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The angiosperm phloem sieve tube system: a role in mediating traits important to modern agriculture

Byung-Kook Ham* and William J. Lucas*

Department of Plant Biology, College of Biological Sciences, University of California, Davis, CA 95616, USA

* To whom correspondence should be addressed. E-mail: [email protected] and [email protected]

Received 19 September 2013; Revised 7 November 2013; Accepted 11 November 2013

Abstract

The plant vascular system serves a vital function by distributing water, nutrients and hormones essential for growth and development to the various organs of the plant. In this review, attention is focused on the role played by the phloem as the conduit for delivery of both photosynthate and information macromolecules, especially from the context of its mediation in traits that are important to modern agriculture. Resource allocation of sug-ars and amino acids, by the phloem, to specific sink tissues is of importance to crop yield and global food secu-rity. Current findings are discussed in the context of a hierarchical control network that operates to integrate resource allocation to competing sinks. The role of plasmodesmata that connect companion cells to neighbour-ing sieve elements and phloem parenchyma cells is evaluated in terms of their function as valves, connecting the sieve tube pressure manifold system to the various plant tissues. Recent studies have also revealed that plasmodesmata and the phloem sieve tube system function cooperatively to mediate the long-distance deliv-ery of proteins and a diverse array of RNA species. Delivery of these information macromolecules is discussed in terms of their roles in control over the vegetative-to-floral transition, tuberization in potato, stress-related signalling involving miRNAs, and genetic reprogramming through the delivery of 24-nucleotide small RNAs that function in transcriptional gene silencing in recipient sink organs. Finally, we discuss important future research areas that could contribute to developing agricultural crops with engineered performance characteristics for enhance yield potential.

Key words: Carbon allocation, epigenetics, phloem long-distance signalling, production agriculture, protein trafficking, yield potential.

Introduction

Acquisition of a vascular system represented a major event in plant evolution, as it potentiated species radiation into a wide range of ecological niches. Production of the xylem from meristematic tissues, termed the cambium, gives rise to an efficient water transport system comprised of files of cells (vessels and/or tracheids) that, during their development, undergo programmed cell death to form a low-hydraulic-resistance conduit from the root to the shoot. An additional important feature was acquisition of the enzymic pathway(s) for cell-wall lignification. This gave rise to xylem cells having greater mechanical strength, thereby allowing for increased

plant height and better competition for sunlight (Lucas et al., 2013).

The second component of the vascular system, the phloem, develops also from the cambium (Fig. 1A). Here, the conduct-ing cells, termed sieve cells in lower vascular plants and sieve elements (SEs) in the advanced angiosperms, are not dead at maturity. In the angiosperms, during SE maturation, each cell in a specific file undergoes a process in which its vacuole and nucleus are degraded, and many of the organelles are either degraded or become greatly reduced in number. At matu-rity, each SE retains its plasma membrane and cytoplasmic

© The Author 2013. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: [email protected]

Journal of Experimental Botany, Vol. 65, No. 7, pp. 1799–1816, 2014doi:10.1093/jxb/ert417 Advance Access publication 24 December, 2013

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1800 | Ham and Lucas

continuity between neighbouring SEs is established by struc-turally modifying plasmodesmata (PD) to form sieve plate pores (Fig. 1B). Such files of enucleate cells form an individ-ual sieve tube (ST), and collectively, all sieve tubes within the plant establish the sieve tube system which functions in the delivery of nutrients (as fixed carbon, amino acids, etc.) to developing tissues (Fig. 1A and B).

Physiological functions of the mature enucleate SEs are maintained by specialized cells, termed companion cells

(CCs). This support is achieved by the intercellular move-ment of macromolecules through the PD that connect CCs to their neighbouring SEs (Fig. 1C). Although it is gener-ally considered that mature SEs lack the capacity for pro-tein synthesis, recent studies on the sieve tube system of the cucurbits (Lin et al., 2009; Ma et al., 2010) identified many ribosomal and associated components involved in protein synthesis. In addition, a comparative analysis between the phloem proteome and the special messenger (m)RNA

Fig. 1. Development of the angiosperm phloem sieve tube system. (A) Schematic of the phloem sieve tube system (red lines) that develops from the vascular cambium (purple lines) to interconnect the mature leaves (functioning as a source of photosynthetically fixed carbon) to distantly located developing organs. Leaves on the lower part of the plant generally deliver resources to the root system, whereas those on the upper region supply the developing leaves and meristems (blue arrows). (B) Development of mature functional enucleate sieve elements (SE) from cells derived from the vascular cambium. During this process, the SE nucleus and vacuole are degraded, and many organelles become greatly reduced in number. In addition, during SE expansion, enzymic deposition and subsequent removal of β-1,3-glucan (callose; yellow) around the plasmodesmata connecting neighbouring SEs leads to the formation of sieve plate pores (SPP). These pores interconnect mature SEs into a functional sieve tube (ST) through which the phloem translocation stream moves driven by a hydrostatic pressure (turgor) gradient. The plasmodesmata connecting SEs to their neighbouring companion cells (CCs) also undergo a structural modification, in which additional plasma membrane-lined (red) cytoplasmic channels (containing endoplasmic reticulum; ER) are added to the CC side of the cell wall (green); SER, sieve element reticulum, a modified form of the ER. (C) Maintenance of the enucleate SEs is achieved by genes expressed in the CCs. The transcripts for these genes are either translated in the CC cytoplasm followed by trafficking into the SE through the connecting plasmodesmata or are trafficked into the SE, as a complex formed between the mRNA and an RNA-binding protein (RBP), where protein synthesis may then occur. These two pathways can also generate proteins that function in long-distance signalling between various plant organs. Note that in this model the ER/SER has been omitted for simplicity.

Phloem transport and agricultural traits | 1801

population identified from Cucurbita maxima (pumpkin) phloem sap indicated a match between a significant number of mRNA species and their cognate proteins. These findings are consistent with a model in which mRNA produced in the CCs is trafficked through the CC–SE PD into the SEs where protein is produced (Fig. 1C). Of interest here is the recent finding that an in vitro wheat germ lysate translation system was found to be inhibited by the presence of tRNA fragments (30–90 bases) isolated from pumpkin phloem sap (Zhang et al., 2009). Hence, it is possible that such tRNA fragments may serve to regulate translation in the enucleate sieve tube system. In any event, either mRNA import into SEs, followed by protein synthesis, or protein synthesis in CCs, followed by trafficking through the PD into the SEs, must occur for the continued functioning of mature SEs over their life span of from weeks to years, without their nuclei.

