Journal of Experimental Botany, Vol. 65, No. 7, pp. 1737–1749, 2014
doi:10.1093/jxb/ert422 Advance Access publication 17 December, 2013
Review paper
Transcription factor-mediated cell-to-cell signalling in plants
Xiao Han, Dhinesh Kumar, Huan Chen, Shuwei Wu and Jae-Yean Kim*
Division of Applied Life Science (BK21plus/WCU Program), Graduate School of Gyeongsang National University, Plant Molecular Biology
& Biotechnology Research Center (PMBBRC), Jinju 660-701, Korea
* To whom correspondence should be addressed. E-mail: [email protected]
Received 20 August 2013; Revised 22 October 2013; Accepted 11 November 2013
Abstract
Plant cells utilize mobile transcription factors to transmit intercellular signals when they perceive environmental
stimuli or initiate developmental programmes. Studies on these novel cell-to-cell signals have accumulated multiple pieces of evidence showing that non-cell-autonomous transcription factors play pivotal roles in most processes
related to the formation and development of plant organs. Recent studies have explored the evolution of mobile transcription factors and proposed mechanisms for their trafficking through plasmodesmata, where a selective system
exists to facilitate this process. Mobile transcription factors contribute to the diversity of the intercellular signalling
network, which is also established by peptides, hormones, and RNAs. Crosstalk between mobile transcription factors
and other intercellular molecules leads to the development of complex biological signalling networks in plants. The
regulation of plasmodesmata appears to have been another major step in controlling the intercellular trafficking of
transcription factors based on studies of many plasmodesmal components. Furthermore, diverse omics approaches
are being successfully applied to explore a large number of candidate transcription factors as mobile signals in plants.
Here, we review these fascinating discoveries to integrate current knowledge of non-cell-autonomous transcription
factors.
Key words: Cell-to-cell communication, intercellular signalling, non-cell-autonomous proteins, plasmodesmata, transcription
factors.
Introduction
Land plants, which are sessile, have evolved developmental
plasticity to cope up with environmental challenges. Cellto-cell communication is one such regulatory event and
represents a crucial step in the development of a complex
signalling network at the multicellular level for establishing
the spatiotemporal regulation that is essential for development. To transmit biological information, cells utilize various types of mobile molecules as a signalling code, including
peptides, hormones, RNAs, and proteins. A large body of
evidence has suggested that non-cell-autonomous proteins
(NCAPs), especially non-cell-autonomous transcription factors (NCATFs), contribute to intercellular communication
by acting as mobile signals capable of carrying information in plant cells. Plant organs such as the shoot meristem,
flowers, and roots are established through cell fate determination processes under the control of NCAPs. This review
describes some of the recent key findings related to NCATFs
involved in plant development and attempts to integrate the
current understanding of the evolution of non-cell-autonomous pathways. In addition, several representative types of
crosstalk between NCAPs and other mobile molecules are
reviewed to answer the question of how NCAPs can facilitate cell-to-cell communication in complicated signalling
networks. This review also provides insights into how plasmodesmal symplasmic channels dexterously regulate NCAP
movements. Finally, this review describes the fascinating
exploitation of recent innovative omics approaches in the
field of NCAP movement.
© 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]
1738 | Han et al.
Mobile transcriptional regulators involved
in developmental plasticity
Diverse plant cell patterning and organ formation processes
are regulated by intercellular signalling to accomplish events
ranging from reproduction to the responses to various environmental stimuli. It is now widely accepted that many developmental processes require mobile transcriptional regulators
to transmit essential positional information in plants (Fig. 1).
Shoot development
The first endogenous NCAP to be discovered was the maize
homeodomain (HD) transcription factor (TF) KNOTTED1
(KN1), which is essential for maintenance of the shoot apical
meristem (SAM). KN1 is expressed in the L2 cells, and the
KN1 protein has also been detected in the outer domain of
expression, suggesting that this protein exhibits movement
within the meristem (Lucas et al., 1995). In addition, KN1 was
shown to move in a tissue-specific manner during Arabidopsis
shoot development. This cell-to-cell trafficking ability of KN1
was found to be required for mutant rescue when expressed
under layer-specific promoters (Kim et al., 2002). The
Arabidopsis KN1 orthologues SHOOTMERISTEMLESS
(STM) and KNAT1/BREVIPEDICELLUS (BP), and their
highly conserved trafficking domains, including the HD,
have been shown to confer a gain-of-trafficking function on
cell-autonomous proteins within the SAM, leaves, and stem
(Kim et al., 2003). Intercellular trafficking of KNAT1 from
the cortex to epidermal cells in Arabidopsis stems is also
essential for the determination of plant architecture and epidermal differentiation (Rim et al., 2009). Another essential
NCAP involved in the maintenance of the Arabidopsis SAM
is WUSCHEL (WUS) (Laux et al., 1996), a HD-containing
TF that triggers downstream signals in the L1 and L2 layers
following trafficking from the organizing centre of the SAM
(Yadav et al., 2011).
Flowering induction and floral organ development
Fig 1. Representative non-cell-autonomous proteins with
functions in plant development. Maintenance of the shoot
apical meristem requires STM and WUS. Flower development
is controlled by LFY, AG, AP3, SEP3, and PI. Leaf growth is
regulated by AN3. TTG, and CPC play important roles in trichome
pattern formation. Long-distance movement of FT and TSF
and local cell-to-cell movement of TFL1 in the meristem control
flowering. Intercellular trafficking of KNAT1 is essential for the
determination of plant architecture and epidermal differentiation.
