Review

Gibberellin Localization and Transport in

Plants
Jenia Binenbaum,1 Roy Weinstain,1 and Eilon Shani1,*

Distribution patterns and finely-tuned concentration gradients of plant hor- Highlights

mones govern plant growth and development. Gibberellin (GA) is a plant Bioactive gibberellin (GA) can form
local accumulation maxima, for exam-
hormone regulating key processes in plants; many of them are of significant
ple in the root elongation zone and in
agricultural importance, such as seed germination, root and shoot elongation, the hypocotyl, which correlate with cel-
flowering, and fruit patterning. Although studies have demonstrated that GA lular growth.

movement is essential for multiple developmental aspects, how GAs are trans- GA movement is necessary at multiple
ported throughout the plant and where exactly they accumulate remain largely developmental stages including germi-
unknown. Here, we summarize recent findings from studies of GA movement nation, root elongation, the transition
to flowering, and flower development.
and localization, and discuss the importance of GA intermediates in long- and
short-distance movement. We further review recently identified Arabidopsis GA Long-distance movement of GA
seems to be mostly restricted to its
transporters and highlight their complex specialization and robust functional
nonbioactive precursors rather than
redundancy in GA transport activity. to bioactive forms.

The Plant Hormone Gibberellin Proteins from the NPF and SWEET
Gibberellin (GA) was first identified in the pathogenic fungus Gibberella fujikuroi, which causes a families were recently identified and
shown to transport GA in planta.
disease in rice called ‘foolish-seedling’. By producing large quantities of GA, the plants become
long and slender, are incapable of supporting their own weight, and are chlorotic and partially Thus far, identified GA transporters
infertile [1]. Further research established GA as a hormone that is essential for many develop- are redundantly robust on the one
hand, but have diverse substrate spe-
mental processes in plants, among them are seed germination; organ elongation and expan-
cificity for bioactive GAs, their precur-
sion through cell growth; trichome development; transition from vegetative to reproductive sors, and other signaling molecules
growth; and flower, seed, and fruit development [2–4]. GA manipulation in agriculture is on the other.
common practice; the best-known contribution of GA manipulations to agriculture is the
introduction of dwarfing alleles into staple crops. This manipulation resulted in one of the
cornerstones of the so-called ‘green revolution’ and led to a massive increase in global wheat
and rice yields. Identification of the genes responsible for these traits showed that the encoded
proteins interfere with the action or production of GA [5]. Among more than 130 GAs identified
in plants, fungi, and bacteria to date, only a subset, namely GA1, GA3, GA4, and GA7 are
thought to function as bioactive hormones [3]. Additional forms of GA that exist in plants are
precursors of the bioactive forms or deactivated metabolites [6].

Gibberellin Localization
The fact that the biosynthesis of active GAs (see Glossary) is a complex, multistepped process
with diverse intermediates [7] (Figure 1) makes it difficult to pinpoint the exact tissue or organ in
which GAs are synthesized and localize to. Studies focusing on the spatial organization of the
GA biosynthesis pathway, characterizing the expression patterns of different GA biosynthetic 1
School of Plant Sciences and Food
enzymes using GUS as a reporter, have led to several insights. First, GA biosynthesis genes are Security, Tel Aviv University, Tel Aviv,
differentially expressed among different tissues, cell types, and developmental stages [8–10]. Israel

Second, several members of the GA3ox family, which catalyze the final step in the synthesis
of bioactive GAs, are expressed in growing and elongating shoot and root organs [8–10]. *Correspondence:
Third, although there are several examples of tissues in which the expression of GA eilonsh@post.tau.ac.il (E. Shani).

