Journal of Experimental Botany, Vol. 66, No. 16 pp. 4999–5013, 2015
doi:10.1093/jxb/erv132 Advance Access publication 4 April 2015
REVIEW PAPER
Regulation of chloroplast development and function by
cytokinin
Anne Cortleven* and Thomas Schmülling*
Institute of Biology/Applied Genetics, Dahlem Centre of Plant Sciences (DCPS), Freie Universität Berlin, Albrecht-Thaer-Weg 6, D-14195
Berlin, Germany
* To whom correspondence should be addressed. E-mail: [email protected]; [email protected]
Received 29 December 2014; Revised 15 February 2015; Accepted 24 February 2015
Abstract
A role of the plant hormone cytokinin in regulating the development and activity of chloroplasts was described soon
after its discovery as a plant growth regulator more than 50 years ago. Its promoting action on chloroplast ultrastructure and chlorophyll synthesis has been reported repeatedly, especially during etioplast-to-chloroplast transition. Recently, a protective role of the hormone for the photosynthetic apparatus during high light stress was shown.
Details about the molecular mechanisms of cytokinin action on plastids are accumulating from genetic and transcriptomic studies. The cytokinin receptors AHK2 and AHK3 are mainly responsible for the transduction of the cytokinin
signal to B-type response regulators, in particular ARR1, ARR10, and ARR12, which are transcription factors of the
two-component system mediating cytokinin functions. Additional transcription factors linking cytokinin and chloroplast development include CGA1, GNC, HY5, GLK2, and CRF2. In this review, we summarize early and more recent
findings of the long-known relationship between the hormone and the organelle and describe crosstalk between
cytokinin, light, and other hormones during chloroplast development.
Key words: Chlorophyll, chloroplast, crosstalk, cytokinin, photomorphogenesis.
Introduction
More than half a century ago when the newly discovered
growth regulator cytokinin was explored for its activities, it
was soon found that its application retards loss of chlorophyll
from detached leaves (Richmond and Lang, 1957) and that it
stimulates, together with light, chlorophyll synthesis (Sugiura,
1963). Stetler and Laetsch (1965) summarized the outcome of
a study exploring the effects of the artificial cytokinin kinetin
on cultured tobacco cells with the notion ‘the possibility that
kinetin exerts a direct effect upon chloroplast differentiation is
considered’. More detailed descriptive studies were published
during the following decades and in recent years details of the
molecular pathways linking cytokinin and chloroplast development have been uncovered. After introducing the acting partners, we summarize the influence of cytokinin on chloroplast
ultrastructure, chlorophyll biosynthesis, and photosynthesis;
report on results obtained from transcriptomic and proteomic
studies; highlight the few known transcription factors mediating cytokinin action on chloroplast development; and end by
describing the interaction between cytokinin, light, and other
hormones. Key facts about the role of cytokinin in regulating
chloroplast development are listed in Box 1.
Chloroplasts, the keys for the plants’
autonomous life
Chloroplasts belong to a family of organelles, the plastids,
that are present in living plant cells, each having their own
Abbreviations: ABA, abscisic acid; ACC, 1-aminocyclopropane-1-carboxylic acid; ALA, 5-aminolevulinic acid; ARF, auxin response factors; Aux/IAA, auxin/
indole-3-acetic acid inducible; BA, 6-benzyladenine; ERF, ethylene response factor; GA, gibberellin; Glu, glutamate; GluTR, Glu-tRNA reductase; GSA-AT, Glu-1semialdehyde aminotransferase; iP, isopentenyl adenine; cZ, cis-zeatin; tZ, trans-zeatin.
© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved.
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5000 | Cortleven and Schmülling
Box 1. Key facts about cytokinin-regulated chloroplast development and function
•• There is no absolute requirement for cytokinin to develop chloroplasts per se but plants with an altered cytokinin status
show altered chloroplast structure and function.
•• The principal action of cytokinin on the chloroplast is that of a modulator required for fine-tuning the organelle’s functions, presumably mediating environmental cues.
•• Cytokinin protects chloroplast functions under high light stress.
•• Cytokinin accelerates the disintegration of the prolamellar body and the development of thylakoid membranes during
the etioplast-to-chloroplast transition.
•• Cytokinin accelerates chlorophyll biosynthesis by promoting ALA synthesis and enhancing POR activity.
•• Cytokinin regulates nuclear and plastid gene expression and alters protein abundance to exert its role on chloroplast
development and function.
•• The action of cytokinin on plastids is mediated mainly by the AHK3 receptor, with AHK2 having an accessory function,
and the transcription factors ARR1, ARR10, and ARR12.
•• The transcription factors CGA1, GNC, HY5, and GLK2 act downstream of the core cytokinin signalling system to
mediate its effect on chloroplast development and to realize crosstalk between cytokinin, light, and other plant hormones.