Angiosperm leaves and their advanced vascular system

The angiosperms serve as major crop species, worldwide, due in large part to the characteristics of their leaf vascular system. The leaves of gymnosperms and other less advanced tracheophytes have limited specialization in their vein pat-terning, resulting in low vein densities per leaf area, being on the order of 1.5 mm mm–2 (Sack and Scoffoni, 2013). In marked contrast, the angiosperms evolved a developmental programme that allowed for the formation of higher vein orders within the lamina of the leaf (Brodribb and Field, 2010; Lucas et al., 2013). This gave rise to main veins (primary and secondary) that serve two functions; namely, the major pathway for xylem water delivery into and phloem export of sugars out of the leaf, and mechanical support—an impor-tant adaptation that allowed for a significant increase in leaf size (Sack and Scoffoni, 2013). The addition of minor veins (third through fifth order) allowed for higher vein densities ranging from 5 to 15 mm mm–2, thus providing an enhanced system for phloem loading of photosynthate. This feature, in conjunction with an increase in stomatal density (Franks and Beerling, 2009), resulted in a significant increase in photo-synthetic capacity and, thus, overall higher plant productiv-ity compared to gymnosperms (Brodribb et al., 2007; Walls, 2011; Sack et al., 2012).

Global control over resource allocation

The processes underlying the loading of photosynthate into the sieve tube system, within the leaf minor veins, and its unloading into developing tissues/organs (termed sinks) have been well characterized (Van Bel, 1993; Patrick, 1997; Ayre, 2011; Lucas et al., 2013). What remains to be resolved is the process(es) by which the plant exerts global control over its sieve tube system, in terms of resource allocation to specific sink organs. The general concept is that a turgor pressure gradient, established between the loading phloem (termed a source region) and sink phloem (termed an unloading or

release region), drives bulk flow through the individual sieve tubes. In this model, more resources would be delivered to highly metabolically active sink organs, as the elevated local consumption of imported sugars in such organs should cause the turgor pressure within the unloading phloem to decrease, thereby enhancing the pressure differential between the source region and these specific sinks (Fig. 2A).

Although intuitively simple to understand, this sink-strength model for resource allocation needs to be consid-ered in the context of a global control system that mediates between abiotic and biotic inputs and endogenous devel-opmental and physiological programmes. Insight into the operation of such a control system has been gained through a number of studies, including those on the regulation of the plant root-to-shoot ratio. Here, environmental inputs, such as water or nutrient (phosphate [Pi], nitrogen, etc.) availability in the soil, exert control over the extent of phloem delivery to shoot versus root sink tissues. These stress conditions gener-ally cause a change in root architecture—notably an increase in lateral root development—with a concomitant increase in root relative to shoot growth (Nibau et al., 2008; Niu et al., 2013). The underlying events that control this shift in resource allocation, towards the root system, ensure that the plant can adapt effectively to the ever-changing local environ-ments within the soil to acquire the essential nutrients and water required for growth at the whole-plant level.

In split-root experiments, in which the root system was divided to allow exposure to Pi-sufficient and Pi-deficient conditions, lateral root development and gene expression associated with Pi acquisition remained unaffected in the region of the root exposed to Pi-deficient conditions provided that the other portion of the root was bathed in Pi-sufficient media (Baldwin et al., 2001; Linkohr et al., 2002; Franco-Zorrilla et al., 2005; Shane and Lambers, 2006; Shane et al., 2008; Thibaud et al., 2010). Similar studies involving local-ized application of jasmonic acid to one part of a split-root system resulted in a rapid reallocation of phloem delivery to the untreated part of the root system (Henkes et al., 2008). Importantly, in this study it was established that this reallo-cation of resources was not directly related to a change in sink strength in the untreated portion of the root. Results of this nature clearly reveal the presence of a signalling net-work, involving both the xylem and the phloem that can exert long-distance control over developmental and physiologi-cal processes. These findings support the hypothesis that a hierarchical control network operates within the body of the plant to regulate resource allocation between competing sinks (Fig. 2B).

The phloem translocation stream contains sugars, amino acids, mineral nutrients, hormones, and unique popula-tions of proteins and small interfering (si) RNA, micro (mi) RNA, and mRNA (Bostwick et al., 1992; Fisher et al., 1992; Lough and Lucas, 2006; Buhtz et al., 2008; Turgeon and Wolf, 2009; Lucas et al., 2013). As phloem long-distance delivery of both proteins and mRNA has been well dem-onstrated (Golecki et al., 1999; Ruiz-Medrano et al., 1999; Xoconostle-Cázares et al., 1999; Kim et al., 2001; Haywood et al., 2005), such signalling macromolecules are plausible


candidates in this hierarchical global resource allocation control system. In this regard, it is noteworthy that graft-ing studies with Arabidopsis demonstrated that IAA18 and IAA28 transcripts, synthesized in the phloem of mature source leaves, are delivered via the phloem into the root sys-tem where they influence root architecture by negatively reg-ulating the development of lateral roots (Notaguchi et al., 2012). It will be interesting to determine whether environ-mental inputs control transcription of these auxin-related genes and if a regulatory system, at the level of the CC–SE PD, might control entry of the transcripts into the phloem translocation stream.

Based on split-root experiments involving Pi-deficiency treatments, it is clear that a signal(s) from the roots must travel through the xylem to the shoots where it then activates a response output that travels through the phloem down into the divided root system to prevent the upregulation of both

lateral root development and Pi transport activity. The signal moving through the xylem could well be the actual concentra-tion of Pi in the transpiration stream or hormones such as cytokinin and/or strigolactone (Franco-Zorrilla et al., 2005; Chiou and Lin, 2011). Recent studies have established that phloem long-distance delivery of miR399 plays an important role in regulating Pi transport across the root (Buhtz et al., 2008; Lin et al., 2008; Pant et al., 2009). Furthermore, func-tional genomics studies are providing insights into the gene regulatory networks that are activated in shoot and root tis-sues in response to nutrient (e.g. Pi-deficiency) stress treatment to the root system (Misson et al., 2005; Calderon-Vazquez et al., 2008; Li et al., 2010; Thibaud et al., 2010; O’Rourke et al., 2013). However, the nature of the phloem-borne signals involved in regulating root architecture in response to abiotic and biotic stresses remains as an important area for future studies.

Fig. 2. Models depicting possible mechanisms that could control resource allocation between competing sink organs. (A) Metabolic activity within a specific sink organ drives the rate of sucrose unloading from the phloem. In this model, the sink region having the highest metabolic activity (sink organ 1; high demand for imported sugars) will generate the largest pressure gradient within its phloem: i.e. a high pressure [P] in the source region and a low P in the SEs in the sink. This would maximize phloem translocation into this sink region of the plant. Thus, an organ in which the cells have low metabolic activity (sink organ 3) will have a relatively high pressure in its SEs, and thus the resultant small pressure gradient will deliver less sugar to this sink region of the plant. (B) Operation of a hierarchical regulatory network that controls resource allocation between competing sinks. The phloem sieve tube system serves as a pressure manifold (thick red lines) to interconnect source regions of the plant (sites of net production of photosynthate) to the various sink organs. Delivery of photosynthate to a specific sink can be adjusted in response to a range of inputs. Endogenous inputs are generated within a sink and include the interplay between developmental/physiological programmes and metabolism. Local environmental inputs (blue arrows), such as nutrient or water availability and hormonal gradients, can have positive or negative effects on endogenous inputs to adjust the process of phloem unloading. A global integrator (purple arrows), involving root-to-shoot and shoot-to-root signals, can adjust or override both endogenous and local input signalling systems to modulate photosynthate delivery to specific sinks. Elucidation of the molecular events underlying these various hierarchical regulatory components will provide important insights into possible ways to manipulate resource allocation to specific sink organs. The phloem sieve tube system serves as a pressure manifold (thick red lines) to interconnect source regions of the plant (sites of net production of photosynthate) to the various sink organs (S1, S2, S3, to Sn). Delivery of photosynthate to a specific sink can be adjusted in response to a range of inputs. Endogenous inputs are generated within a sink and include the interplay between developmental/physiological programmes (D) and metabolism (M).