410 Trends in Plant Science, May 2018, Vol. 23, No. 5 https://doi.org/10.1016/j.tplants.2018.02.005
© 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-
nd/4.0/).
biosynthesis genes co-localizes with GA perception genes (e.g., in inflorescence meristem Glossary
and developing leaves), there are also examples where these two groups do not overlap GA ion-trap mechanism: the
(e.g., GA-biosynthesis genes are not expressed in the aleurone cells of the endosperm but GA phenomena describing the trapping
signaling genes are) [10]. Such spatial separation between genes involved in GA biosynthesis of gibberellin (GA) inside cells as a
result of local pH differences. In the
and perception suggests the requirement for GA movement. Finally, levels of expression of low pH environment of the apoplast,
genes constituting the GA biosynthetic pathway itself do not always coincide [8,9]. For GA, which is a weak acid, is mostly
example, the expression of the late stage GA biosynthesis genes AtGA3ox1 and AtGA3ox2 in its protonated, neutral form and is
thus able to diffuse through cell
in germinating embryos is spatially different from that of the early GA biosynthesis gene AtCPS.
membranes into cells (depending on
This and other examples [11,12] suggest that the location of GA precursors could play an the GA form; see Box 1). Once
important role in regulating GA responses. inside the cell, in the higher pH
environment of the cytosol, GA is
mostly deprotonated and negatively
A recent study, combining mathematical and experimental approaches, compared the
charged and, as a result, its diffusion
putative GA response, represented by the expression pattern of the SCR3 GA responsive out of the cell is restricted.
gene (pSCR3:GUS reporter) and GA perception sites, represented by the expression GA conjugate: a gibberellin (GA)
pattern of GA perception proteins (GID1 and DELLA). The study demonstrated that molecule coupled to another low
molecular weight component by
alternating temperatures act as an instructive signal in the embryonic root tip in Arabidopsis
covalent binding through either the
dormant seeds [13]. The modeling nicely showed that the process of dormancy break in the carboxyl or a hydroxyl group on the
seed is defined by the distribution of the plant hormones GA and abscisic acid (ABA) [14]. GA. The conjugation may be
This spatial separation of ABA and GA responses suggests that crosstalk between ABA and reversible and, while present, it
affects the physical, chemical, and
GA is non-cell-autonomous and is controlled at the level of hormone movement between biological properties of GA.
spatially separated signaling centers [13]. Active GAs: a small subset of
gibberellins (GAs), out of the over
It should be noted that the observations and interpretations regarding GA localization are 100 known forms, that are
biologically active in plants and can
limited by several factors. First, the spatiotemporal resolution of the studies, using GUS promote the association between the
reporters or mRNA expression, is relatively low. It would be constructive to increase the GID1 receptor and the DELLA
resolution of such studies through dynamic monitoring of fluorescent reporters. Second, only proteins. The major bioactive GAs in
plants are GA1 and GA4. Active GAs
a few of the GA biosynthesis genes families, and only a few members from those families, have
commonly have a hydroxyl group on
been analyzed so far. In order to draw a comprehensive map of the spatial distribution of GA C-3b, a carboxyl group on C-6, and
biosynthesis, a concurrent characterization of the whole pathway will be required. Third, studies a lactone between C-4 and C-10.
to date have usually analyzed expression of GA biosynthetic genes at the mRNA level. As it is GA7 and GA3 are biologically active
but are present at minor levels, or in
possible that these enzymes are subjected to post-translational modifications and non-cell-
specific species in higher plants.
autonomous movement, it will be important to examine their localization as translational GA intermediates: precursors of
fusions. The ultimate goal should be to generate specific sensors that will provide a readout gibberellin (GA) that are not
for the enzyme family activity. This would allow a specific readout of the final enzymatic biologically active. They are products
of biosynthetic steps in the active GA
biochemical activity and overcome redundancies. It is reasonable to assume that GA localiza-
biosynthetic pathway.
tion is also regulated by catabolism, conjugation, and transport steps [15]. Thus, expression GA efflux transporters:
patterns of GA biosynthesis genes will not necessarily enable identification of all sites of active transporters capable of transporting
GA localization and response. gibberellin (GA) from inside the
cytosol to the apoplast (GA
exporters). No such proteins have
In order to overcome several of the limitations illustrated above, a novel fluorescence resonance been identified so far, yet their
energy transfer-based GA biosensor (termed GPS1) was developed. The GPS1 biosensor, existence is predicted in order to
constructed by fusing GID1 variants to the DELLA N-termini, showed an increased emission overcome the GA ion-trapping
mechanism.
ratio in response to nanomolar concentrations of GA4 [16]. With the exception of a few GA influx transporters: proteins
limitations such as nonreversible response to GA4, phenotypic hypersensitivity to a GA with the activity to transport
biosynthesis inhibitor, and a limited response to GA3 and GA1, this biosensor should be a gibberellin (GA) across the plasma
membrane from the apoplast into the
useful new tool for identifying GA response sites. For example, GPS1 revealed that GA
cytosol (GA importers).
response is higher in the elongation zone compared with the root meristematic zone. GA Nitrate transporter 1/peptide
localization correlated with cell length when GA4 was exogenously applied, suggesting that transporter family (NPF): a large
rapid transport or catabolism of GA in the root may generate local GA gradients independently transporter family divided into eight
subfamilies. These proteins are active
of GA biosynthesis. In addition, the GPS1 sensor indicated that high levels of GA4 in the

