouse-ear (me) mutant. Tomato me is a chromosome rearrangement mutant in which the LeT6 (a homeobox transcription factor) RNA is over expressed in the vascular tissues. The leaflets of me plants display ovate phenotype compared with serrate leaflet in the wild type. The graft experiments performed with wild-type scion on me mutant stock demonstrate that the phenotype of me is graft-transmissible. In addition, the detection of LeT6 RNA from the scion apex is coincident with phenotypic alteration in the scion, consistent with the notion that RNA might serve as a long-distance information macromolecule and regulate plant development. However, in me mutant, the over production of LeT6 RNA in the vascular tissues only occurs at low light conditions, therefore, the detection of graft-transmissible phenotypic alterations in the scion is limited in this condition. This limitation greatly impairs the potential application of tomato me system for the further analyses.
Previous Accomplishments and Future Projects
To develop a robust system suitable for analyzing the parameters involved in pRNA trafficking, a pumpkin pRNA CmGAIP (a homologue of Arabidopsis GIBBERELLIC ACID INSENSITIVE, GAI) has been selected and engineered to produce a mutant (Cmgaip) equivalent to the Arabidopsis gai-1 allele. The resultant construct was then transformed to tomatoes and Arabidopsis. Graft experiments showed that the Cmgaip RNA could be detected in the wild-type scion apex that grafted onto tomato and Arabidopsis transformants. These results indicate that the long-distance movement of pRNA is a general phenomenon that occurs in these plant species. In plants, the phloem trafficking is regulated by the sink-source status. Therefore, the translocation of pRNA might partly reflect the direction of phloem transport. As the scion growth, the earlier developed leaf will eventually begin to contribute to the phloem translocation stream. This will gradually dilute the signal derived from stock. Consistent with this notion, the Cmgaip RNA detected from the scion apex is gradually reduced. Further analysis the leaves along the scion axis revealed that the leaves of the scion also display a gradient phenotype. The earlier developed leaves always produced mutant phenotype, but the later developed leaves displayed less mutant or even wild type like phenotype. These results reflect the dynamic trafficking of pRNA in the phloem, and suggest that the delivery of RNA through phloem might contribute in fine-tuning of developmental programs.
To test whether the delivery of RNA through the phloem is simply followed the mass-flow process, RT-PCR was next performed to detect the Cmgaip RNA from different sink tissues of the wild-type scions grafted onto tomato Cmgaip transformants. The results showed that although Cmgaip RNA was detected in scion apex and flower, it was not detected in another strong sink tissue, fruit. This result suggests that selective rather than passive transport mechanisms might involve in RNA long-distance trafficking. Although most of the pRNA tested so far is expressed in CC, but not all of the RNA in the CC is subjected to phloem translocation. The simplest hypothesis is that the motifs within pRNA might contribute to their trafficking. To test this hypothesis, transformants that carry P35S-GFP transgene were generated. RT-PCR failed to detect GFP RNA from wild-type scion that grafted onto P35S-GFP transformant, suggesting that the RNA trafficking is regulated by specific sequence.
Taken together, we have built up a working model to elucidate the movement of pRNA from CC into SE (Figure). Currently, we are designing experiments to test this model. In the future, the knowledge derived from this project will likely provide a platform for the development of new directions for plant biotechnology.
Figure. The model of pRNA long-distance trafficking.
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