Studies originating with plant viruses led to the concept that plasmodesmata potentiate the cell-to-cell trafficking of viral and endogenous proteins and nucleoprotein complexes. In this article, we develop the theme that, at the tissue/organ level, cell-to-cell trafficking of information molecules enables non-cell-autonomous control over a range of processes, whereas at the organismal level, the phloem serves as an information superhighway. The capacity to deliver proteins and
nucleoprotein complexes, over long distances, allowed for the development of a viral surveillance/resistance mechanism, as well as the integration of processes at the whole-plant level.
Addresses
*Section of Plant Biology, Division of Biological Sciences, University of California, One Shields Avenue, Davis, CA 95616, USA;
e-mail: [email protected]
†Department of Vegetable Crops, Faculty of Agriculture, Hebrew University of Jerusalem, POB 12, 76100 Rehovot, Israel
Current Opinion in Plant Biology1999, 2:192–197 http://biomednet.com/elecref/1369526600200192 © Elsevier Science Ltd ISSN 1369-5266
Abbreviations
GFP green fluorescent protein
MP movement proteins
TMV tobacco mosaic virus
Introduction
Over the course of evolution, viruses have developed com-plex relationships with their biological hosts. Indeed, through such interactions, viruses may well have contributed to this evolutionary process through horizontal gene transfer, or by the development of host-based information processing cascades aimed at viral detection and interdiction. In the lat-ter case, the host may have adapted this viral surveillance system for the regulation of endogenous genes. Research on plant virus–host interactions is currently providing consider-able insight into the mechanism(s) by which viruses move within plants, the manner in which plants carryout viral sur-veillance and the strategies viruses have evolved to overcome the host detection system. Of equal importance, these viral studies have provided a foundation for experi-ments aimed at elucidating the mechanism by which plants exert supracellular control over physiological and develop-mental processes. The emerging picture is that, at the cellular or tissue level, plasmodesmata play a central role in the trafficking of information macromolecules (proteins and nucleic acids), whereas at the organismal level, the phloem acts as the conduit for delivery of equivalent macromolecules to distant organs. These paradigms are providing insights into novel regulatory pathways that may underlie the manner in which resources, including photosynthate, are allocated on a whole-plant basis.
Plasmodesmata control the cell-to-cell trafficking
of viral and endogenous macromolecules
Over the past decade, results from a wide range of genetic, molecular and cellular studies have provided support for the hypothesis that plant viruses infect cells within a tissue by moving cell to cell through plasmodesmata [1,2]. This process is mediated by viral encoded non-structural pro-teins, termed movement proteins (MP). These MP have the capacity to bind to viral nucleic acids in a non-sequence-specific manner, interact with plasmodesmata to induce an increase in molecular size exclusion limit, and to mediate the transport of viral nucleic acids into neighbor-ing cells through plasmodesmata. Direct evidence that viral MP can move from cell to cell was gained through the use of microinjection techniques. Furthermore, experi-ments in which particle bombardment or viral infection was employed to introduce transcripts encoding MP:GFP (green fluorescent protein) constructs, have confirmed these results [3,4], and provided additional insight into the complexity of the interaction of the MP within cells under-going viral infection [5,6,7•,8].
The hypothesis that the genes encoding these viral MP were acquired from the plant genome, and the corollary that plants utilize plasmodesmata to control physiological and developmental processes by transporting endogenous macromolecules from cell to cell [9], provided a connection between the evolution of the virus and its plant host. A number of lines of experimental evidence now provide sup-port for this general concept. Direct evidence that plasmodesmata mediate the cell-to-cell transport of endoge-nous proteins was provided by microinjection experiments employing proteins ranging from transcription factors involved in meristem [10] or floral development [11] to the unique class of proteins that are translocated in the phloem sieve tube system [12••,13]. Additional evidence for a role of
plasmodesmata in protein transport has been gained through the use of somatically stable periclinal chimeras [14]. These studies, involving the Antirrhinum majus MADS-box transcription factors, DEFICIENS and GLOBOSA, provided evidence consistent with the hypothesis that there is a polarity in the cell-to-cell trafficking of these proteins from the inner to the outer layers of the developing petal.