Plasmodesmata and the sieve tube system

The phloem sieve tube system serves effectively as a pressure manifold, running throughout the body of the plant, in which the SE–CC, CC–phloem parenchyma, and SE–phloem paren-chyma PD represent gated valves. For those species that load photosynthate via an apoplasmic step (Lucas et al., 2013), it is critical that the CC–phloem parenchyma and SE–phloem parenchyma PD valves be closed to prevent the loss of the sugars uploaded into the CC–SE complexes present within the source leaf minor veins. In practical terms, this means that the size exclusion limit of the cytoplasmic microchan-nels within these PD must be below the size of the sugars and amino acids being loaded for delivery to distant organs (Fig. 3A).

A similar condition holds for the long-distance region of the pathway, except that here, release of some sugars and amino acids is needed to sustain metabolism within tissues such as the vascular cambium and establish starch reserves in parenchyma storage tissues. Within the unloading regions located in the various sinks, many species use a PD symplas-mic pathway for nutrient delivery into the surrounding tissues (Lalonde et al., 2003; Stadler et al., 2005; Zhang et al., 2006, 2007; Lucas et al., 2013). Thus, PD along the phloem can serve both as pathways for the trafficking of important infor-mation macromolecules, as described above, and as valves having the capacity to control local resource release (alloca-tion) (Fig. 3A). The important question is, how might a plant exert control over these PD valves?

An increase in PD size exclusion limit and, hence, perme-ability, can be achieved through interactions with specific classes of proteins, the first identified being the movement proteins used by many plant viruses to mediate the cell-to-cell trafficking of their infectious RNA/DNA (Wolf et al., 1989; Lucas, 2006). Unfortunately, although it has been established that such movement protein-induced increases in PD size exclusion limit involve protein–protein interactions, which can be blocked by mutations engineered within the move-ment protein, the molecular mechanism(s) underlying these changes largely remain to be elucidated (Benitez-Alfonso et al., 2010).

A number of endogenous proteins have also been shown to have the capacity to increase PD size exclusion limit (Lucas et al., 1995; Xoconostle-Cázares et al., 1999; Lucas et al., 2009; Maule et al., 2011). Furthermore, recent studies have identified PD-localized proteins that have the capacity to reg-ulate PD size exclusion limit (e.g. BG_ppap, Levy et al., 2007; PDLP1, Thomas et al., 2008; PDCB1, Simpson et al., 2009; RGP2, Zavaliev et al., 2010; GSL8, Guseman et al., 2010; PDLP5, Lee et al., 2011). It is noteworthy that these proteins are either involved in deposition or removal of β-1,3-glucan (callose) that is deposited around each orifice of the PD or in regulating this level of PD callose (Fig. 3B and C).

Another mechanism shown to alter PD size exclusion limit involves disruption to actin filaments. Treatment of cells with either cytochalasin D or profilin causes an increase in PD size exclusion limit (Ding et al., 1996). It has also been shown that the movement protein of Cucumber mosaic virus can interact

with and sever actin filaments, which gives rise to an increase in PD size exclusion limit (Su et al., 2010). That NET1A, a member of a plant-specific super family of actin-binding pro-teins, has been localized to PD, both at the orifice and along the length of the PD plasma membrane (Deeks et al., 2012) further supports the notion that changes in PD size exclusion limit can occur by this mechanism, either by viral movement protein action or through interaction with plant regulatory proteins.

Collectively, these findings establish that plants have evolved an array of mechanisms by which to modulate PD permeability. Studies have shown that such changes in PD, within source leaves, can influence the ability of plants to export sugars from these leaves, thereby leading to a decrease in root-to-shoot ratio (Zavaliev et al., 2010). Here, it is inter-esting to note that the maize tdy2 mutant, which accumulates high levels of sugars and starch in specific regions of its source leaves, was recently cloned and shown to encode for a callose synthase (Slewinski et al., 2012). A range of studies suggested that the defect in sugar export from source leaves was located to the CC–SE PD. However, the mechanism underlying this loading defect in tdy2 has yet to be elucidated.

A similar phenotype, in terms of accumulation of car-bohydrates in source leaves and an associated increase in shoot biomass, has been described for transgenic plants overexpressing phloem-specific NHL26 (Vilaine et al., 2013). The NHL26 membrane protein was detected within PD of phloem cells, and high levels in the CCs were shown to inhibit sucrose entry into the SEs, possibly through an increase in CC–SE PD callose or by some form of sugar sensing within the CCs between NHL26 in the PD and sugar movement into the SEs (Vilaine et al., 2013).

Plasmodesmal function, including modulation of size exclusion limit, can also be regulated by a range of cellu-lar processes and signalling molecules, including changes in redox state, reactive oxygen species (ROS), etc. (Burch-Smith and Zambryski, 2012). In terms of PD function in controlling unloading of sugars and subsequent movement out into sink tissues, studies on the gat1 mutant, which encodes an allele of thioredoxin-m3 from plastids, established that restriction of GFP exit from the unloading phloem into root sink tissues was highly correlated with an increase in ROS and PD callose (Benitez-Alfonso et al., 2009). Hence, GAT1 may well play an important role in controlling the size exclusion limit of SE–CC PD to adjust sugar unloading in sink tissues. Further sup-port for the role of ROS in regulating nutrient flux through root PD was provided by studies integrating mathematical modelling of the symplasmic pathway with fluorescence recovery after photobleaching experiments (Rutschow et al., 2011).

A possible link between PD permeability and plant metab-olism was suggested based on genetic studies on KOBITO1, which encodes a glycosyltransferase-like protein (Kong et al., 2012). Importantly, various mutants displayed an increase in protein movement cell to cell without any detectable changes in callose around the PD orifice. Finally, studies on transgenic plants expressing a dominant negative form of PDGLP1 (Ham et al., 2012) indicated that, in roots, this PDGLP1-GFP


Fig. 3. Plasmodesmata play a central role in controlling phloem delivery of nutrients to sink tissues. (A) Plasmodesmata interconnect the cytoplasm of mesophyll, bundle sheath, and phloem parenchyma cells to that of the companion cell–sieve element (SE) complex. In the source region of apoplasmic loading plants, the size exclusion limit (SEL) of plasmodesmata connecting phloem parenchyma cells to companion cells must be downregulated to prevent the back diffusion of sucrose loaded across the plasma membrane into the companion cell–SE complex. The same condition holds for the long-distance transport region of the phloem. However, here, at times, there is a need to adjust the plasmodesmata SEL to allow the release of sugars, either for local metabolism or for use in carbohydrate storage. In sink regions, control over plasmodesmata permeability can act as an important regulatory site to control phloem unloading. (B and C) Control over plasmodesmata permeability through enzymic adjustments in the level of callose. The opposing activities of an integral plasma membrane enzyme, glucan synthase-like (GSL), and a β-1,3-glucanase anchored to the outer surface of the plasma membrane at the neck region of plasmodesmata can control the actual size exclusion limit of the plasmodesmata cytoplasmic microchannels.


fusion protein causes a shift in root architecture. Based on phloem unloading studies, the observed change from primary to lateral root growth appeared to be accomplished through a shift in competition for resource allocation away from the primary root in favour of the lateral roots. The inability of this PDGLP1-GFP to form stable PD-localized protein complexes (Ham et al., 2012) may well have prevented the targeted delivery of either proteins or RNA complexes out of the unloading phloem into the surrounding meristematic cells. It will be important in future studies to elucidate the mechanism by which PDGLP1/2 influences primary root growth through phloem-mediated allocation of resources between the primary and lateral root meristems.