Trends in Plant Science, May 2018, Vol. 23, No. 5 411

elongating hypocotyl depend on darkness [16]. GPS1 should find broad utility in exploration of in transporting a variety of substrates
GA distribution and transport mechanisms and is expected, for example, to shed light on GA such as nitrate, peptides, amino
acids, dicarboxylates, glucosinolates,
distribution in known and novel GA transporter loss-of-function and gain-of-function lines and phytohormones.
reported for the nitrate transporter 1/peptide transporter family (NPF) and SWEET SWEET: members of the SWEET
families [17,18] (further discussed below). transporter family are thought to
mainly transport sucrose but also
have different biological functions.
A different approach to address the question of GA distribution and accumulation sites utilized SWEET13 and SWEET14 have
fluorescently labeled versions of GA3 and GA4 (termed GA-Fl). Combining imaging of GA-Fl recently have been identified as the
localization with information on transporter expression levels and genetics showed that the only non-NPF gibberellin
transporters.
TEMPRANILLO (TEM) proteins play an essential role not only in GA biosynthesis [19] but also in
regulating GA distribution in the mesophyll, which, in turn, regulates epidermal trichome
formation [20]. In roots, GA-Fl accumulated specifically in the elongating endodermal cells
of Arabidopsis roots [21]. The localization of GA-Fl in the elongating cells is consistent with GFP-
RGA levels [22] and with other studies indicating that GA activity is necessary for root elongation
and gravitropic response [23,24], but only partially overlaps with GPS1 signal, which was not
restricted to the endodermis [16]. Since GA-Fl is highly specific to NPF3 [25], it is possible that it
represents only a subset of GA forms. Alternatively, it is possible that GPS1 and GA-Fl report on
GA levels on different sites within the cell; whereas GPS1 mainly responds to nuclear GA4
levels, the fluorescent GA4 reports on transport and localization of exogenously applied GA that
eventually localizes to the vacuole [21]. Since the GA response was shown to be restricted to
the root endodermis cell layer [26,27], it will be important to evaluate the distribution of active
GA and its precursors at a cellular resolution, as has been successfully carried out for the plant
hormones auxin and cytokinin [28,29].

Cells entering the elongation zone increase in length by approximately 10-fold over 5 hours.
Such a rapid expansion is expected to result in a rapid intracellular dilution of GA, practically
reducing its effective concentration. Modeling of this process suggests a correlation between
GA distribution and root cell growth, and the study authors posit that cellular GA levels
decrease at the elongation zone due to cytosolic dilution [30]. Independent analyses of the
GPS1 sensor response and GA-Fl distribution indicate that GA levels are higher in the root
elongation zone compared with the meristematic zone; therefore, GA is probably either
synthesized locally or imported from surrounding tissues to compensate for dilution.

Gibberellin Movement
The first reports on GA mobility in higher plants appeared almost 50 years ago, establishing the
presence of GA in the phloem sap and demonstrating the ability of GA to move through this
medium [31–33]. Since the early work was published, efforts to characterize and quantify GA
movement in plants have been ongoing [3]. GA moves inside the plant in both acro- and basi-
petal directions, though it is more efficient in the former case [34–36]. GA movement is clearly
essential for several developmental processes [37].

One of the biological activities of GA is the induction of hypocotyl xylem expansion following the
flowering transition in Arabidopsis. GA3ox1 mRNA, which encodes the enzyme that catalyzes
the final step in active GA biosynthesis, is upregulated in the shoot but not in the hypocotyl after
transition to flowering. ga1-3 mutant plants are impaired in GA biosynthesis and do not show
expansion of the hypocotyl xylem following transition into the flowering stage. When wild type
scions were grafted on top of ga1-3 rootstocks the xylem expansion was restored. This work
indicates that GA is a mobile shoot-derived signal that triggers xylem expansion in the hypocotyl
[38]. In tobacco plants, defoliation resulted in a phenotype similar to paclobutrazol treatment (a
GA biosynthesis inhibitor): short internodes, lower cambial activity, and fiber differentiation in