TMO7 is required for MONOPTEROS-dependent embryonic root
initiation. In seedling roots, cell fate determination is regulated by
mobile TFs such as AHL4, SHR, and UBP1. Root hair cells are
determined through the cooperation of CPC, TRY, GL3, EGL3,
and ETC. Red arrow: phloem-based long-distance movement.
Various important aspects of flower development are
controlled by mobile transcriptional regulators. During
the process of transforming the SAM into an inflorescence meristem, the SAM transition is initiated by the
mobile signals FLOWERING LOCUS T (FT) (Corbesier
et al., 2007; Jaeger and Wigge, 2007) and TWIN SISTER
OF FT (TSF) (Yamaguchi et al., 2005), members of the
CETS (CENTRORADIALIS-TERMINAL FLOWER
1-SELFPRUNING) gene family in Arabidopsis, and orthologous proteins HEADING DATE 3A (HD3A) (Tamaki et al.,
2007), SINGLE FLOWER TRUSS (SFT), and FT-Like 2
(FTL2) (Lin et al., 2007; Yoo et al., 2013) in rice, tomato,
and squash, respectively. FT is loaded into the phloem
sieve tube for long-distance transport from the leaf blade
and cotyledons, unloaded at the proximal SAM unloading zone, and travels in a cell-to-cell manner to the SAM
(Corbesier et al., 2007; Jaeger and Wigge, 2007). Arabidopsis
TERMINAL FLOWER 1 (TFL1) and its tomato orthologue SELFPRUNING, which are also NCAPs, play a role
in the maintenance of the indeterminate meristem. Thus,
the ratio of FT to the antagonistic TFL1/SELFPRUNING
protein specifies the transition of the SAM into an inflorescence meristem (Lifschitz et al., 2006; Conti and Bradley,
2007; Shalit et al., 2009). With a mature inflorescence meristem, the TFL1 protein travels to the epidermal cell layer and
acts to inhibit the floral identity genes LEAFY (LFY) and
APETALA1 (AP1). Interestingly, LFY, which is expressed
in peripheral cells (anlagen) in the inflorescence and floral
meristems, is indirectly required for TFL1 protein movement, thus establishing a TFL1–LFY feedback loop. LFY
is also known as an NCAP; however, the biological significance of LFY movement is not clear because the patterns of
LFY RNA and LFY protein expression in wild-type plants
Intercellular signalling by transcription factors | 1739
are similar (Sessions et al., 2000). One possible explanation
for this intricate phenomenon is that the non-cell-autonomous action of the LFY protein functions as a safe-guard
mechanism to ensure complete conversion of a meristem
into a flower. In addition, MADS domain transcription
factors AGAMOUS (AG), AP3, PISTILLATA (PI), and
SEPALLATA3 (SEP3), which serve as fundamental regulators of floral organ identity, are able to travel from cell to
cell (Urbanus et al., 2010a), reinforcing the conclusion that
cell-to-cell communication mediated by mobile transcription
factors is an essential process for tissue patterning and organ
formation.
Organ size determination and epidermal patterning
Coordinated proliferation between clonally distinct cell layers
is essential for determining the size and shape of plant organs
and has been proposed to be regulated in a non-cell-autonomous manner during leaf development (Savaldi-Goldstein
and Chory, 2008; Bai et al., 2010; Kawade et al., 2010).
Recently, the transcriptional coactivator ANGUSTIFOLIA3
(AN3), which is responsible for typical compensation for a
decrease in cell proliferation via enhanced post-mitotic cell
expansion in leaf primordia, was shown to travel from leaf
mesophyll cells to epidermal cells in Arabidopsis. An interference with AN3 movement resulted in defective proliferation
in leaf epidermal cells and abnormal leaf sizes and shapes,
as shown in an an3 mutant, indicating that the intercellular
movement of AN3 from subepidermal cells to epidermal cells
is an essential process for AN3 function (Kawade et al., 2010;
Kawade et al., 2013).
The most recent model for epidermal trichome and hair cell
patterning in Arabidopsis relies on a bidirectional intercellular
feedback circuit in which mobile TFs participate (Schellmann
et al., 2002; Bernhardt et al., 2005; Kurata et al., 2005). This
model has been well established through studies examining
root hair patterning, with similarities being observed between
shoot and root epidermal cell development. During root hair
patterning, a WEREWOLF (WER)–GLABRA3 (GL3)–
ENHANDER OF GLABRA3 (EGL3)–TRANSPARENT
TESTA GLABRA1 (TTG1) complex forms in non-hair
cells and induces the expression of GLABRA2 (GL2) and
CARPRICE (CPC), which are a repressor and an activator
of hair cell fate expression, respectively (Koshino-Kimura
et al., 2005; Ryu et al., 2005). While GL2 specifies a nonhair cell fate, CPC travels to the neighbouring hair cells, and
replaces WER in binding to the GL3–EGL3–TTG1 complex
(Kurata et al., 2005). Then, the CPC-based complex inhibits GL2 expression, resulting in hair cell fate. GL3–EGL3
expression is activated by the CPC-based complex, and these
proteins subsequently travel to non-hair cells, forming a lateral feedback loop (Bernhardt et al., 2003). In addition to the
proposed simultaneous movement of CPC (or the CPC complex) and GL3–EGL3, it has been shown that CPC, GL3,
and EGL3 exhibit autoinhibitory activity related to their own
transcription, possibly enabling pattern determination to
cease and tip-directed growth to commence. The CPC homologues ENHANCER OF TRY AND CPC 2/3 (ETC2/3) and
TTG1 can also travel from cell to cell (Bouyer et al., 2008;
Wester et al., 2009).