The finding that viral MP compete with endogenous pro-teins for the cellular machinery involved in their cell-to-cell transport [15••] offers an additional line of evidence in
sup-port of the hypothesis that viral MP genes were acquired from the plant genome. Furthermore, as plasmodesmata are engaged in the selective transport of regulatory molecules, these results suggest that competition between MP and such plant proteins may form the basis for some aspects of plant symptom development in response to viral infection. Finally,
Connections between virus movement, macromolecular
signaling and assimilate allocation
strong evidence for the connection between non-cell-autonomous control over developmental and physiological processes in plants and viral movement was recently provid-ed by the identification of a plant paralog to a viral MP that potentiates transport of mRNA into the phloem [16••]. In
contrast to KNOTTED1 [10], this plant paralog from pump-kin, CmPP16 [16••], has the capacity to bind with RNA and
mediate its cell-to-cell transport in a non-sequence-specific manner, a property similarly displayed by the MP of RNA viruses. Collectively, these findings support the hypothesis that plasmodesmata evolved the capacity to mediate the transport of information molecules, thereby establishing the capacity for non-cell-autonomous — or supracellular — con-trol over tissue and organ development. As in other biological systems, plant viruses evolved the capacity to exploit this unique plant system to achieve the ability to replicate and move from cell to cell within this host.
Inter-organ communication mediated by
macromolecules transported by the phloem
The operation of an inter-organ information superhighway, involving the phloem, has gained considerable support from recent studies on both the systemic movement of viruses and the translocation of endogenous signaling molecules [17•].
The long-distance transport of viruses and endogenous macromolecules (proteins and RNA molecules) also involves their trafficking through the plasmodesmata that interconnect companion cells to the functional sieve tube system [11,12••,18••,19,20]. A consistent feature associated with
pro-tein transport through plasmodesmata is an increase in the size exclusion limit of the microchannels through which this trafficking occurs. This change in plasmodesmal size exclu-sion limit, from 850 Da to values ranging from 20–50 kDa, normally permits the diffusion of fluorescently labeled reporter molecules, such as fluorescein isothiocyanate labeled dextrans. Interestingly, the high level of macromolecular transport that occurs through the plasmodesmata connecting the nucleate companion cells and their associated enucleate sieve elements [12••] results in a sufficient increase in
plas-modesmal size exclusion limit to permit the entry of GFP (27 kDa) into the phloem translocation stream [20]. It is also at this cellular boundary that CmPP16, and its orthologs in other plant species, appears to mediate the delivery of mRNA into the phloem translocation stream [16••,21].
It has long been recognized that plant viruses (either as viri-ons or nucleoprotein complexes) are delivered into developing (sink) tissues by the phloem and that their egress into the surrounding cells, via plasmodesmata, results in the establishment of a systemic infection. The concept of the phloem as a vehicle for the long-distance delivery of endoge-nous proteins and RNA molecules, however, has only recently come under scrutiny. Support for this hypothesis has now been provided by a number of lines of experimental evi-dence. Grafting studies provided irrefutable evidence that proteins and RNA move within the phloem from stock to scion tissues [16••,19]. Such experiments, performed on
transgenic tobacco plants expressing the nitrate (or nitrite)
reductase gene, demonstrated that the cosuppression state (post-transcriptional gene silencing), activated in the stock, could be transmitted to the scion [22••]. Importantly, the
activation of this state is highly sequence-specific, consistent with the concept that RNA is being delivered, via the phloem, into developing leaves, where it activates post-tran-scriptional gene silencing. Results of this nature provide a solid foundation for the hypothesis [17•,22••] that plants
uti-lize the phloem for the establishment of systemic acquired silencing. Furthermore, treatments that elevate the mRNA level of an endogenous gene, within a specific tissue, can also activate the SAS state, resulting in the silencing of the target gene in developing organs [23].
Parallel studies performed on transgenic plants expressing a range of foreign or viral gene constructs revealed that viral infection also induces post-transcriptional gene silencing, and that this state can similarly be transmitted, via the phloem, to developing leaves [24–26,27••,28]. These
find-ings implicated the phloem in the operation of a general RNA surveillance mechanism that functions within the body of the plant to identify and degrade specific tran-scripts, including those of viral origin [17•]. In this regard, it
is interesting to note that viruses appear to have evolved strategies to interdict, or suppress, this plant RNA surveil-lance system [29••,30••,31]. Here, it is important to stress
that certain viruses — such as cucumber mosaic virus — have acquired the capacity to block systemic acquired silencing directed at endogenous and viral transcripts [32]! Clearly, it will be important to ascertain whether the plant utilizes an equivalent anti-systemic acquired silencing sys-tem to suppress the silencing of endogenous genes.