412 Trends in Plant Science, May 2018, Vol. 23, No. 5

 Trans - geranylgeranyl diphosphate
CPS

PlasƟd
Ent - copalyl diphosphate
GA KS

Ent - kaurene
GA9 KO

Ent - kaurenoic acid

KAO

Endoplasmic reƟculum
Ent - 7α-hydroxykaurenoic acid
KAO

GA12 aldehyde
KAO

GA12
COOH COOH GA13ox

OH

GA GA53
COOH COOH

GA20ox

HO
HO OH

GA12 GA15 GA44

GA20 COOH COOH
GA20ox
COOH COOH

CHO OH
CHO

GA24 GA19 Cytosol

COOH COOH
COOH COOH
GA20ox
OH
O
CO
O
CO GA9 GA20 COOH

COOH
GA3ox OH
O
CO
O
CO GA4 GA1 HO
COOH
HO
COOH

Non-C13-hydroxylaƟon Early-C13-hydroxylaƟon

Figure 1. Gibberellins are Mobile Signaling Molecules in Plants. Illustration of a schematic plant (left) and gibberellin (GA) biosynthesis pathway (right). Arrows
indicate documented long-distance movement of mobile GAs. The arrows are color-coded to correlate with GA forms shown in the biosynthetic pathway. Root-to-
(See figure legend on the bottom of the next page.)

Trends in Plant Science, May 2018, Vol. 23, No. 5 413

the stem. The defoliated plants had a significantly lower GA content in the internodes than
control plants [39]. Another example of an organ dependent on an external source of GAs are
the petals, which are dependent on the anthers as their GA source. This dependence was
demonstrated in Arabidopsis [40] and in Petunia [41], and anther expression patterns of GA3ox
genes were also observed in tobacco [42] and rice [43]. These pieces of evidence support the
hypothesis that GA is a mobile plant hormone and that its movement is required for certain
processes necessary for plant growth and development. It should be noted, however, that in
several of these studies, it was difficult to elucidate which form of GA was the mobile entity since
the intervention in GA biosynthesis was performed at very early stages of the pathway.

Other studies perturbed GA biosynthesis at later stages of the pathway in an effort to elucidate
the mobile entity. A study carried out in Pisum sativum showed that it is possible to rescue the
phenotype of GA biosynthetic enzyme mutants by grafting them onto wild type plants.
Quantification of endogenous GA levels in grafted versus nongrafted plants, using gas chro-
matography coupled to mass spectrometry (GC-MS), showed that nongrafted mutant plants
had low GA content and that grafting of mutant with wild type plants increased GA content in
the shoot [36]. Focusing on GA forms that showed the highest mobility in this experiment, the
transport of radiolabeled [3H] forms of GAs, GA19, GA20, and GA1 was further evaluated in the
grafted plants. Results pointed to GA20 as the major mobile form in P. sativum. Regnault et al.
conducted a series of grafting experiments using Arabidopsis thaliana mutant plants compro-
mised at different stages of GA biosynthesis and identified GA12 as the major GA form
transported over a long distance through the vasculature [44]. GA12 moved through the xylem
in a root-to-shoot manner and in the phloem in a shoot-to-root direction to regulate plant
growth [45] (Figure 1).

In addition to long-distance movement, there is evidence of short-distance movement involving

other forms of GA. In Cucumis sativus flowers, expression of GA20ox is relatively high in the
ovaries and relatively low in the sepals and petals, while the expression of GA3ox shows an
exactly opposite trend. Accordingly, GC-MS analysis revealed that the ovaries had relatively
high levels of the GA precursors GA12, GA15, and GA9 and that the sepals and petals had
relatively high levels of the bioactive GA4 [11]. Deuterated GA was used in order to support the
claim that GA9 is produced in the ovaries and is transported to the sepals and petals where it is
converted into GA4. Injection of deuterated GA9 and GA4 into the ovaries resulted in a large
amount of deuterated GA4 in the sepals and the petals. This trend was not seen when other
forms of deuterated GA were injected into ovaries. Thus, GA9 is naturally produced in the
ovaries and moves to the sepals and the petals, where it is converted into a bioactive GA4 to
regulate organ growth [11]. Further comprehensive studies that correlate the expression
pattern of each of the GA biosynthetic pathway genes to direct measurements of GA levels
are needed to explore additional developmental stages in which GA movement is fundamental.