Root development
NCAPs play a vital role in root radial cell patterning, vascular tissue patterning, and the transition from proliferation
to differentiation. During root radial patterning, root endodermis cell fate is determined by SHORT-ROOT (SHR), a
member of the GRAS gene family, whose transcripts are
generally expressed only in the stele, although SHR protein
is also detected in the endodermis (Nakajima et al., 2001).
This movement is also required for asymmetric cell division during the formation of the adjacent ground tissue
(endodermis, cortex, and middle cortex) (Helariutta et al.,
2000; Nakajima et al., 2001). To understand the pattern of
SHR movement, several studies have focused on the downstream targets of SHR and SHR-interacting proteins, such
as SCARCROW (SCR) (Gallagher et al., 2004; Sena et al.,
2004), JAKDOW (JKD), MAGPIE (MGP) (Welch et al.,
2007), and SHR-INTERACTING EMBRYO LETHAL
(SIEL) (Koizumi et al., 2011). Interestingly, SCR plays a special role in regulating SHR movement by forming SHR–SCR
complex, sequestrating SHR into endodermal cell nuclei and
thereby inhibiting further movement to the cortex cell layer
(Cui et al., 2007).
The procambium/cambium tissue provides cells for establishing the xylem and phloem, and the organization of these
tissue types is tightly controlled by the NCAP AT-HOOK
MOTIF NUCLEAR-LOCALIZED PROTEIN 4 (AHL4).
AHL4 travels from the procambium to the xylem in the root
meristem, and its movement is required for vascular tissue
patterning (Zhou et al., 2013). When the intercellular movement of AHL4 is impaired, AHL4 cannot complement the
xylem phenotype observed in the ahl4 mutant. AHL4 movement is facilitated by its interaction with AHL3.
Growth in multicellular organisms requires maintaining
the proper balance between cell proliferation and differentiation. A study was performed to identify TFs that regulate
the first stages of the transition from cellular proliferation to
differentiation by analysing root map gene expression data
(Brady et al., 2007). Approximately 100 TFs show increased
gene expression at the boundary between the meristematic
and elongation zones. Among these TFs, a T-DNA insertional line harbouring an insertion in the UPBEAT1 (UPB1)
gene, a member of the bHLH TF family, showed increased
primary root growth. In addition, UPB1 has been revealed to
display different distribution patterns in terms of mRNA and
protein expression (Tsukagoshi et al., 2010). UPB1 travels
from lateral root cap cells or vascular tissue to cells in the root
elongation zone. As a consequence, different downstream targets of UPB1 operate a process that controls the balance of
reactive oxygen species, which determine the cell proliferation
and cell elongation zones where differentiation begins. Taken
together, the findings described above indicate that NCAPs
exhibit a prominent role in regulating the appropriate spatial
and temporal occurrence of developmental programmes by
controlling coordinated expression of a set of genes.
1740 | Han et al.
Evolutionary aspects of NCAPs and the
plasmodesmal trafficking pathway
Evolutionary conservation of NCAPs in flowering and
floral organ development
compared to ft mutants, suggesting the existence of an FTIPindependent FT movement pathway(s). A critical motif in
Arabidopsis FT and its Cucurbita moschata homologue FTL2
for the execution of their non-cell-autonomous nature was
A good example of the evolutionary conservation of NCAP
functioning is provided a group of genes involved in floral organ
determination. For example, Antirrhinum FLORICAULA
(FLO) is required for the transition from vegetative to reproductive development through its intercellular movement
in the floral meristem, which has also been observed for its
Arabidopsis orthologue LFY (Carpenter and Coen, 1995).
Analysis of periclinal chimeras demonstrated that when FLO
was only expressed in a single cell layer, expression of the
downstream target genes DEFICIENS (DEF) and PLENA
(PLE) was also detected in all of the examined periclinal chimeras (Hantke et al., 1995). The MADS-domain Antirrhinum
majus TFs DEF and GLOBOSA (GLO), which are required
for establishing petal and stamen identity, have been known
to travel towards the epidermis in the floral meristem, and it
has been proposed that the control of the intradermal trafficking of these proteins could play a role in maintaining the
boundaries of their expression domains (Perbal et al., 1996).
In Arabidopsis, the orthologues of these two proteins, AP3
and PI, cannot travel through the secondary plasmodesmata
(PD) between the L1 and L2 cell layers (Jenik and Irish, 2001;
Urbanus et al., 2010b). However, photobleaching assays
showed that both proteins can travel through the primary PD
between epidermal cells, indicating evolutionary conservation in the maintenance of the boundaries of their expression
domains (Urbanus et al., 2010a).
FT and its orthologues have been shown to function as florigens in diverse plant species including Arabidopsis (Navarro
et al., 2011; Yamagishi and Yoshikawa, 2011; Yamagishi
et al., 2011), rice (Kojima et al., 2002), tomato (Lifschitz
et al., 2006), Cucurbita maxima (Lin et al., 2007), Pharbitis
nil (Hayama et al., 2007), Populus deltoids (Böhlenius et al.,
2006), and Vitis vinifera (Sreekantan et al., 2006; Carmona
et al., 2007). These observations suggest an ancestral origin
of NCAPs that function in the same developmental process.