•• Cytokinin regulates plastid division acting through CRF2 on the level of PDV2, which is a rate-limiting factor for plastid
division.
characteristics and function. Chloroplasts contain chlorophyll and are the sites of photosynthesis where light energy
capture and conversion take place. They are not only important for this energy conversion process, but they are also
involved in the biosynthesis of many essential primary and
secondary metabolites (Lopez-Juez and Pyke, 2005).
Like all plastids, chloroplasts develop from proplastids that
are present in the immature cells of plant meristems. If a leaf
is grown in darkness, proplastids develop into etioplasts, which
have a semi-crystalline structure, called the prolamellar body,
consisting of lipids and the NADPH-dependent protochlorophyllide oxidoreductase (Armstrong et al., 1995; Vinti et al.,
2005). Upon illumination, the prolamellar body is dispersed,
thylakoid membranes are formed, and the protochlorophyllide
accumulated in the dark is converted to chlorophyll and a fully
functional chloroplast is formed. The transition from etioplast
to chloroplast is part of the de-etiolation process (for review:
von Arnim and Deng, 1996) and coincides with the biosynthesis of chlorophyll (for review: Tanaka and Tanaka, 2007).
Chlorophyll biosynthesis (Fig. 1) is a side-branch of the
tetrapyrrole biosynthesis pathway and starts with the production
of 5-aminolevulinic acid (ALA). ALA is synthesized from glutamate (Glu) via Glu-tRNA synthetase, Glu-tRNA reductase
(GluTR), and Glu-1-semialdehyde aminotransferase (GSA-AT)
followed by enzymatic steps resulting in the synthesis of protoporphyrinogen IX (Proto IX), which is a common precursor
of haem and chlorophyll. Haem biosynthesis continues on a
separate branch, which is not detailed further here (for review:
Tanaka and Tanaka, 2007).The chlorophyll branch inserts Mg2+
into Proto IX for chlorophyll a biosynthesis, followed by the
chlorophyll cycle, which refers to the interconversion between
chlorophyll a and chlorophyll b. Within the chlorophyll branch,
the NADPH:protochlorophyllide oxidoreductase encoded by
the POR genes converts protochlorophyllide into chlorophyllide.
Importantly, this is the first step in chlorophyll biosynthesis that
requires light. As a consequence, chlorophyll intermediates accumulate during growth in the dark, especially protochlorophyllide. When dark-grown (etiolated) seedlings are exposed to light,
protochlorophyllide is immediately converted to chlorophyllide
followed by a lag phase during which no additional chlorophyll
can be synthesized. The reason for the lag phase is the necessity to form the enzyme system responsible for ALA synthesis
de novo before greening can continue (Castelfranco et al., 1973).
Once chlorophyll a and b are formed and properly incorporated
into the thylakoid membranes and associated photosystems,
chloroplasts are fully functional and can photosynthesize.
Cytokinin, a versatile plant hormone
Cytokinin is a plant hormone originally discovered because of
its ability to induce, together with auxin, cell division (Miller
et al., 1956). Numerous physiological and developmental processes regulated by cytokinin are known today, ranging from
the cell cycle, activity of root and shoot meristems, regulation
of sink strength, and shoot and root branching, to floral transition and leaf senescence (for review: Werner and Schmülling,
2009; Hwang et al., 2012; El-Showk et al., 2013; Kieber and
Schaller, 2014). More recently, cytokinin has also been shown
to play a role in responses to environmental cues, such as
drought, cold, and osmotic and high light stress (for review:
Argueso et al., 2009; Ha et al., 2012; Vanstraelen and Benková,
2012). Cytokinin action was linked to chloroplast behaviour
soon after its discovery, as will be detailed further below.