Tanaka et al. showed that the sex determining mechanism in Lygodium japonicum ferns
involves GA movement [12]. They proposed a model in which early-maturing prothalli in a
colony express GA-biosynthetic genes, with the exception of GA3ox, thus producing only the
GA9 intermediate. GA9 is modified into an antheridiogen by methyl esterification of its C6
carboxyl group before being secreted to the outside environment. In this transmission process,

shoot and shoot-to-root movement of GA12 in Arabidopsis [45] and GA20 in Pisum sativum (blue) [36]. GA9 movement from the ovaries to the sepals and petals was
shown in Cucumis sativus flowers (red) [11]. Movement of GA from leaves to stem was demonstrated in tobacco and Arabidopsis [38,39] and from stamens to petals in
Arabidopsis [40] and Petunia [41] (black); in these cases, the exact form of mobile GA is not clear.

414 Trends in Plant Science, May 2018, Vol. 23, No. 5

there is the possible involvement of transporters. Subsequently, antheridiogens are incorpo-
rated into neighboring late-maturing prothalli in a colony, which express high levels of GA3ox.
The antheridiogen is then hydrolyzed into the bioactive GA4 to regulate antheridial and
archegonial formation [12].

Importantly, these studies in Arabidopsis, cucumber (C. sativus), pea (P. sativum), and ferns
[11,12,36,44], covering different tissues and developmental processes, point to distinct GA
intermediates, rather than active GA, as the mobile entity (Figure 1). However, evidence for
mobility of bioactive GA also exists [34,46]. If plants are using one form of GA to transport GA
signals over short or long distances, the nature of this GA could be different between plants.
Alternatively, since GA participates in regulation of numerous cellular, developmental, and
adaptive growth responses, plants may be making use of their palette of GA forms (in its entirety
or just partially), generated throughout its biosynthetic pathway, to refine and tune their GA
responses. Under such a hypothesis, different GA forms will serve as specific carriers of the GA
signal to regulate distinct processes in specific locations or developmental stages. Such use of
different GA forms within the plant would subsequently call for a well-orchestrated mechanism
of biosynthesis, catabolism, and transport to regulate such a diverse network of GA signal
carriers.

Gibberellin Transporters
Similar to auxin, GA is subjected to the ion-trap mechanism, limiting its ability to move out of
cells [47] (Box 1). The existence of GA efflux transporters is therefore predicted in order for
GA to effectively move locally, both at the tissue and cellular level [47]; however, GA efflux
transporters have not yet been discovered. It is possible that GA efflux carriers act redundantly
and are therefore difficult to identify.

By contrast, in the past few years, a number of Arabidopsis GA influx transporters have been
identified [44]. A cDNA library screen carried out in a modified yeast two-hybrid system, based
on the GA perception complex GID1/DELLA that allowed yeast growth only in the presence of
GA, was able to reveal GA import activity for the nitrate transporter 1/peptide transporter family
(NPF) [48]. The screen, covering 45 out of the 53 NPF family members, identified several NPF
transporters as being capable of importing different active GAs across the yeast membrane
[49]. This finding indicates that different GA transporters might have varying affinities for the
wide range of GA forms. Unfortunately, the method does not allow testing the transport activity
of inactive GA intermediates. Several of the tested proteins were able to import other plant
hormones (e.g., ABA) in addition to GAs (discussed below). Almost all subfamilies of the NPF
transporters contained at least one member that could transport GA in this assay. Interestingly,
there was no clear correlation between GA transport activity and the arrangement of the
proteins on the phylogenetic tree, emphasizing that the biological relevance of such biochemi-
cal experiments must be further studied. The transport activity of GA transporters in planta may
be affected by post-translational regulation, which may not take place in heterologous systems.

A separate study showed that several of the NPF members indeed act as GA transporters in
planta. NPF3 was identified in a T-DNA insertion screen for mutants defective in accumulation
of both GA3-Fl and GA4-Fl in the root endodermis elongation zone [25]. Overexpression of
NPF3 caused enhanced accumulation of GA3-Fl in all cells of the root. Transport assays in
Xenopus oocytes demonstrated that NPF3 can import unmodified active (GA3, GA4, GA1) as
well as intermediate (GA8, GA19, GA20) GAs across membranes [25]. NPF3 expression is
upregulated by the lack of nitrogen and by high levels of ABA, and is downregulated in response
to high levels of GA [25,50]. The npf3 mutants have impaired hypocotyl elongation and seed

Trends in Plant Science, May 2018, Vol. 23, No. 5 415

 Box 1. Gibberellin Membrane Permeability
The ability of gibberellin (GA) forms (bioactive and intermediates) to diffuse through membranes will significantly influence
their movement characteristics and pattern. The ion-trap mechanism predicts that following entrance into the relatively
alkaline environment of the cytosol (pH 7.2), weak acids such as GA will be trapped in their charged forms with a
limited ability to diffuse out of the cytosol. Thus, exit will require the activity of GA efflux transporters.