Evolutionary aspects of proteins involved in
plasmodesmal trafficking pathways
During the evolution of NCAPs, how many plasmodesmal
trafficking pathways have occurred in plants? While LFY
has been suggested to travel in a cell-to-cell manner via diffusion, several selective types of NCAP trafficking have also
been reported. The selective non-cell-autonomous modes of
trafficking involve a specific interaction between an NCAP
and the components of a non-cell-autonomous protein pathway (Kim, 2005; Kim et al., 2005). FT movement, especially
FT loading from companion cells to sieve elements, is at least
partly dependent on an endoplasmic reticulum membrane
protein, FT-INTERACTING PROTEIN 1 (FTIP1), in
Arabidopsis (Fig. 2A). Accordingly, ftip1 null mutants exhibit
late flowering under long days but still shows early flowering
Fig. 2. Proteins involved in non-cell-autonomous pathways with
functions in intercellular protein movement. (A) FTIP facilitates
intercellular trafficking of FT. (B) Trafficking of TMV movement
protein and CmPP16 through plasmodesmata requires the
phosphorylation and glycosylation of NCAPP1. (C) KN1 undergoes
unfolding (through an unknown chaperone X) and refolding to
pass through the plasmodesmata channel. In recipient cells,
KN1 refolding is accomplished by the chaperonin CCT8. (D) In
donor cells, the movement of SHR is promoted by SIEL, which
interacts with SHR. In recipient cells, SHR interacts with SCR
to form a complex that directly enters the nucleus. Whether the
movement of non-cell-autonomous transcription factors such as
TMO7, CPC, and AGL21 is controlled by SIEL1 or not has not
been determined. Red arrows, cell-to-cell movement; blue arrows,
interaction/activation.
Intercellular signalling by transcription factors | 1741
determined through analyses employing a zucchini yellow
mosaic virus expression vector (Yoo et al., 2013). In this
study, FT trafficking was distinguished between phloem
loading and post-phloem unloading. A protein analysis performed on phloem sap collected from just beneath the vegetative apex of C. moschata plants indicated that all of the
mutant proteins tested maintained their ability to travel to
the phloem sieve tubes, possibly suggesting the existence of
a non-selective mechanism. However, a number of FTL2/FT
mutants could not be detected in the post-phloem zone in
immunolocalization studies. The fact that FTIP is expressed
in the leaf phloem, but not in the proximal SAM unloading
zone, suggests that a putative FTIP-independent selective
mechanism for FT trafficking exists during the post-phloem
unloading step. These results, together with the confirmation
of FT and FTL2 movement in microinjection and trichome
rescue studies in Arabidopsis, indicates the existence of several
pathways for FT/FTL2 movement: (i) FTIP-mediated selective movement; (ii) FTIP-independent non-selective movement in phloem loading; and (iii) FTIP-independent selective
movement in the post-phloem unloading step (Fig. 2A and
Fig. 3).
In another independent screen of plasmodesmalenriched cell-wall proteins, it was shown that NON-CELLAUTONOMOUS PROTEIN PATHWAY PROTEIN 1
(NCAPP1), an endoplasmic reticulum/PD-localized intercellular pathway component, is a component of the NCAP
translocation machinery and facilitates the movement of
CmPP16 (Lee et al., 2003) (Fig. 2B). A dominant-negative
form of NCAPP1Δ1–22 blocks the capacity of CmPP16 and
TMV movement proteins to modulate plasmodesmal channel
size, but not that of KN1 and cucumber mosaic virus movement proteins, again suggesting diverse pathways of NCAP
movement.
The long-standing notion that partial unfolding or refolding
of KN1 (Kragler et al., 1998) could enhance its movement was
experimentally verified in a recent genetic mutant screen that
identified a class-II chaperonin subunit, CHARPERONINCONTAINING TCP1 8 (CCT8), which is responsible for the
required refolding of KN1 and STM following their transport
into destination cells (Xu et al., 2011) (Fig. 2C). In addition
to KNOX HD proteins, CCT8 facilitates the movement of
TRANSPARENT TESTA GLABROUS1 (TTG1) (Bouyer
et al., 2008; Xu et al., 2011) and the spreading of the Oilseed
rape mosaic virus (ORMV) in Arabidopsis (Fichtenbauer
et al., 2012). However, the movement of SHR and AN3, two
other NCAPs, is not influenced by CCT8, suggesting that at
least two groups of NCAPs have evolved.
In the case of SHR, the intercellular trafficking of this
protein occurs through a conserved mechanism in both
Arabidopsis and rice. The movement of SHR from the stele
to the neighbouring cells requires SIEL and intact microtubules (Koizumi et al., 2011; Wu and Gallagher, 2013). SIEL
null mutants are embryonic lethal, and hypomorphic alleles
of this gene result in defects in root patterning and reduce
SHR movement (Fig. 2D). In root cells, SIEL localizes to
the nucleus and the cytoplasm, where it is associated with
endosomes. Interestingly, microtubule malfunction results in
mislocalization of the SIEL protein. SIEL also interacts with
intercellular trafficking TFs including CPC, TARGET OF
MP7 (TMO7), and AGAMOUS-LIKE 21 (AGL21), but not
with LFY and STM (Koizumi et al., 2011). However, whether
SIEL is required for the intercellular trafficking of these TFs
remains to be tested.