Phloem delivery of tuberization signals controls potato yield

Potato tubers serve as an important food source, worldwide. Tubers develop from underground juvenile lateral shoots, called stolons, whose development is regulated by a short-day photoperiod, which is sensed by the aboveground vegetative tissues (Fig. 4A). Thus, although stolons are present during the long days of summer, they do not function as strong sinks until a short-day-induced signal, originating in the mature source leaves, moves through the phloem to upregulate the process of tuberization (i.e. activation of radial growth in the stolon).

Fig. 4. Phloem delivery of tuberization signals controls potato yield. (A) Potato plants grown under long-day (LD) photoperiodic conditions do not develop tubers, whereas those grown under short-day (SD) conditions receive tuberization signals from the source leaves and subsequently produce tubers. (B) Under SD conditions, StBEL5 and StPOTH1 mRNAs are transported from companion cells (CCs) into the sieve tube system by RNA-binding proteins (RBPs). These transcripts then move long distance through the phloem in the form of ribonucleoprotein complexes (RNPc), which are delivered to underground stolons where they enter meristematic cells to initiate tuber formation. (C) Production of StSP6A transcripts in source CCs of SD-grown potato plants allows StSP6A protein to enter the sieve tube system for delivery to underground stolons. After moving through plasmodesmata from the sieve tube system into meristematic cells, StSP6A protein enters the nucleus where, in conjunction with a transcription factor like FLOWERING LOCUS D (FD), it activates an autoregulatory loop, leading to enhanced levels of StSP6A and induction of the tuberization pathway (TP); N, nucleus. (D) Relationship between stolon StBEL5 and StSP6A protein levels and tuber yield.


The nature of the phloem-mobile tuber-inducing signal(s) has been the subject of intense study. An important compo-nent in this signalling pathway was identified as the mRNA encoded by the Solanum tuberosum (St) BEL5 gene, a BEL1-like transcription factor (Banerjee et al., 2006; Hannapel, 2010). Binding of StBEL5 to the target genes in the stolon is facilitated through its interaction with its Knox partner, StPOTH1, and recent studies have provided evidence that both StBEL5 and StPOTH1 transcripts are able to move long distance through the phloem (Banerjee et al., 2006; Mahajan et al., 2012) (Fig. 4B). Interestingly, although transcription of StBEL5 occurs in the phloem cells of mature source leaves under long-day photoperiodic conditions, mRNA entry into the long-distance sieve tube system is facilitated by a short-day photoperiod (Banerjee et al., 2006). In addition, both 5′ and 3′ untranslated regions of this mRNA are essential for both entry into the sieve tube system and targeting to stolon tips (Banerjee et al., 2009).

Trafficking of the StBEL5 transcripts through the CC–SE PD would be mediated by a non-cell-autonomous RNA-binding protein (Xoconostle-Cázares et al., 1999; Ham et al., 2009; Li et al., 2011) that likely binds to sequence elements within these untranslated regions. Hence, photoperiodic con-trol over long-distance transport of StBEL5 transcripts may be achieved through short-day control over synthesis of the requisite RNA-binding protein. Alternatively, as transgenic potato plants overexpressing StBEL5 under a leaf-specific promoter produce tubers under long-day conditions (Banerjee et al., 2006), it may be that the RNA-binding protein is consti-tutively expressed, but under long-day conditions, the level of StBEL5 mRNA in CCs of normal potato plants is not suffi-cient to form a stable phloem-mobile RNA–protein complex. In summary, experiments with a range of transgenic potato plant lines clearly established a direct relationship between phloem delivery of StBEL5 mRNA, its level of accumulation in stolons, and tuber yield (Hannapel, 2010) (Fig. 4B and D).

Additional phloem-mobile signalling agents can also con-tribute to tuber induction (Jackson, 1999). These include a paralogue of FLOWERING LOCUS T (FT; Rodriguez-Falcon et al., 2006; Abelenda et al., 2011; Navarro et al., 2011) and possibly miR172 (Martin et al., 2009). Irrefutable proof for the ability of FT to serve as a phloem-mobile tuber-inducing signal was obtained from studies in which Hd3a, the FT orthologue in rice, was expressed in transgenic potato driven by a vascular-specific promoter (Navarro et al., 2011). Here, the wild-type control potato line only developed tubers under short-day conditions; however, the Hd3a:GFP trans-genic lines formed tubers when grown under long-day photo-periodic conditions. Furthermore, Hd3a:GFP scions grafted onto wild-type stocks also resulted in tuber formation, when plants were grown under long-day conditions. Finally, fluo-rescence associated with Hd3a-GFP was detected in the sto-lons of these heterografted plants.

Tissue expression analysis of potato FT orthologues revealed that, for plants transferred to tuber-inducing short-day conditions, StSP6A transcript levels were first elevated in leaves and subsequently in stolons. Confirmation that StSP6A is the potato paralogue of FT was obtained by studies in

which overexpression of this gene (StSP6Aox) caused tuberi-zation in plants grown under noninductive long-day condi-tions (Navarro et al., 2011). The time-dependent appearance of StSP6A transcripts in stolons, following transition to a short-day photoperiod, was not due to transcript delivery from the source leaves. Rather, through an elegant series of experiments using grafting between wild-type, StSP6Aox, and StCOox plants, results were obtained consistent with phloem delivery of StSP6A into the stolon to cause the acti-vation of an autoregulatory loop that upregulates StSP6A expression in the stolons to drive tuber induction (Fig. 4C and D).

The control of tuberization by multiple phloem-mobile inputs likely underscores the acquisition of tubers as an important avenue for vegetative reproduction. The operation of these parallel signalling systems also provides an excellent example of the critical role played by the phloem in mediat-ing between perception in the source leaves of an environ-mental input (a change in day length for tuber induction) and activation of an agriculturally important developmental programme in a distantly located plant organ (the under-ground stolon). Furthermore, this knowledge opens the door to an enhancement in production (yield potential) for potato plants being grown under a range of environmental condi-tions (Kloosterman et al., 2013).