Furthermore, the ability of different GA forms to diffuse from the acidic apoplast (pH 5.5) into cells is not identical. In two
recent studies [25,50], transport of a range of GA forms was analyzed in yeast and in oocytes, and an interesting and
consistent trend emerged: GAs formed through the non-C13-hydroxylation pathway (see Figure 1 in main text)
accumulate to a significantly higher extent in cells lacking any transporter compared with GAs formed through the
early C13-hydroxylation pathway. This observation suggests that C13-hydroxylated GAs have a much lower ability to
diffuse through cellular membranes. For example, GA9 (a GA intermediate) and GA4 (a bioactive GA), both from the non-
C13-hydroxylation pathway, diffused into cells more readily than their respective parallels, GA20 and GA1 from the early
C13-hydroxylation pathway. Based on these observations, one might speculate on different transport mechanisms
occurring in plants that produce mainly GAs through the non-C13-hydroxylation pathway (e.g., Arabidopsis) and plants
producing mainly GAs of the C13-hydroxylation pathway (e.g., Pisum sativum). Influx carriers would be of particular
importance for C13-hydroxylated GA.

The pKa of the C13 allyl alcohol is not low (ca. 15.5), which appears to rule out an enhanced ion-trap effect;
however, it has a pronounced effect on the molecules’ polarity (as can be observed in any reverse- or normal-
phase affinity chromatography separation). It would therefore be constructive to comprehensively examine the
correlation between logP values of GA forms and their abilities to diffuse through membranes. This analysis will
establish whether cellular permeability depends mostly on chemical-physical properties or whether structural
recognition (binding to sugars, proteins, lipids, etc.) and retention takes place. Interestingly, GA12, the immediate
precursor for both the early- and non-C13-hydroxylation pathways, showed a significantly higher ability to freely
penetrate into cells than any other of the tested GA forms, accumulating at an approximately 10-fold higher
concentration. It could be speculated that the high membrane permeability of GA12 is a characteristic that allows it
to serve as a long-distance GA mobile signal. It is important to note that both studies of GA transport [25,50] were
conducted on systems lacking a cell wall, the existence of which might have a more pronounced effect than that of
the membrane, potentially eliminating the observed differences in cellular permeability of GAs arising from the
different pathways.

germination under conditions of low nitrate availability [50]. Furthermore, the Bassel group has
combined mathematical and experimental approaches to show that NPF3 overexpressing-
seeds have a greater propensity to break dormancy in response to variable inputs compared
with wild type seeds. This supports their model that increased hormone transport rates further
sensitize seeds to low-temperature oscillations [13]. Work carried out on the glucosinolate
transporter Arabidopsis NPF2.10 (GTR1) [51], suggest that it can transport certain forms of GA
both in yeast and in Xenopus oocytes [52]. npf2.10 mutant plants have reduced fertility under
consistent light, which was restored by GA treatment [53].

The only transporters so far to have been linked to GA transport that are not members of the
NPF family are SWEET13 and SWEET14. The SWEET transporter family members are thought
to mainly transport sucrose and are expressed in different organs and at different develop-
mental stages [54–56]. For example, AtSWEET11 and AtSWEET12 localize to the plasma
membrane and are responsible for sucrose export to the phloem [57]. AtSWEET13 and
AtSWEET14 were identified in a modified yeast two-hybrid screen as potential GA transporters
(the same screen described earlier for NPFs [49]). Xenopus oocytes injected with SWEET13
and SWEET14 RNAs were able to import several GA forms with varying efficiencies. Both
proteins localize to the plasma membrane and are expressed in the anthers, the vascular tissue
in leaves and roots, and at the junctions of stems and petioles. During seed development,
promoter activity of AtSWEET13 and AtSWEET14 was detected in embryonic cotyledons [58].
The double mutant had reduced fertility and produced fewer seeds per silique compared with
the wild type plants. Seeds and seedlings of the double mutant were larger than those of wild

416 Trends in Plant Science, May 2018, Vol. 23, No. 5

type plants [58]. Interestingly, both the SWEET13, SWEET14, and NPF3 GA transport activity
was not restricted to active GAs or a specific intermediate form, as yeast and oocyte experi-
ments showed they transport both types [25,50,58].