Evolutionary scenario of intercellular trafficking domains
Plants have evolved cell-autonomous and non-cell-autonomous TFs. Based on the concept that PD evolved from simple
algal PD lacking a central desmotubule into highly complex
PD, we previously proposed a hypothetical sequential evolutionary scenario: (i) sophistication of PD structure via the
insertion of endoplasmic reticulum into the centre of the PD;
(ii) evolution of cell-autonomous proteins (CAPs) from initial NCAPs in nature; and (iii) the emergence of a mechanism for selective NCAP trafficking (Lucas et al., 2009). To
develop cell-autonomous TFs, several strategies—including (i) increases in protein size, (ii) the formation of complexes through protein-protein interaction, (iii) subcellular
Fig. 3. Hypothetical evolutionary scenarios for PD-mediated
protein trafficking pathways. NCATFs are introduced into plants
at different evolutionary stages. The Dof intercellular trafficking
motif could have functioned as a molecular template during the
evolution of non-cell-autonomous protein pathways in lower
multicellular plants. However, homeodomain (HD) transcription
factors might have gained a selective trafficking function during the
evolution of non-vascular land plants. Potential mechanisms for
intercellular FLOWERING LOCUS T (FT) movement are presented
for flowering plants.
1742 | Han et al.
sequestration or anchoring, and (iv) control at the protein
expression level—have been proposed (Rim et al., 2011).
According to these hypotheses, selective trafficking of TFs
might have evolved following the establishment of the land
plant PD system. Indeed, KNOX family proteins, which
share a HD that has been shown to be pivotal in cell-to-cell
movements, seem to fall within this category. It is evident
that the movement ability of closely related KNOX HD proteins is quite conserved in diverse plant species (Fig. 3). For
example, HDs found in KNOX family transcription factors
in Arabidopsis, Zea mays, and Physcomitrella show intercellular trafficking activity in Arabidopsis (Kim et al., 2003, 2005;
Chen et al., 2013). However, the HD of the GSM1 KNOX
protein of a unicellular alga, Chlamydomonas reinhardtii,
exhibits little intercellular trafficking activity compared
to the KNOX HD observed in other plant species, including Physcomitrella. One possible evolutionary scenario that
might explain this finding is that land plants have developed
a unique mechanism during evolution allowing them to efficiently transport KNOX HD proteins through endoplasmic
reticulum-containing complex PD (Chen et al., 2013).
Recently, a plant-specific TF family member, Dof4.1, was
shown to be a NCATF, and its Dof-domain-spanning region
(the intercellular trafficking motif) is necessary and sufficient
for cell-to-cell trafficking (Lee et al., 2006; Chen et al., 2013).
An evolutionary analysis suggested that an ancestor of the
intercellular trafficking motif served to foster diverse selectivity among Dof NCATFs (Fig. 3). When Dof of unicellular
Chlamydomonas algae, which do not exhibit plasmodesmata,
was tested, it was found to show a movement ability in higher
plants similar to that observed in Physcomitrella patens,
Nicotiana tabacum (tobacco), and Glycine max (soybean).
This study therefore led a totally new concept, indicating that
during evolution, higher plants might have developed a plasmodesmal pathway for the selective trafficking of non-cellautonomous Dof TFs, where the Dof intercellular trafficking
motif might have served as a molecular template.
The intercellular trafficking signal domains of many
NCATFs, including Dof, KNOX, and GRAS (Gallagher and
Benfey, 2009), include a DNA-binding domain. It is interesting to speculate that similar mechanisms or related interacting partners exist between the DNA binding motifs and
intercellular trafficking motifs of these proteins. Given that
knowledge of the molecular details of evolutionarily related
NCATFs among different species is limited, further exploration of this field in the future is of interest.
NCAPs, peptides, RNAs, and hormones:
the major mobile messengers involved in
intercellular signalling networks
Cells respond not only to signals from NCATFs but also to the
cumulative systemic information coming from other intercellular molecules, such as small peptides, RNAs, and hormones,
at the same time. These molecules exploit different mechanisms
to accomplish cell-to-cell trafficking, but the crosstalk among
them incorporates the entire intercellular signalling network.
Signalling integration between NCATFs and mobile
peptide ligands
In the shoot apical meristem, WUS-mediated shoot meristem homeostasis is essential (Mayer et al., 1998; Yadav and
Reddy, 2012). In Arabidopsis, wus mutants failed to maintain
the shoot meristem and plant growth, whereas when WUS
was ectopically expressed, the meristem was expanded, which
is a phenotype reminiscent of the fasciation observed in lossof-function mutants for each of the three CLAVATA genes
(CLV1, CLV2, CLV3) found in Arabidopsis. CLV3 belongs
to a large gene family referred to as the CLE family, for
CLAVATA3/ESR-related, and encodes a protein processed
into a post-translationally modified 13-amino acid arabinosylated glycopeptide (Ohyama et al., 2009). CLV3 peptides
are secreted from the L1/L2 cells and travel to the L2/L3
cells, where they bind to the CLV1 receptor-like complex
(Fletcher et al., 1999). This functional complex leads to signalling that eventually inhibits the expression of WUS in the
SAM. Strikingly, CLV3 expression is positively promoted by
WUS, which is indeed one of the NCATFs expressed in the
organizing centre (Schoof et al., 2000; Yadav et al., 2011).
