Journal of Experimental Botany, Vol. 65, No. 7, pp. 1799–1816, 2014
doi:10.1093/jxb/ert417 Advance Access publication 24 December, 2013
Review paper
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 sugars and amino acids, by the phloem, to specific sink tissues is of importance to crop yield and global food security. 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 neighbouring 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 delivery 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-hydraulicresistance 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 conducting 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 maturity, 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.
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1800 | Ham and Lucas
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.
continuity between neighbouring SEs is established by structurally modifying plasmodesmata (PD) to form sieve plate
pores (Fig. 1B). Such files of enucleate cells form an individual 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 movement of macromolecules through the PD that connect CCs
to their neighbouring SEs (Fig. 1C). Although it is generally considered that mature SEs lack the capacity for protein 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
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 patterning, 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 important 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 photosynthetic capacity and, thus, overall higher plant productivity 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 sinkstrength model for resource allocation needs to be considered in the context of a global control system that mediates
between abiotic and biotic inputs and endogenous developmental 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 generally 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 environments 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; FrancoZorrilla et al., 2005; Shane and Lambers, 2006; Shane et al.,
2008; Thibaud et al., 2010). Similar studies involving localized 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 reallocation 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 network, involving both the xylem and the phloem that can exert
long-distance control over developmental and physiological 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 populations 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 demonstrated (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
1802 | Ham and Lucas
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).
candidates in this hierarchical global resource allocation
control system. In this regard, it is noteworthy that grafting 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 system where they influence root architecture by negatively regulating the development of lateral roots (Notaguchi et al.,
2012). It will be interesting to determine whether environmental 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 concentration 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, functional genomics studies are providing insights into the gene
regulatory networks that are activated in shoot and root tissues 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.
Phloem transport and agricultural traits | 1803
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 parenchyma 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 microchannels 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 symplasmic 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 information macromolecules, as described above, and as valves
having the capacity to control local resource release (allocation) (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, permeability, 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 movement 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 regulate 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 proteins, 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 interesting 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 carbohydrates 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 cellular 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 support 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 metabolism 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
1804 | Ham and Lucas
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.
Phloem transport and agricultural traits | 1805
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.
1806 | Ham and Lucas
The nature of the phloem-mobile tuber-inducing signal(s)
has been the subject of intense study. An important component in this signalling pathway was identified as the mRNA
encoded by the Solanum tuberosum (St) BEL5 gene, a BEL1like 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 shortday 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 RNAbinding 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 control 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 constitutively expressed, but under long-day conditions, the level of
StBEL5 mRNA in CCs of normal potato plants is not sufficient 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 contribute to tuber induction (Jackson, 1999). These include a
paralogue of FLOWERING LOCUS T (FT; RodriguezFalcon 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 tuberinducing 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 transgenic lines formed tubers when grown under long-day photoperiodic conditions. Furthermore, Hd3a:GFP scions grafted
onto wild-type stocks also resulted in tuber formation, when
plants were grown under long-day conditions. Finally, fluorescence associated with Hd3a-GFP was detected in the stolons of these heterografted plants.
Tissue expression analysis of potato FT orthologues
revealed that, for plants transferred to tuber-inducing shortday 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 tuberization in plants grown under noninductive long-day conditions (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 activation 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 mediating between perception in the source leaves of an environmental input (a change in day length for tuber induction)
and activation of an agriculturally important developmental
programme in a distantly located plant organ (the underground stolon). Furthermore, this knowledge opens the door
to an enhancement in production (yield potential) for potato
plants being grown under a range of environmental conditions (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 represent 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 presence 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 promoter or a shoot apical meristem-specific promoter did result
in floral induction. As CO controls FT expression under
Phloem transport and agricultural traits | 1807
long-day conditions (Samach et al., 2000) these findings provided 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 longday 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 signal, 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, heterografting 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 meristem 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 shortday 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, heterografting 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 spectroscopy methods to phloem sap collected from these heterografted 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 (RuizMedrano 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 generation of a wide range of transgenic Arabidopsis plants carrying 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 employing P35S:RFP:FT (or PSUC2:RFP:FT) stocks and ft-10 scions,
flowering time was shortened relative to the ft-10:ft-10 homografts 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 experiments 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 diffusion. However, a recent study reported identification of an
FT-interacting protein, FTIP1 that was suggested to function, 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
1808 | Ham and Lucas
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 regulatory system (Fig. 5A).