Cell-to-cell and long-distance transport of
macromolecules: integration of physiological
and developmental processes
The intriguing question raised by these recent discoveries is whether the plant also employs the phloem to deliver protein and/or RNA, to distant organs, in order to integrate physio-logical processes, such as photosynthate production in source leaves, with its overall developmental program. By analogy with the viral-induction and spread of post-transcriptional gene silencing/systemic acquired silencing, initiated from a small group of mesophyll cells in a source leaf [33•], the
delivery of RNA (e.g. encoding a transcription factor) from source leaves, via the phloem, into a unique set of cells in the meristem, could conceivably activate localized post-tran-scriptional gene silencing. Absence of the encoded protein may induce a switch in the developmental program(s) con-trolling a single process, or it may initiate a cascade of events resulting in conversion of the shoot apical meristem into an inflorescence meristem. Alternatively, delivery and transla-tion of mRNA molecules may also be used to integrate events occurring in distantly-located plant organs.
been provided by studies on photoassimilate partitioning/allocation. It has long been recognized that the allocation of resources to various plant organs is a complex process governed by the activities of source leaves, the vas-cular tissue along the transport pathway and the various importing plant organs [34]. The combined growth rate of all new organs cannot, for any length of time, exceed the photo-synthetic capacity of the source tissues to supply the assimilate needs of the plant [35]. Thus, assimilate allocation to the various sink organs must be controlled by a system that receives input signals from the whole plant. Historically, dif-ferences in sugar concentration along the plant vascular system were thought to serve as a key factor in the control of assimilate allocation. The utilization rate of the transported sugar, in a specific organ, was considered to be one of the major parameters involved in modulating the driving force for sugar transport towards a particular organ [36]. Although this simple biochemical/biophysical feedback system may operate at the cellular level, its inadequacies at the organ and whole-plant levels are only now being appreciated [37].
Evidence for the involvement of macromolecular traf-ficking in the control of photosynthate partitioning was first provided by studies performed on transgenic tobac-co plants expressing the MP of tobactobac-co mosaic virus (TMV); expression was driven by the 35S-CaMV promot-er. Quite unexpectedly, compared with control lines, higher levels of starch and sugars accumulated in source leaves of these TMV-MP expressing plants [38]. Consistent with these findings, export of photoassimi-lates, during the day, was lower in TMV-MP expressing lines compared with control tobacco plants [39]. A similar influence on source leaf carbon metabolism was also observed for transgenic tobacco expressing the MP of potato leafroll luteovirus [40]. An elegant series of exper-iments conducted by Tecsi et al.[41–43] on the influence of viral infection, per se, on photosynthetic capacity and carbohydrate partitioning, demonstrated that viral-induced alterations in leaf metabolism were not due to virus replication. Interestingly, the alterations in carbohy-drate metabolism that were detected, during virus infection, were spatially regulated and extended beyond the cells where viral replication was taking place. This complex spatial and temporal response, within source tis-sues, may well reflect the operation of a supracellular control system. Collectively, these studies raised the pos-sibility that viral MP interact (compete) with host proteins, presumably at the level of macromolecular traf-ficking through plasmodesmata, to alter this putative endogenous metabolic control network [44].
At the whole plant level, constitutive expression of the TMV-MP results in a significant alteration in photoassimi-late allocation. The most dramatic effects were observed on biomass production in the lower stem and root system of MP-expressing plant lines; the root mass was ~50% below that of control lines, yielding a 2-fold decrease in root-to-shoot ratio [45]. Grafting studies revealed that
expression of the TMV-MP, in scion tissues, induces the same reduction in photoassimilate allocation to the lower stem and roots of the stock. Transgenic plants expressing various mutant forms of the TMV-MP displayed either enhanced effects on carbon allocation and plant growth, or, in the case of a carboxy-terminal deletion mutant, a normal phenotype was fully restored. These studies provide addi-tional support for the hypothesis that the influence of the TMV-MP on carbon allocation is the result of protein–pro-tein interaction within the source tissue.