This indicates that WUS builds an autoregulatory feedback
loop through the CLV3 peptide signal via precise intercellular
movements to maintain a constant cell number in the shoot
meristem (Fig. 4A).
Signalling integration between NCATFs, hormones,
and mobile miRNAs
During root development, NCATFs, hormones, and mobile
miRNAs occur in the same places to establish a complicate
intercellular signalling network. A genome-wide direct target
analysis illustrated that a cytokinin catabolism enzyme, cytokinin oxidase 3, is directly upregulated by SHR (Cui et al.,
2011). It is well established that cytokinins can repress PIN
gene expression (Ruzicka et al., 2009; Zhang et al., 2011). By
downregulating cytokinin synthesis, SHR might indirectly regulate PIN proteins, which are the major efflux transporters for
auxin. In addition, auxin, which is a plant morphogen, has been
shown to undergo cell-to-cell trafficking under various circumstances and to exert multiple functions in plant development.
These findings may be included in a signalling pathway model
in which NCATF and hormones would function together to
fine-tune spatial and temporal signalling for programming
cell patterns in plants. Yet another layer of interaction comes
into play when the regulation of miRNA165/166 by SHR and
SCR is considered (Carlsbecker et al., 2010) (Fig. 4B). In an
elegant study, both shr and scr mutants were shown to reduce
miR165/166 levels, without affecting the small RNA biogenesis
pathway in the root tip. Surprisingly, these miRNAs have been
shown to behave in a non-cell-autonomous manner: they are
generated in the endodermis and spread to the stele to control PHABULOSA (PHB) (Miyashima et al., 2011). In turn,
PHB has been extensively studied to examine its definitive role
in regulating the PIN-mediated auxin flow during embryogenesis and vascular patterning (Izhaki and Bowman, 2007; Zhou
et al., 2007; Smith and Long, 2010).
Intercellular signalling by transcription factors | 1743
hypophysis precursor, thus representing an MP-dependent
intercellular signal involved in embryonic root specification
(Fig. 4C). MP controls hypophysis specification by sending
two mobile factors whose transport or expression is activated
by MP. How these two signals converge during this process
remains to be determined. Taking these findings together,
it appears that plant organs have found ways to manipulate
developmental aspects by integrating pathways that are individually employed by intercellular regulators in each plant.
Signalling integration between NCATFs and
mobile mRNAs
Fig. 4. Cooperation between different mobile intercellular signals.
The cell-to-cell communication network is composed of diverse
intercellular signalling molecules, including transcription factors,
peptides, hormones, and RNAs. (A) In the shoot apical meristem,
the WUS-CLV regulatory loop includes a mobile transcription
factor and a peptide ligand. (B) In roots, miRNA165/166 functions
as a reciprocal signal from the endodermis to the stele in response
to SHR signalling. CK, cytokinin, (C) Both TMO7 and auxin are
controlled by MONOPTEROS during embryo development.
(D) FT, FT mRNA, and ATC mRNA function synergistically or
antagonistically to regulate flowering. Yellow arrows, intercellular
trafficking; other arrows, activation; blocked arrows, inhibition.
The cell types found in plant root are initially specified early
during embryogenesis. Auxin plays a pivotal role in embryonic root initiation, including the specification of the hypophysis, which is the uppermost cell in the suspensor and the
precursor of the root cap and the quiescent centre (Hamann
et al., 1999). The auxin-dependent TF AUXIN RESPONSE
FACTOR 5 (ARF5)/MONOPTEROS (MP) functions noncell-autonomously in embryonic cells (Hardtke and Berleth,
1998) and drives hypophysis specification by activating polar
auxin transport from the embryo to the hypophysis precursor.
Auxin, itself, is one of the signals involved; however, its accumulation is neither restricted to only the uppermost suspensor
cell nor sufficient to promote hypophysis specification (Weijers
et al., 2006). Recently, additional MP-dependent mobile signals were identified through microarray analysis. TMO7,
encoding a basic helix–loop–helix (bHLH) transcription factor, was shown to be required for MP-dependent embryonic
root initiation (Schlereth et al., 2010). This gene is expressed
in the hypophysis-adjacent embryo cells and travels to the
As shown for RNA-binding NCAPs such as CmPP16 and
viral movement proteins, KN1/KN1 mRNA travels from cell
to cell and FT/FT mRNA/Arabidopsis CENTRORADIALIS
homologue (ATC) mRNA moves systemically through the
phloem. While KN1 mRNA is transported through the formation of KN1 RNA-KN1 protein complexes (Lucas et al.,
1995), how FT mRNA/ATC mRNA enters the phloem stream
is not clear. As KN1 mRNA is undetectable in L1 cells in the
SAM, where KN1 protein is detected, the biological function
of KN1 mRNA is not clear. In contrast, FT mRNA and ATC
mRNA act non-cell-autonomously to stimulate or inhibit
floral initiation, respectively (Huang et al., 2012; Lu et al.,
2012) (Fig. 4D). Thus, the balance between FT (florigenic)
and ATC (antiflorigenic) impacts flowering time. Interestingly,
independent of FT protein trafficking, the trafficking of FT
RNA alone is sufficient to promote flowering. This conclusion
is supported by the three lines of evidence: (i) expression of
the cell-autonomous protein marker red fluorescent protein
(RFP)-FT in leaf companion cells stimulates flowering; (ii)
removal of RFP-FT RNA from the meristem by introducing
FDp::amiR-FT delays flowering; and (iii) surprisingly, translation-free FT mutants can still induce flowering in tobacco.