Phloem and the vegetative-to-floral transition

The switch from the vegetative to reproductive state in plants is of paramount importance to agriculture, as seeds repre-sent one of the major sources of food for both animals and humans. A central role for the phloem in exerting control over this developmental transition has long been known, largely due to pioneering grafting studies which revealed the pres-ence of an unknown photoperiod-controlled signal, termed florigen, whose delivery to the apex caused the onset of floral induction (Zeevaart, 1976; Lang et al., 1977).

FLOWERING LOCUS T identified as a florigenic signal

Progress in the quest to identify the molecular nature of the phloem-mobile florigenic signal was made possible by molecular genetic studies performed with Arabidopsis. In this plant, mutants in either CO or FT do not flower or are greatly delayed in flowering time. Interestingly, expression of both genes was detected in the phloem of leaves rather than within the plant apex (Takada and Goto, 2003; An et al., 2004). Furthermore, expression of a CO:GFP construct, driven by a CC-specific promoter active in source leaves of co mutant plants, restored flowering, but fluorescence associated with the CO-GFP produced in source CCs was confined to these phloem cells (An et al., 2004). A similar result was obtained when CO production was restricted to the small minor veins in source leaves (Ayre and Turgeon, 2004). In contrast, expression of FT under either a CC-specific source leaf pro-moter or a shoot apical meristem-specific promoter did result in floral induction. As CO controls FT expression under


long-day conditions (Samach et al., 2000) these findings pro-vided strong support for the hypothesis that, in Arabidopsis, FT acts as a component of the phloem-mediated florigenic signalling system.

Florigen was deemed to be a universal signal capable of promoting flowering in all plants (Zeevaart, 1976). The role of FT as this universal, graft-transmissible signal was tested by Lifschitz et al. (2006). In tomato, a day-neutral plant, the orthologue of FT is SFT (SINGLE-FLOWER TRUSS) and the sft mutant exhibits a late-flowering phenotype. Grafting P35S:SFT scions onto sft tomato stocks restored flowering and later removal of the scion returned the stock to the sft phenotype. Importantly, grafting tomato P35S:SFT scions onto Maryland Mammoth tobacco stocks grown under long-day conditions similarly resulted in flowering in this short-day plant. Finally, transgenic Arabidopsis plants expressing SFT under a leaf-specific promoter exhibited a very early flowering phenotype. Importantly, reverse-transcription PCR analysis failed to detect the presence of SFT transcripts in mRNA samples extracted from P35S:SFT graft-reverted receptor sft shoots. Collectively, these findings provided strong support for the notion that FT protein, or an SFT-derived mobile sig-nal, serves as the universal florigenic signal.

To test whether FT is the actual long-distance signal, experiments were conducted on both Arabidopsis and rice and involved use of a number of different promoters to drive expression of FT, or tagged FT-GFP, and Hd3a, or tagged Hd3a-GFP. Use of the CC-specific SUC2 promoter, which is active not only in source leaves but along the length of the phloem pathway (Martens et al., 2006), meant that direct analysis of Arabidopsis ft-7 transgenic PSUC2:FT:GFP plants only provided information regarding the ability of the FT-GFP to move beyond the terminal phloem in the plant apex (Corbesier et al., 2007). However, heterograft-ing studies between ft-7 scions and PSUC2:FT:GFP stocks indicated the presence of FT-GFP in phloem tissues located beneath the scion apex. As might have been expected by the absence of detectable FT-GFP signal in the apex proper, the capacity of this graft-transmitted signal in floral induction was weak.

Parallel studies conducted on transgenic rice plants expressing PHd3a:Hd3a:GFP established an early heading (flowering) phenotype and, importantly, Hd3a-GFP signal could be detected in vascular tissue located beneath the mer-istem as well as within the lower regions of the apex/shoot apical meristem (Tamaki et al., 2007). Direct analysis of FT within the phloem sap was carried out on a cucurbit species, Cucurbita moschata (squash) that flowers only under short-day conditions (Lin et al., 2007). Ectopic expression of FT or CmoFT, in mature source leaves was highly effective at floral induction of long-day-grown plants. In addition, het-erografting studies demonstrated efficient transmission of the florigenic signal from day-neutral flowering C. maxima (pumpkin) stocks to C. moschata scions being kept under long-day conditions. Application of quantitative mass spec-troscopy methods to phloem sap collected from these hetero-grafted floral-induced C. moschata scions identified a clear signal associated with CmFT.

Considering that the phloem has been shown to mediate the long-distance trafficking of certain mRNA species (Ruiz-Medrano et al., 1999; Kim et al., 2001; Haywood et al., 2005; Banerjee et al., 2006; Mahajan et al., 2012; Notaguchi et al., 2012), there is no a priori reason why FT transcripts might not also move to the plant apex. However, most studies have failed to detect evidence for movement of FT mRNA through the phloem (Lifschitz et al., 2006; Lin et al., 2007; Tamaki et al., 2007; Notaguchi et al., 2008). Consistent with these findings, transgenic plants in which artificial miRNAs that target FT mRNA are expressed in CCs have been shown to cause late flowering, but not when they were expressed in the meristem (Mathieu et al., 2007; Tamaki et al., 2007).

Experiments based on viral vectors suggested that the first 100 nucleotides of the FT RNA may act as a cis element to mediate its long-distance movement, as well as that of viral RNAs, in Nicotiana benthamiana (Li et al., 2009). These observations were further investigated through the genera-tion of a wide range of transgenic Arabidopsis plants carry-ing various FT permutations being driven either by the 35S or the SUC2 promoter (Lu et al., 2012). These transgenic lines were used as stocks onto which were grafted either WT or ft mutant recipient scions. In heterografting studies employ-ing P35S:RFP:FT (or PSUC2:RFP:FT) stocks and ft-10 scions, flowering time was shortened relative to the ft-10:ft-10 homo-grafts and RFP-FT transcripts could be amplified from apical tissues collected from these florally induced scions.

As the RFP-FT protein does not move cell to cell, these results are consistent with the hypothesis that the RFP-FT transcripts detected in the scion were responsible for the observed formation of flowers on the scions. Unfortunately, this study failed to provide direct evidence for the presence of RFP-FT protein in the apex/ shoot apical meristem (Lu et al., 2012). In addition, it is unfortunate that more experi-ments were not carried out using the FT promoter, as concern can always be raised with results obtained employing the 35S promoter or the SUC2 promoter, due to their rather strong ubiquitous activity along the length of the phloem.

Cell-to-cell trafficking of FT

The FT protein has a molecular mass of 20 kDa and, thus, as the CC–SE PD size exclusion limit is on the order of 60 kDa, potentially FT could enter the sieve tube system by diffusion through the PD microchannels. To address this mode of FT movement, Yoo et al. (2013) tested the ability of a wide range of c-Myc-tagged FT mutants to enter and exit the phloem translocation stream. Western blot analysis, performed on phloem sap collected from just beneath the vegetative apex of long-day-grown C. moschata plants, established that all FT mutant proteins tested had retained the capacity to enter the sieve tube system. These findings are consistent with the hypothesis that FT moves through the CC–SE PD by diffu-sion. However, a recent study reported identification of an FT-interacting protein, FTIP1 that was suggested to func-tion, at the level of the CC–SE PD, to regulate FT entry into the phloem (Liu et al., 2012). It is possible that FT entry into the phloem can occur through CC–SE PD both by the


selective and diffusion pathways. Expressing high levels of FT in CCs, as was the case in the Yoo et al. (2013) movement assay system, may simply have overcome the FTIP1 regula-tory system (Fig. 5A).