Parallel floral induction and immunolocalization studies 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 floral 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, various 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 movement from the terminal phloem into the apex/shoot apical 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 preparation; 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 agedependent 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.
Phloem transport and agricultural traits | 1809
mature leaves and the vegetative apex. However, along the
phloem long-distance pathway the CC–SE complexes functions 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 knockout mutant scions and 35S:ATC transgenic stock plants
resulted in a significant late-flowering phenotype in the scions. Western analysis of protein samples extracted from atc2 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 heterografts. 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 interest, 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 shortday growth conditions, where flowering occurs only after a
prolonged growth period. Hence, these very interesting findings 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 transgenic plants had a significant reduction in the product of this
enzyme, trehalose-6-phosphate (T6P), and a delay in flowering. 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, consistent 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 flowering 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 overlaying 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 synthesized 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 bacterial T6P-catabolizing enzyme or TPS1. Here, a reduction in
T6P was highly correlated with a significant increase in flowering 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 meristem (Fig. 5C).
Phloem-mediated systemic epigenetic
regulation of development
The cellular pathways involved in gene silencing (RNA interference, 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 processes 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 developmental and physiological processes are also epigenetically regulated 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 interfering RNA (siRNA), trans-acting siRNA, and miRNA as
1810 | Ham and Lucas
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 delivery of silencing signals was elegantly established by grafting 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 species. Analysis of these siRNAs indicated that the 21-nt class,
present in phloem collected from virus-infected plants, contained 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 silencing of endogenous genes in distantly located tissues and
organs. Analysis of phloem exudates from other plant species confirmed the presence of siRNA and miRNA populations (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 molecules (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 double-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 pumpkin phloem exudates probed with various forms of siRNA
identified a small RNA-binding protein, PSRP1, that displayed 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 cellto-cell transport. This protein was also shown to preferentially 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 system, delivered to the shoot through the xylem, with shootderived 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 phloemmobile 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 transpiration stream (Chiou and Lin, 2011).
Additional nutrient-stress-induced miRNAs have been
reported, including those upregulated by both macronutrients 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 population 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 identified 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 tissues 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) construct 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-throughput sequencing data obtained from these grafted roots indicated that the 23- and 24-nt sRNAs were the main size class
and these graft-transmissible sRNAs were found to be associated with chromatin silencing, with the majority being associated 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)
Phloem transport and agricultural traits | 1811
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 findings 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 mechanisms 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 connection 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 programmes (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 controls carried out with wild-type tomato lines. Control strategies based on expression of siRNAs designed to target genes
involved in critical developmental programmes have also
been conducted. Here, transgenic tobacco plants expressing 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
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 RNAdirected DNA methylation (Rd-DM) or chromatin remodelling.
In the past decade, considerable progress has been made in
terms of advancing the understanding of the evolutionary
events and developmental programmes involved in forming 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 availability of powerful omics tools, should accelerate the elucidation 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
1812 | Ham and Lucas
the hierarchical control systems employed by plants to allocate 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 conduit for the transport of RNA, including mRNA, miRNA,
21- and 24-nt siRNA, and various other forms of noncoding 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 remodelling. The current knowledge, although in its infancy, provides 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 assembled. 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 physiological 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 supported by National Science Foundation (grant number IOS0752997) and the USDA National Institute of Food and
Agriculture (grant number NIFA 201015479).
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