These findings suggested that non-translatable FT mRNA
is mobilized to the meristems, possibly facilitating the longdistance transport of the tobacco FT protein (Li et al., 2011).
Plasmodesmata: symplasmic channels
for NCAPs
Regulatory components of the PD
NCATFs travel through plasmodesmata, which are intercellular symplasmic channels that interconnect the cytoplasm
of neighbouring cells in plants. Such intercellular trafficking
can be achieved through either selective or diffusion-based
non-selective movements through PD. The former involves
an interaction between NCATFs and other non-cell-autonomous pathway proteins and/or protein unfolding/folding
by chaperon proteins, as shown for the KN1 and STM proteins required for shoot meristem development (Lucas et al.,
1995; Sessions et al., 2000; Kim et al., 2002; Xu et al., 2011).
The latter is dependent on the molecule size/dimension of
NCATFs, as proposed for the movement of LFY and GFP.
The PD size exclusion limit is measured using symplasmic
tracers, such as GFP fusion proteins and fluorescent dyes of
1744 | Han et al.
different sizes (e.g. 8-hydroxypyrene 1,3,6-trisulphonic acid
and carboxy fluorescein). Several regulators of the PD channels have been identified and localized in PD (e.g. callose
synthases, beta-1,3-glucanases, PD CALLOSE-BINDING
PROTEINs (PDCBs), PD-LOCALIZED RECEPTORLIKE PROTEINs (PDLPs), and PD-GERMIN-LIKE
PROTEINS (PDGLPs); Fig. 5; Levy et al., 2007; Simpson
et al., 2009; Amari et al., 2010; Guseman et al., 2010; Lee
et al., 2011; Vaten et al., 2011; Ham et al., 2012). In addition,
PD regulators have been observed at diverse subcellular locations, including within mitochondria (INCREASED SIZE
EXCLUSION LIMIT OF PLASMODESMATA 1 (ISEI)),
chloroplasts (ISE2, GFP ARRESTED TRAFFICKIG 1
(GAT1), SUCROSE EXPORT DEFECTIVE 1 (SED1);
Benitez-Alfonso et al., 2009), and nuclei (DECREASED
SIZE EXCLUSION LIMIT OF PLASMODESMATA
1 (DSE1); Fig. 5; Burch-Smith and Zambryski, 2010). In
the case of GAT1, ISE1, and ISE2, mutant forms of these
proteins result in altered ROS levels, causing a decrease or
increase in the PD size exclusion limit. Although the upstream
signalling pathways involved in PD regulation include diverse
components such as reactive oxygen species (ROS), salicylic
acid and biotic/abiotic stimuli, the final phenotype associated with mutant forms of most regulators is correlated with
PD callose level or PD structure. For example, ISE1, ISE2,
GAT1, and DSE1 are likely involved in the modification of
PD structure and function. In addition, the expression of
GSL, PDBG, PDCB, PDLP, ISE1, ISE2, GAT1, and SED1
affects the deposition of PD callose.
Callose: an important component of PD regulation
The most compelling evidence of the role of endogenous callose levels in protein movement was provided by studies involving a mutation of chorus (chor)/glucan synthase-like8 (gsl8)
that results in excessive proliferation of stomatal lineage cells
mediated by SPEECHLESS (SPCH) (Guseman et al., 2010).
Intercellular diffusion of SPCH, which does not normally
travel in a cell-to-cell manner, was observed. This suggests that
restriction of symplasmic TF movement is required for the
proper segregation of cell fate determinants during stomatal
development. More recently, a semi-dominant allele (cals3-d)
of the gene CALLOSE SYNTHASE-3 (CALS-3) was found
to be defective in GFP unloading from the phloem and in the
patterning and specification of the phloem and the xylem tissues via ectopically producing callose at PD, hinting at a link
between a reduced PD aperture and NCATF signalling (Vaten
et al., 2011). Consistent with these results, overexpression of
PD-localized beta-1,3-glucanase was able to rescue this mutant
phenotype. The most stunning aspect of this study was that
the movement of SHR was reduced in a cals3-d mutant background. As described above, SHR can travel from its original
location in stele tissue to the neighbouring endodermis, initial
cells and QC. However, in cals3-d plants, the root endodermis
showed lower levels of SHR, suggesting that movement of
SHR from the stele was reduced. In accord with this finding,
cals3-d plants exhibited disturbed pattering of the ground tissue and vasculature. Surprisingly, the intercellular movement
of established mobile RNAs miRNA165/66 was abolished following ectopic callose synthesis in cals3-d mutants. The observation that this dysregulation of callose synthase affected the
cell-to-cell trafficking of endogenous miRNAs suggests that
the PD transport of RNAs might be controlled by a regulatory mechanism similar to that observed for NCAPs. Thus, it
is tempting to speculate that a cellular level of callose that is
capable of forming symplasmic domains may act as an endogenous regulator of macromolecular transport.
Toolboxes for examining intercellular networks
Fig. 5. Subcellular symplasmic regulation network. Intercellular
trafficking through plasmodesmata can be controlled by several
components during development or by biotic/abiotic stress.