Parallel floral induction and immunolocalization stud-ies performed on these c-Myc-tagged FT mutants identified a number of amino acid residues that are essential for both cell-to-cell movement through the post-phloem zone and flo-ral induction. A combination of microinjection and trichome rescue assays established that wild-type FT can interact with PD to mediate its trafficking cell to cell. Furthermore, vari-ous FT mutants that were dysfunctional in moving out from the phloem into the apex were also unable to rescue trichome

formation. Collectively, these results indicate that FT move-ment from the terminal phloem into the apex/shoot api-cal meristem occurs on the selective PD pathway (Fig. 5A). Further evidence in support of this mode of trafficking by FT was provided by coimmunoprecipitation studies using c-Myc-FT and a C. moschata PD-enriched cell-wall prepara-tion; three FT-interacting proteins were identified (Yoo et al., 2013). It will be interesting to perform mutant analysis of these putative FT-interacting proteins to test their role in FT delivery into the shoot apical meristem.

At first sight, a nonselective mode for FT entry into the sieve tube system seems inconsistent with the central role played by FT as an environmental agent that signals between

Fig. 5. Phloem-mediated control over flowering. (A) Florigenic FLOWERING LOCUS T (FT) signalling. Photoperiodic control over FT expression in companion cells (CCs) located in the lamina, petiole, and stem (white dashed circles) results in FT entry into the phloem either by diffusion through the CC–SE plasmodesmata, or by FT INTERACTING PROTEIN 1 (FTIP1)-mediated delivery to and trafficking through the CC–SE plasmodesmata (left image). FT moves from the terminal phloem to the meristem, through the selective plasmodesmata trafficking pathway, where it forms a florigen activation complex with FLOWERING LOCUS D (FD) and a 14-3-3 protein (Taoka et al., 2011) that then binds to the APETALA1 (AP1) promoter to initiate the vegetative meristem (VM) to inflorescence meristem (IM) transition; FB, floral bud; SE, sieve element. (B) Antiflorigenic ATC signalling. Expression of Arabidopsis thaliana CENTRORADIALIS (ATC), an FT homologue, in CCs of SD-grown plants results in phloem long-distance delivery of both ATC transcripts and ATC protein. In the apex, ATC interacts with FD to repress AP1 expression, thereby acting as an antiflorigenic signalling agent to preventing flowering. (C) FT-independent trehalose-6-phosphate (T6P) signalling pathway. Expression of TREHALOSE-6-PHOSPHATE SYNTHASE 1 (TPS1) in the vegetative apex allows for the synthesis of T6P from sucrose delivered by the phloem. High levels of T6P serve to activate the age-dependent flowering pathway (ADFP). The linear relationship between sucrose and T6P concentrations in the apex (right graft) serves as an integrator between the capacity of the plant to produce photosynthate and flowering time.


mature leaves and the vegetative apex. However, along the phloem long-distance pathway the CC–SE complexes func-tions as a symplasmic domain in which molecular exchange with the surrounding cells is highly regulated (Knoblauch and van Bel, 1998; Itaya et al., 2002; Ding et al., 2003) (Fig. 3A). Thus, in this region of the phloem, FT would be confined to the CC–SE complexes. It is also important to note that here also the SE conduit volume is much larger than that of the CCs. Hence, FT sequestered in CCs should have a minimal effect on the efficacy of the florigenic signalling process.

Phloem movement of an antiflorigenic signal

A recent study revealed that expression of Arabidopsis ATC, a member of the TFL-clade of FT homologues, also occurs in the phloem but not in the apex (Huang et al., 2012). Growth under long-day conditions of heterografted atc-2 knock-out mutant scions and 35S:ATC transgenic stock plants resulted in a significant late-flowering phenotype in the sci-ons. Western analysis of protein samples extracted from atc-2 scions grafted onto either act-2 or wild-type stocks failed to detect ATC in the atc-2:atc-2 homografts, but ATC was routinely detected in the atc-2 scions from WT:atc-2 hetero-grafts. Thus, as with TFL1 (Conti and Bradley, 2007), ATC appears to function as a non-cell-autonomous protein, but unlike TFL1, this ATC appears to have acquired the ability to enter and move through the phloem. Of significant inter-est, parallel RNA analyses failed to detect ATC transcripts in the atc-2:atc-2 homografts, but transcripts were detected in WT:atc-2 heterografts.

Expression of ATC is strongly upregulated under short-day growth conditions, where flowering occurs only after a prolonged growth period. Hence, these very interesting find-ings suggest that both ATC protein and ATC transcripts may well serve as a long-distance antiflorigen signal through a possible interaction with FLOWERING LOCUS D (FD) in the shoot apical meristem (Fig. 5B). Evidence for translation of phloem delivered ATC transcripts, within the receiving shoot apical meristem, would provide further support for this model. Finally, as FT, TFL, and ATC are highly conserved, sequence swapping studies should provide insight into the cis-acting motif(s) in the ATC transcript that is presumably responsible for its observed phloem mobility.

Trehalose-6-phosphate, sucrose sensing, and floral induction

As the phloem carries sugars to the vegetative apex, a role for sugar signalling in floral induction has long been in contention (Lejeune et al., 1993). Studies on Arabidopsis grown under noninductive short-day conditions revealed the presence of an FT-independent pathway, as both ft-1 and wild-type plants flowered earlier when they were grown under a combination of short-days and high light intensity (King et al., 2008). Insights into the molecular nature of this FT-independent floral induction pathway was recently provided by studies in which an artificial miRNA (amiR) approach was employed to knockdown TREHALOSE-6-PHOSPHATE SYNTHASE

1 (TPS1) transcript levels (Wahl et al., 2013). These trans-genic plants had a significant reduction in the product of this enzyme, trehalose-6-phosphate (T6P), and a delay in flower-ing. As sucrose levels increased in these silenced plants, this implicated a role for T6P in sugar sensing and regulation of flowering time.

Flowering time was only slightly delayed in ft-10 mutant plants transformed with the 35S:amiR-TPS1 construct, con-sistent with the finding that FT expression was down-regulated in mature leaves of 35S:amiR-TPS1 plants. Furthermore, as transgenic plants carrying both the 35S:amiR-TPS1 and 35S:FT constructs exhibited an almost normal flower-ing time phenotype, this indicated that in source leaves T6P acts upstream of the FT signalling pathway. A second site of action for T6P was identified within the plant apex where T6P was found to function independently from FT (Wahl et al., 2013).