A relatively large number of proteins have been implicated so far in GA transport, and further
studies are needed to characterize their functions in planta. Such a multitude of GA transporters
might imply that an intricate and complex system is responsible for managing the diverse
network of GA forms to result in proper GA signaling. In addition, there may be functional
redundancy among these transporters, and this may be hampering efforts to identify GA
transporters through genetic approaches.

Specialization and Robust Functional Redundancy in Gibberellin Transport

Activity
Multiple members of the NPF and the SWEET transporter families have been shown to function
as plant hormone transporters [48,59–65]. The NPF family is evolutionarily conserved but is
expanded and diversified, counting 53 members in Arabidopsis, 90 members in tomato, 93 in
rice, and 79 in maize [66] (Figure 2). Most of the Arabidopsis NPFs localize to the plasma
membrane, yet at least two members, AtNPF8.4 and AtNPF8.5, are localized on the vacuole
tonoplast [66,67]. In most plant species, NPFs have evolved to transport nitrate as well as
specialized metabolites and hormones. Arabidopsis NPF6.3/NRT1.1 and NPF4.6/AIT1 encode

SWEET10
SWE

9
EET
ET1
SW

SW
7
EE

T1
5
T1

SW EE
1

EE SW
T1
2 16
EET
SW
SWEET
14
SWEET2
13
SWEET
SWE
ET 1
T6
EE SW
SW EE
T7

T3
SW
EE

E T8

SWEET4
SW

EET
SWE

GA transport acvty: 0.8

Heterologous system and in planta

2.0
Heterologous system
No acvity
Not tested

Figure 2. Identified Gibberellin Transporters. Phylogenetic trees of known gibberellin (GA) transport protein; the NPF and SWEET families in Arabidopsis. Color-
coded circles indicate GA transport activity. Heterologous systems include yeast and Xenopus oocytes [25,49,50,53,58]. The NPF and SWEET proteins also transport
additional diverse substrates (reviewed in [66,69]).

Trends in Plant Science, May 2018, Vol. 23, No. 5 417

for transporters of auxin and ABA, respectively [48,64]. Recent work showed that various NPF
proteins can transport GA, ABA, and jasmonic acid-isoleucine (JA-Ile) [49,53]. This complexity
makes it difficult to assess the biological roles of distinct NPF proteins.

It is still not sufficiently understood how a transporter can recognize such a diverse substrate
range. An interesting example of this diversity is the dual-affinity-specificity nitrate and auxin
transporter NRT1.1 [64]. The dual sensor/transceptor model proposes that the nitrate-sensing
function of NRT1.1 is due to its dual transport activity. The nitrate signal transduced by NRT1.1
is a nitrate-dependent modification of auxin transport in lateral root development [64]. Chiba
et al. reported that among the 45 tested Arabidopsis NPF members, 18 were able to transport
more than one GA [49] (Figure 2). Although biochemical assays in yeast and oocytes have
suggested putative substrates for 32 of the Arabidopsis NPF members, the biological relevance
of such activity is not entirely clear.

A significant amount of the work carried out on the NPF family did not expose observable
phenotypes in the single-gene loss-of-function mutants. The lack of phenotype might suggest
that these genes are not essential under the considered developmental and environmental
stage. Alternatively, these single mutants might be partly or completely compensated by the
activity of other genes. Due to their large number and strong sequence similarities, it is
reasonable to assume that there is very prominent genetic redundancy in the NPF family
[17]. For example, whereas the single npf2.10 and npf2.11 (gtr1 and gtr2, respectively) mutants
did not display an evident phenotype, the npf2.10 npf2.11 double mutant did not accumulate
glucosinolates in seeds and had a more than 10-fold overaccumulation in source tissues such
as leaves and siliques [68].

Similarly to the NPF family, the SWEET transporters are evolutionarily conserved and transport
a diverse range of sugars, including hexose, sucrose, and fructose [69] (Figure 2). Thus, the
diversity in specialization exists at the single protein level (e.g., NPF3 transports GA and ABA
[25,49]) and at the family level (e.g., SWEET13 transports GA and SWEET17 transports
fructose [54,58,69,70]). Evidently, both NPFs and the SWEETs are extreme examples of gene
expansion that provide redundantly robust responses and diverse specialization in plant
hormone transport.

