| Higher plants have evolved a communication system that enables coordination of developmental and environmental cues between different organs.This communication is achieved by long-distance signaling that takes place in the vasculature tissue.Phloem,one of the major components in the vasculature system,has long been recognized as a tissue that transports carbohydrates and amino acids.In recent years,it has been found that this tissue is also a conduit for signals,e.g.,mRNAs,small RNAs,proteins,small peptides,and hormones.Among which,the physiological functions of some long-distance mobile mRNAs have been demonstrated.For example,the development of sink tissues such as tomato young leaves,potato tubers and Arabidopsis roots are partially controlled by the long-distance mobile mRNAs generated in the source tissues.Heterograft represents grafts in which one species/genotype was used as a scion and another as a rootstock.In recent years,a few heterograft systems combined with RNA-seq technology were used to demonstrate that large-scale migration of mRNAs from leaf to root,or vice versa,exists.This variability,with over an order of magnitude differences in the number of mRNAs transported,suggests a great deal of species specificity and that not all the mobile mRNAs are functional.However,the mechanistic elucidation of mRNA movement in these researches remains preliminary.A computational analysis suggested that most of the identified mRNA species are mobile as a consequence of local abundance in companion cells.To what extent this correlation drawn from mathematic modeling method applies to the natural plant systems needs further experimental verification.Another research discovered that among the large number of mobile mRNAs identified in the Arabidopsis heterografts,11.4% of them harbored a tRNA-like structure(TLS).Further experimental evidence supported the hypothesis that not only does this motif impart stability and mobility to mRNAs,but also mRNAs with this structure can be translated into proteins after transport.However,how general this mechanism can be applied to other plant heterograft systems remains unknown.In this study,we used a Nicotiana benthamiana/tomato heterograft system and a transgenic approach to elucidate the mechanisms of long-distance mRNA movement.The main results obtained were as follows:(1)For the first time,the N.benthamiana/tomato heterograft system was established and applied to study the mRNA transport process from source leaves to root.Compared to the most published systems,the relatively large genome sequence and larger heterogeneity difference between the two species allowed confident and exhaustive identification of mobile mRNAs.The larger size of the graft partners was another advantage that allows to answer more general questions.A total of 183 mobile mRNAs were identified from this system.By comparing their abundance in the leaf and root,we found that the overall mRNA abundance in the leaf is not a good indicator of transcript mobility to the root.In this system,the none of the 100 most abundant mRNAs in the N.benthamiana scion could move to the root;and only 3 of the 183 most abundant transcripts in leaf moved to the root.This lack of correlation is in conflict with the grapevine graft system in which 17 of the most abundant 33 leaf mRNAs were mobile.This large difference between the two systems led us to hypothesize the the cell origins for the synthesis of mobile mRNAs may differ between herbaceous and woody species.The abundance of most of the mobile mRNAs identified in this system was very low and 14.7% of which showd higher abundances in the root that leaf,which indicated the movement of these mRNAs were under regulation.(2)We overexpressed two non-mobile mRNAs in the source companion cells of potatoes and grafted them onto WT rootstock.We found that increasing the expression levels of non-mobile mRNAs in the companion cells does not promote their mobility,which suggested that the mRNA movement is a regulated process.(3)We designed the long-stem heterograft to study the accumulation pattern of shoot born mRNAs during their movement to the root.The analysis revealeda total of 1,096 mobile mRNAs.The numbers of mRNAs decreased from the high stem to low stem and root.Although the abundance of most mRNAs decreased during the shoot-to-root transport,10 mRNAs showed higher abundance in the root than in the high stem and low stem.In addition,we found that the majority of the mobile mRNAs harboring the TLS motif did not arrive in the tomato root.Only 23 of the 122 mobile transcripts having this motif that successfully passed the graft junction were detected in the roots.These results indicate that the mRNA movement has both regulated and unregulated components.(4)Establishment of a split-shoot heterografting system.We identified 58 N.benthamiana mRNAs in the mature potato leaves and demonstrated that some mobile mRNA can move downward,and then upward into new parts of the shoot,which indicated a mRNA cycling existed.However,the movement path remains unclear.In conclusion,these findings suggest that the long-distance mRNA movement is a complex process.Identifying the core mobile mRNAs across species and the elucidation of the physiological roles associated with this movement are challenging but remains an important task for future research.Although the origin of the mobile mRNAs moving from the shoot to the root has not been confirmed,one of the most likely location is the phloem companion cells.In addition to being the cellular origin of mobile mRNAs,phloem companion cells have been indicated to be an active metabolism machinery for other components such as small RNAs,signaling peptides,plant hormonal compounds,proteins,etc.Therefore,understanding companion cell specific cellular processes have been pursued for a long time by scienties.However,it has been a challenge to accurately study companion cells due to its small size and deeply buried characteristics.In this study,a TRAP method combined with a cell specific transgenic approach were used.We efficiently identified the companion cell specific translatome and provided accurate reference data to study the companion cell physiology.The biological proecesses of the companion cell differed significantly from other leaf cells.The companion cells have important physiological roles,including proceeding energetic energy metabolism;transporting of organics,mineral nutrients,proteins and RNAs;G-protein coupled plant signal transduction;regulation of plant circadian clock and photo period;transcriptional controlling and regulation of plant long-distance mineral signlling. |
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