In 12-day-old Arabidopsis seedlings, TPS1 expression was detected in the flanks of the meristem in a pattern overlay-ing that of vascular development and extending down into the protophloem. Measurements of T6P and sucrose levels in meristems dissected from 30-day-old short-day-grown plants which had subsequently been subjected to long-day inductive conditions revealed a linear relationship between the phloem-delivered sucrose and T6P, presumably synthe-sized in the apex. A direct test for the role of TPS1 and T6P in floral induction was carried out by using the shoot apical meristem-specific CLV3 promoter to express either a bacte-rial T6P-catabolizing enzyme or TPS1. Here, a reduction in T6P was highly correlated with a significant increase in flow-ering time, whereas in contrast, plants expressing TPS1 in their meristems exhibited a very early flowering phenotype. Taken together, these findings establish that, in Arabidopsis, T6P can serve to integrate the physiological input of sucrose availability to developmental programming within the meris-tem (Fig. 5C).

Phloem-mediated systemic epigenetic regulation of development

The cellular pathways involved in gene silencing (RNA inter-ference, RNAi) have largely been elucidated (Baulcombe, 2004; Brodersen and Voinnet, 2006). Numerous studies also describe the initiation and subsequent local and long-distance movement of sequence-specific silencing signals through PD and the phloem, respectively (Palauqui et al., 1997; Voinnet et al., 1998; Brosnan et al., 2007; Dunoyer et al., 2010a). Much of the early work in this area was focused on the pro-cesses involved in virus infection and the defence mechanisms employed by the host plant to interdict the spread of these invading pathogens (Dougherty et al., 1994; Waterhouse et al., 1998). Subsequent work established that plant develop-mental and physiological processes are also epigenetically reg-ulated through various RNAi pathways (Alvarez et al., 2006; Marín-González and Suárez-López, 2012). Collectively, these studies established the involvement of 21–24-nt small inter-fering RNA (siRNA), trans-acting siRNA, and miRNA as


the agents that target homologous sequences in endogenous transcripts, or viral RNA, for degradation (Melnyk et al., 2011a; Molnar et al., 2011).

The phloem as a systemic pathway for interorgan deliv-ery of silencing signals was elegantly established by graft-ing studies involving transgenic tobacco lines overexpressing genes for nitrate or nitrite reductase (Palauqui et al., 1997). Experiments performed with cucurbits, in which phloem exudate can be readily collected, identified a population of siRNA that fell into two size classes, namely 21- and 24-nt in length (Yoo et al., 2004). In healthy control plants, the predominant size class was 24 nt, whereas in virus-infected plants the 21-nt class became the major small RNA spe-cies. Analysis of these siRNAs indicated that the 21-nt class, present in phloem collected from virus-infected plants, con-tained both sense and antisense sequences homologous to the genome of the infecting virus.

Phloem exudates from control plants contained both siRNA and miRNA species (Yoo et al., 2004), consistent with the involvement of the phloem in mediating silenc-ing of endogenous genes in distantly located tissues and organs. Analysis of phloem exudates from other plant spe-cies confirmed the presence of siRNA and miRNA popula-tions (Buhtz et al., 2008; Pant et al., 2009; Buhtz et al., 2010; Varkonyi-Gasic et al., 2010; Rodriguez-Medina et al., 2011). Collectively, these studies provide support for the hypothesis that phloem-mobile siRNAs, either as single-stranded mol-ecules (Yoo et al., 2004) or duplexes (Dunoyer et al., 2010a), function as the long-distance silencing agents. However, one cannot exclude the possibility that either long RNAs or dou-ble-stranded RNAs also contribute to systemic silencing (Yoo et al., 2004; Brosnan et al., 2007).

Currently little is known concerning the molecular basis for either cell-to-cell or long-distance transport of the mobile silencing signals. A screen carried out on fractionated pump-kin phloem exudates probed with various forms of siRNA identified a small RNA-binding protein, PSRP1, that dis-played the ability to bind specifically to siRNA (Yoo et al., 2004). Microinjection studies indicated that PSRP1 can increase PD size exclusion limit and mediate its own cell-to-cell transport. This protein was also shown to preferen-tially bind to and traffic single-stranded siRNA. Functional homologues in other plant species for the pumpkin PSRP1 have yet to be identified. Advances in the understanding of long-distance delivery of small RNA silencing agents must therefore await identification of the proteins that form stable si/miRNA complexes that travel in the phloem translocation stream.

Role of phloem-mobile small RNA signals in systemic stress responses

Nutrient stress signalling to establish homeostasis in plants involves the integration of input signals from the root sys-tem, delivered to the shoot through the xylem, with shoot-derived response signals, translocated to the roots through the phloem. Recent studies have begun to unravel the nature of these vascular-transmitted signalling agents, and a role

for small RNAs is emerging. The best characterized phloem-mobile small RNA is currently miR399 that is involved in Pi stress signalling (Chiou and Lin, 2011). Here, a root-derived signal induces the upregulation of MIR399 expression in the leaves, followed by loading of miR399 into the phloem for delivery to the root system of the Pi-stressed plants (Lin et al., 2008; Pant et al., 2008). Within the root, miR399 directs the cleavage of PHO2 transcripts (Chiou et al., 2006), thereby reducing the level of PHO2, an ubiquitin-conjugating E2 enzyme. This allows for an increase in Pi uptake into and across the root system for delivery into the xylem transpira-tion stream (Chiou and Lin, 2011).

Additional nutrient-stress-induced miRNAs have been reported, including those upregulated by both macronutri-ents such as sulphur and micronutrients such as copper and iron (Jones-Rhoades and Bartel, 2004; Kawashima et al., 2009; Buhtz et al., 2010). Interestingly, MIR395 is expressed in CCs within source leaves (Kawashina et al., 2009) and analysis of phloem exudates collected from sulphur-stressed Arabidopsis plants revealed that miR395 was present in the phloem and could cross a graft union (Buhtz et al., 2010). However, MIR395 is also expressed in CCs located in the root phloem, which raises the question as to the role of the phloem-borne miR395 in sulphur homeostasis. An imposed copper stress causes an increase in the level of miR398 within shoot tissues and it has also been detected in phloem exudates (Buhtz et al., 2008, 2010). However, the role of miR398 in the phloem in terms of its targeting of copper/zinc superoxide dismutase transcripts (Yamasaki et al., 2007) requires further study.

Phloem-mobile sRNAs direct transcriptional gene silencing in target tissues

As mentioned above, the pumpkin phloem sap siRNA pop-ulation collected from uninfected plants was distributed around the 24-nt size class which is known to be involved in the transcriptional gene silencing pathways (Chen, 2009; He et al., 2011). Sequence analyses revealed the presence of iden-tified 24-nt sRNAs derived from transposable elements and it was suggested that these phloem-mobile agents would likely be involved in transcriptional gene silencing within target tis-sues in sink organs (Yoo et al., 2004).