On average, 65% of annotated genes in plant genomes have a duplicate copy [71]. In
Arabidopsis, 80% of all protein coding genes belong to families with at least two members
[71]. In some cases, two genes have redundant functions in a subset of developmental or
cellular processes but have independent functions in others [72,73]. In order to elucidate the
functions of the genes in question, it is necessary that mutations will generate visible pheno-
types. In recent years, several novel gene silencing/knockout technologies have emerged, the
most prominent being artificial microRNA-mediated inhibition of gene expression (amiRNA)
[74,75] and CRISPR/Cas9 knockout [76,77]. Using these technologies to target regions
conserved across gene families, it is possible to inhibit expression of multiple genes at once
[73]. Developmental processes in plants are regulated by an interlaced network of plant
hormone responses. One might speculate that due to the agonist/antagonist nature of these
hormones, it is possible that some hormone transporters have gained the ability to transport
more than one substrate, either in a symport or in an antiport fashion. This type of multi-
substrate recognition has been demonstrated in other transport systems [78–81]. As most
work demonstrating multihormone transporter specificity in plants has been done in heterolo-
gous systems, it is possible that in their proper biological context, the roles of transporters are
more defined.

418 Trends in Plant Science, May 2018, Vol. 23, No. 5

Concluding Remarks and Future Perspectives Outstanding Questions
Plants, as sessile organisms, rely on finely-tuned responses to their hormones and other Where are gibberellins (GAs) biosyn-
signaling molecules to mediate adaptive growth and developmental processes. It is therefore thesized and how much overlap do
their synthesis sites have with GA per-
crucial for plants to tightly regulate their hormones’ signaling pathways. The data accumulated ception proteins in the spatiotemporal
in the studies reviewed here indicate that GA is a mobile signal in plants and that a number of space?
developmental and adaptive growth processes require mobility of GA to take place properly. In
addition to biosynthesis, metabolism, and perception, the evidence suggests that spatial Where do bioactive GAs localize and
accumulate at the plant tissue level?
control is an additional layer of regulation that plants exert over their GA signaling. Several
GA forms, both bioactive and not active, have been implicated as the mobile GA in different
Is there an active and regulated long-
plant species and biological processes. It is currently not clear whether different plant species distance GA transport mechanism in
are committed to distinct GA forms as their mobile GA signal or whether plants utilize multiple plants? If so, how does it differ from
GA forms as mobile signals, regulating their mobility in a contextual manner. Further compre- local GA transport mechanisms?
hensive studies that correlate the full GA biosynthetic pathway gene expression and direct
How specific are the different GA
measurements of GA levels are needed to address this fundamental question. In addition, GA
transporters? Are there GA12- and
conjugates could be an additional factor affecting GA localization and transport. GA glucosyl GA9-specific transporters in plants?
conjugates have been well documented [82], however, data on their localization in plants or
their transport is scarce and requires further investigation. What is the biological importance of
the multisubstrate transport activity
observed in biochemical assays for
The discovery of GA import transporters complements the evidence for GA movement,
several GA transporters?
supporting the existence of an active regulation mechanism that directs GA distribution and
flow in plants to ensure appropriate GA signaling. Evidence for the biological importance of Does subcellular localization of bioac-
most GA transporters identified so far remains weak, primarily because of poor genetic tive and intermediate GA contribute to
support. There is a strong need to combine biochemical transport assays with solid loss-of- regulation of GA signaling?

function and gain-of function developmental phenotypes to reveal the biological roles of
Which, if any, roles do GA-conjugates
these proteins in GA transport and responses [83]. The putative functional redundancy play in GA movement and distribution,
within GA transporter families identified so far will probably necessitate experiments in and how are the GA conjugates rec-
which expression of multiple family members is inhibited or knocked-out simultaneously to ognized and transported?
unveil their distinct roles. Finally, reinforced efforts are needed to identify the predicted, yet
currently missing, GA efflux carriers (see Outstanding Questions). Are there GA exporters, as predicted
by the ion-capture mechanism?

Acknowledgments Can agriculturally important traits,

We thank Daria Hudiakov for graphical assistance. This work was supported by grants from the Israel Science Foundation such as seed germination or transition
(1832/14 to E.S. and 596/17 to E.S. and R.W.), the German Israeli Foundation for Scientific Research and Development (I- to flowering, be manipulated through
236-203.17-2014 to E.S.), the Human Frontier Science Program (HFSP—RGY0075/2015 to E.S.) and by European interventions in GA movement?
Research Council Starting Grants (757683- RobustHormoneTrans to E.S. and 679189-GAtransport to R.W.).

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