GSL, glucanase, PDLP, and PDCB regulate callose at the
plasmodesmata to change the plasmodesmal channel. PDGLP
controls plasmodesmata via an unknown mechanism. The
chloroplast-localized proteins SED1, GAT1, and ISE2 and the
mitochondrion-localized protein ISE1 control plasmodesmata
by modifying ROS signals and nuclear signalling. The nuclear
WD-repeat protein, DSE1, downregulates plasmodesmata
channel through unknown mechanism. Arrows and dotted arrows,
regulation and potential regulation, respectively.
Based on the tremendous advances in the understanding of
the importance of NCAP movement during plant development in the last decade, novel technologies have been rapidly
conceived and are being explored to study such scenarios at
the genomics/proteomics level. One of the potentially most
promising approaches for addressing intercellular regulation
in individual cell types is based on macromolecular expression
profiling. In Arabidopsis, a gene expression map of specific
root cells has been developed using fluorescence-activated cell
sorting, which involves cell-specific GFP marker-based cell
sorting and transcriptomics/proteomics analyses (Birnbaum
et al., 2003; Brady et al., 2007). Using this approach, it was
shown that among 2000 proteins identified in Arabidopsis
roots, only 35% were found in only a single root cell population, while the remained 65% were detected in more than two
cell types (Petricka et al., 2012). Strikingly, the Pearson correlation coefficient between the root cellular proteome and transcriptome is significantly low for a given cell type (0.19–0.36).
Intercellular signalling by transcription factors | 1745
Considering such inconsistencies between RNA and protein
expression profiles, it seems possible that there might be several NCAPs, especially among the 65% of proteins that were
detected in multiple cell types. Indeed, it was previously
reported that 25% of the TFs tested undergo post-transcriptional regulation via microRNA-mediated mRNA degradation or intercellular protein movement (Lee et al., 2006).
Another approach that has advanced the comprehension
of NCAPs is high-throughput omics analyses. Using this
approach, studies in a number of plant species, including
Populus (Dafoe et al., 2009; Abraham et al., 2012), pumpkin (Lin et al., 2009; Cho et al., 2010), Agrilus planipennis
(Whitehill et al., 2011), white lupin (Rodriguez-Medina et al.,
2011), rice (Aki et al., 2008), Chelidonium majus (Nawrot
et al., 2007), and cucumber (Walz et al., 2004), have shown
that transcripts, proteins, and small RNAs are present in the
phloem sap. In pumpkin phloem sap, a minimum of 1209
non-redundant proteins were identified. These proteins are
distinct from the observed total protein profiles and travel in
a source-to-sink direction, exhibiting a wide range of functions, as observed in the case of proteins with functions in
RNA binding, signal transduction/plant defence, protein
synthesis, the cell cycle, cell fate, and metabolism (Lin et al.,
2009). Likewise, a phloem proteomics approach resulted in
the identification of a group of proteins involved in the control of embryonic development and flowering. In addition,
through phloem sap proteomics analyses, TFs such as BTF3b,
HAP5A, HAP5B, MBF1B, and ZFP30 have been identified
in pumpkin and rice (Aki et al., 2008; Lin et al., 2009). The
future issue facing this field is elucidation of the biological
significance of these phloem macromolecules and the roles
they play in the integration of whole-plant signalling.
Other technologies, including those deployed in reporter
gene expression and tagged protein localization studies, have
been applied for the large-scale screening of NCATFs, regarding their potential to coordinate specific intercellular regulatory
networks (Rim et al., 2011). These researchers quantitatively
investigated almost 70 Arabidopsis TFs, among which 22,
including proteins belonging to the homeobox, GRAS, and
MYB protein families, were shown to exhibit an extensive
capacity for cell-to-cell movement as well as discrete trafficking patterns. Information gleaned from the application of these
technologies can be directly applied to novel screens for regulators that exhibit movement and fluxes in various tissues related
to different cellular processes at a cellular resolution.
Concluding remarks
In plants, direct cell-to-cell communication through the
movement of TFs, together with diverse mobile signals
including peptide ligands, RNAs, and hormones, regulates
cell specification and patterning, associated with great developmental plasticity. The big picture concerning the evolution
of non-cell-autonomous pathways together with components
that regulate the transport of NCATFs has begun to emerge.
Although there have been significant findings concerning the
probable convergence of diverse intercellular regulators in
signalling networks across cell types, the mechanistic details
of how such adaptations are implemented on a molecular
scale to control a specific developmental output remains to
be determined. As NCATFs are divided among multiple protein families, it will be interesting to determine how many
distinct non-cell-autonomous pathways have evolved and
what components are shared among them. Adapting new
emerging technologies, including genomics, proteomics, and
bioinformatics methods, to be used alongside conventional
approaches will be required to develop a complete picture of
the non-cell-autonomous communication network through
the PD. Conducting studies to obtain a comprehensive understanding of intercellular signalling via mobile TFs represents
a large challenge to shed light on the mechanisms underlying
the organization and evolution of multicellular plants.
Acknowledgements
This research was supported by the Basic Science Research
Program through the National Research Foundation of
Korea (NRF) funded by the Ministry of Education (NRF2013R1A1A2007230) and by the Next-Generation BioGreen
21 Program (SSAC, grant PJ009495), Rural Development
Administration, Republic of Korea. The authors thank their
colleagues for technical assistance, comments, and advice.
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