Experimental support for this hypothesis was provided by studies in which Arabidopsis (C24 genotype background) scions, expressing both a GFP inverted repeat (GF-IR) con-struct and a GFP (G) construct (GxGF-IR), were grafted onto Columbia (Col)-dcl2,3,4 rootstocks that were defective in the production of 22-, 23-, and 24-nt sRNA from double-stranded RNA GFP (Molnar et al., 2010). Analysis of high-through-put sequencing data obtained from these grafted roots indi-cated that the 23- and 24-nt sRNAs were the main size class and these graft-transmissible sRNAs were found to be associ-ated with chromatin silencing, with the majority being associ-ated with transposons. DNA methylation analysis of chosen genomic loci revealed that these sites were hypermethylated in the Col-dcl2,3,4 rootstocks grafted onto C24 scions, compared to homografted Col-dcl2,3,4 (scion):Col-dcl2,3,4 (rootstock)


plants. These findings are consistent with the hypothesis that phloem-delivered endogenous 24-nt sRNA can enter the root system and guide RNA-directed DNA methylation (Molnar et al., 2010; see also Melnyk et al., 2011b) (Fig. 6).

In-depth analysis of two endogenous inverted repeats in the Arabidopsis genome, IR71 and IR2039, also indicated that the siRNAs generated from these loci can move across a graft union and that 24-nt siRNA mediated in the RNA-directed sequence-specific DNA methylation in recipient root cells (Dunoyer et al., 2010b). Of equal importance, these studies revealed that such IR loci are dynamic in nature, with sRNA from the IR2039 locus being detected in only Col of the 18 Arabidopsis accession studies. Small RNAs from the IR71 locus were detected in 16 accessions. Thus, IR2039 most likely represents a very recent event in the Col genome. These find-ings support a model in which the formation of endogenous IR loci, with cell-type-specific expression patterns, can serve as a mechanism to sense or respond to local environmental inputs that reflect discrete ecological niches (Dunoyer et al., 2010b).

Parasitic plants as vulnerable ‘scions’

Plants within some families have developed specialized mech-anisms to become parasitic on other plants; they represent a ‘natural’ scion, as these plants develop phloem and xylem connections to the host vascular system to obtain water and nutrients. As such, parasitic plants can cause major losses to certain crops worldwide. Recent studies have shown that, as

with heterografted plants, by establishing a phloem connec-tion with their host, these parasitic plants receive not only nutrients but also a range of information molecules, including various forms of RNA (Roney et al., 2007; David-Schwartz et al., 2008). This finding may well allow for the development of a novel strategy to control such invasive plants through targeted delivery through the phloem of specially designed sRNAs that could target genes in the parasite that would disrupt important physiological and/or developmental pro-grammes (Yoder et al., 2009).

Support for this control strategy was recently provided by studies on transgenic tomatoes engineered to produce siRNA designed to silence the gene for mannose 6-phosphate reductase in parasitic broomrape (Orobanche aegyptiaca) (Aly et al., 2009). As broomrape uses this enzyme to produce physiologically important levels of mannitol, their growth on these transgenic tomatoes was reduced compared to the con-trols carried out with wild-type tomato lines. Control strate-gies based on expression of siRNAs designed to target genes involved in critical developmental programmes have also been conducted. Here, transgenic tobacco plants express-ing siRNAs against dodder (Cuscuta pentagona) KN1-Like genes involved in haustoria development compromised the ability of this parasitic plant to form vascular connections to its tobacco host (Alakonya et al., 2012). These siRNAs were carefully designed to target sequences specific to the dodder KN1-Like genes, and thus, only reduced the growth of dodder plants growing on the transgenic tobacco host plants. Collectively, these studies indicate that control over plant parasitism may well be achieved by silencing a pyramid of parasite genes involved in various aspects of physiology, growth, and development.

Conclusions

In the past decade, considerable progress has been made in terms of advancing the understanding of the evolutionary events and developmental programmes involved in form-ing the plant vascular system (Lucas et al., 2013). Both the xylem and phloem play pivotal roles in delivering water, essential nutrients, and signalling molecules to the various plant organs. The concept of integrating both physiological and developmental programming, at the whole-plant level, through long-distance signalling involving root-to-shoot (xylem) and shoot-to-root (phloem) pathways is now well established. Although there is a growing body of evidence that these xylem- and phloem-borne signals mediate in activation or modulation of local genetic programmes, the identities of only a few of these agents and their downstream targets have been identified. The emergence of genomics databases for an ever-increasing number of plant species, along with the avail-ability of powerful omics tools, should accelerate the elucida-tion of these vascular-based signalling programmes and their target gene regulatory networks.

The global agricultural community is presently faced with meeting the challenge of increasing the food supply to feed a growing world population. Meeting this challenge will require that plant biologists advance their understanding of

Fig. 6. Long-distance control over gene expression is mediated by phloem delivery of small RNAs. Small RNAs (21–24 nt) generated in the companion cells (CCs) of source leaves enter the sieve tube system (STS) for delivery to distantly located sink organs, such as the root. Following unloading from the phloem, the 21-nt small RNAs and miRNA target transcripts within the recipient cells for degradation by the RNA interference pathway. The 24-nt small RNAs enter the nucleus where they activate transcriptional gene silencing (TGS) of target genes by either RNA-directed DNA methylation (Rd-DM) or chromatin remodelling.


the hierarchical control systems employed by plants to allo-cate carbon and nitrogen resources to competing sink organs. Knowledge of this nature would facilitate the engineering of various crop plants, in terms of increased yield potential and, of equal importance, sustainable yields under nonoptimal plant growth conditions.

It is now clear that the angiosperm phloem system has the machinery to utilize the enucleate sieve tube system as a con-duit for the transport of RNA, including mRNA, miRNA, 21- and 24-nt siRNA, and various other forms of noncod-ing RNA to sink tissues. The diversity of this phloem RNA population is quite remarkable; however, the roles of only a very small number of these long-distance signalling agents have been studied. Importantly, current studies have revealed that such phloem-borne RNA species can participate in sink organs to adjust developmental events, alter physiological programmes and mediate in transcriptional gene silencing, either through DNA methylation or chromatin remodel-ling. The current knowledge, although in its infancy, pro-vides a clear vision as to the importance of these signalling agents as integrators of whole plant physiology, growth, and development.

These RNA species are likely to move throughout the phloem translocation stream in the form of RNP complexes. Important questions remain as to their protein composition as well as how and where such RNP complexes are assem-bled. Furthermore, nothing is currently known regarding the molecular mechanism(s) by which a particular RNP complex could be targeted to specific cells located in a sink organ. This process would require recognition at the level of the SE–CC PD in the specific sink organ, followed by targeted cell-to-cell trafficking from this exit site to the recipient cells. Gaining an understanding of these processes would clearly pave the way for developing systems capable of delivering engineered (novel) transcripts or RNAi/transcriptional gene silencing agents with the aim of controlling specific developmental and/or physiological programmes in certain target tissues/organs.

Knowledge of the developmental programmes and physio-logical functions of the phloem is vital to understanding plant form and function, as well as for achieving a stable global food supply. Contributions from individual scientists will be important; however, to attain these critical goals in a timely manner, the scientific community might be well served by coordinating research programmes through the development of international working groups. Support for such activities could be provided by a cooperative between national and international funding agencies. This approach could utilize a set of model plants as the platform for a highly integrated and coordinated programme on plant vascular biology.

Acknowledgements

Work in the authors’ laboratory on phloem biology is sup-ported by National Science Foundation (grant number IOS-0752997) and the USDA National Institute of Food and Agriculture (grant number NIFA 201015479).

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