Alterations in photoassimilate allocation often reflect the adaptation of the plant to environmental stresses. In this regard, it is of interest to note that exposure of TMV-MP expressing tobacco plants to nutrient deficiency (N, P or K), or to water stress, revealed that although nutrient depriva-tion elicits a significant effect on root-to-shoot ratio, it does not alleviate the influence of the TMV-MP on resource allo-cation. In marked contrast, an imposed water stress treatment completely over-rides the influence of the TMV-MP on the root-to-shoot ratio [46]. These findings are consistent with the TMV-MP interacting with a complex control system involving input signals from the whole plant.
Studies conducted on transgenic potato plants expressing the TMV-MP provided additional insight into the com-plexity of this putative assimilate control system. When expressed under the control of a phloem-specific (rolC) promoter, the TMV-MP elicited a daily accumulation of carbohydrates in source leaves and an alteration in the ratio between the tuber and the shoot dry weight [47••]. In
con-trast, TMV-MP expression within mesophyll and bundle sheath cells (driven by the nuclear photosynthesis ST-LS1 promoter) caused a reduction in the overall level of carbo-hydrates and an enhancement in the rate of sucrose export from potato source leaves [48]. Importantly, these effects were only manifiested in mature plants in which tubers had been initiated, suggesting that the influence of the TMV-MP is under developmental control.
That virally-encoded proteins can exert a profound effect on sugar metabolism and photoassimilate allocation, at sites distant from their expression, clearly implicates the traf-ficking of information molecules between source leaves and distant sink organs. Such a long-distance communica-tion system may utilize a new class of signaling macromolecules (proteins and/or RNA) to co-ordinate pho-tosythesis and carbon/nitrogen metabolism in source leaves with the complex growth requirements of the plant under the prevailing environmental conditions [44,45,49]. As viral MP and endogenous proteins, capable of moving from cell to cell, compete at the level of the plasmodesmata [15••],
the effects of the TMV-MP may represent interference at a pivotal step(s) in this supracellular control network.
Conclusions and future directions
realize that plasmodesmata are not ‘simple holes’. During the past five years, information provided by a wide range of studies has established the paradigm that plasmodesma-ta mediate the selective transport of proteins and nucleoprotein complexes. This unique capacity potentiat-ed the development of a novel supracellular level of control that functions at both the tissue/organ and organis-mal level. Although the role of the phloem in the delivery of nutrients and hormones has long been appreciated, its function as an information superhighway is a very recent discovery. It is now fully apparent that plasmodesmata and the phloem play a central role in post-transcriptional gene silencing and RNA surveillance, which underlies the phe-nomenon of systemic acquired silencing. What remains to be established is the extent to which cell-to-cell and long-distance transport of RNA are involved in epigenetic events that enable the plant to optimize its interaction with an ever changing environment. The recent finding that plant viruses have acquired the capacity to interdict the host RNA surveillance system may well lead to the dis-covery of an equivalent plant system responsible for resetting a given epigenetic programme. Finally, it will be interesting to learn the nature of the relationship between plasmodesmal mediated signaling involving macromole-cules and the signal cascades controlled by the plant’s slate of small hormones.
These emerging paradigms, in conjunction with the discov-ery that expression of viral MP in plants can induce precise changes in carbon metabolism and photoassimilate alloca-tion, now provide a conceptual foundation for future studies aimed at elucidating the communication network responsi-ble for integrating photosynthetic productivity with resource allocation at the whole-plant level. Such informa-tion will surely provide an understanding of how plants coordinate the essential physiological functions performed by distantly-separated organs [50••,51]. Identification of the
proteins involved in mediating and controlling cell-to-cell transport, especially at the companion cell-sieve element boundary, will provide an important first step towards achieving this goal.
Acknowledgements
Research in the authors’ laboratories was supported by grants from the National Science Foundation (IBN-94-06974 to WJ Lucas), the Department of Energy Division of Energy Biosciences (DE-FG03-94ER20134 to WJ Lucas) and the United States-Israel Binational Agricultural Research Development Fund (IS-2385-94C to S Wolf and WJ Lucas).
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest ••of outstanding interest
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