Metabolism and signalling of cytokinin has been elucidated
in decent detail in Arabidopsis thaliana. Cytokinins are synthesized de novo via an enzymatic cascade encoded by a set
of three gene families, including isopentenyltransferase (IPT),
cytokinin trans-hydroxylase (CYP735A) and cytokinin nucleoside 5-monophosphate phosphoribohydrolase (LOG) genes (for
review: Sakakibara, 2006). Degradation of cytokinin is catalysed
by cytokinin oxidase/dehydrogenases encoded by CKX genes
(Schmülling et al., 2003).The cytokinin signal is perceived by
sensor histidine kinases, which are predominantly located in the
endoplasmic reticulum (Caesar et al., 2011; Lomin et al., 2011;
Wulfetange et al., 2011). There are three cytokinin receptors in
A. thaliana, named AHK2, AHK3, and CRE1/AHK4 (Inoue
et al., 2001; Suzuki et al., 2001) (Fig. 2), which have distinct
functions (Higuchi et al., 2004; Nishimura et al., 2004; Riefler
Cytokinin function in chloroplasts | 5001
Light
CK
AHK2
AHK3
CRE1/AHK4
CRF2
AHPs
A-type
ARRs
GNC
CGA1
HY5
GLK2
chloroplast
development
Fig. 1. Role of cytokinin in the chlorophyll biosynthesis pathway. A simplified
scheme of chlorophyll biosynthesis is shown. It starts with the conversion
of glutamate to 5-aminolevulinic acid and is followed by the assembly of a
porphyrin structure resulting in protoporphyrinogen IX. Chlorophyll biosynthesis
continues with the incorporation of Mg into protoporphyrinogen IX resulting
eventually in the synthesis of chlorophyll. Genes and the corresponding
enzymes catalysing distinct reactions of chlorophyll synthesis are indicated
in green (left-hand side) and blue (right-hand side), respectively. Arrows point
to compounds and enzymatic steps of the chlorophyll biosynthesis pathway
that are known to be influenced by cytokinin. Numbers above the arrows
correspond to the following studies: 1, Sugiura (1963); 2, Banerji and Lalorya
(1967); 3, Knypl (1969); 4, Beevers et al. (1970); 5, Fletcher and McCullag
(1971); 6, Fletcher et al. (1973); 7, Buschmann and Sironval (1978); 8, Ford
et al. (1979); 9, Parthier et al. (1981); 10, Lew and Tsuij (1982); 11, Dei (1985);
12–14, Masuda et al.(1992, 1994, 1995); 15, Kuroda et al. (1996); 16,
Kusnetsov et al. (1998); 17, Kuroda et al. (2001); 18, Higuchi et al. (2004); 19,
Brenner et al. (2005); 20, Riefler et al. (2006); 21, Yaronskaya et al. (2006);
22, Werner et al. (2008); 23, Argyros et al. (2008); 24, Hedtke et al. (2012);
25, Brenner et al. (2012); 26, Bhargava et al. (2013); 27, Cortleven et al.,
unpublished results. Numbers printed in bold refer to studies involving mutants
or transgenic lines with a lowered cytokinin status, while standard numbers
refer to studies using exogenously applied cytokinin (this figure is available in
colour at JXB online).
et al., 2006; reviewed by Heyl et al., 2012). The cytokinin signal
is transmitted from the receptors by a two-component signalling
system via histidine phosphotransfer proteins (named AHPs
in Arabidopsis) (Hutchinson et al., 2006) to type-B response
regulators (type-B ARRs), which are transcription factors and
regulate cytokinin response genes (Sakai et al., 2001; Argyros
et al., 2008). Among the direct target genes of type-B ARRs are
B-type
ARRs
ARR1
ARR10
ARR12
PDV2
chloroplast
division
SIGs
regulation of
plastid genes
Fig. 2. Cytokinin signalling pathways of Arabidopsis regulating
chloroplast development and function through transcription factors. In this
schematic overview, components of the core cytokinin signalling pathway
(two-component signalling system) involved in transducing the cytokinin
signal to transcription factors regulating chloroplast development and
function are marked in bold. These include the cytokinin receptors AHK2
and AHK3 as well as several B-type response regulators (ARR1, ARR10,
ARR12). It is not clear whether CRF2 acts downstream of B-type ARRs
or is directly activated through a side path of the cytokinin signalling
system (Rashotte et al., 2006). Links that have not been demonstrated
experimentally are indicated by dashed lines. GNC and CGA1 regulate
several aspects of chloroplast development as well as plastid division
(Richter et al., 2010; Köllmer et al., 2011; Chiang et al., 2012). HY5
is absolutely required for cytokinin-induced root greening, and GLK2
overexpression requires HY5 for maximal root greening (Kobayashi et al.,
2012). CRF2 increases the level of PDV2, which is required for plastid
division (Okazaki et al., 2009). Sigma factors (SIGs) are involved in the
regulation of plastid transcription and are needed to mediate the full
transcriptional response of plastid genes to cytokinin (Borsellino, 2011).
For further information see text.
some encoding type-A ARRs acting as negative regulators of
the pathway (Hwang and Sheen, 2001; To et al., 2004) (Fig. 2).
Several studies reporting the transcriptomic response to cytokinin have been published, including three meta-analyses analysing and summarizing the outcome of these studies (Brenner
et al., 2012; Bhargava et al., 2013; Brenner and Schmülling,
2015). A number of the cytokinin-regulated genes linking the
hormonal activity to chloroplast function have been identified
in this way (see below). However, among all the genes regulated
by cytokinin there is only a rather low number that appear to
have a role in regulating chloroplast function.
Of interest in an evolutionary context is that among the
multicellular eukaryotes only land plants possess a two-component signalling system similar to the one used to transmit
the cytokinin signal, while comparable systems are abundant
in prokaryotes and single-celled eukaryotes such as yeast. It
has been suggested that cytokinin metabolism and signalling
genes have been delivered through the endosymbiont pathway
via horizontal gene transfer from cyanobacteria, the plastids’
ancestors (Gruhn and Heyl, 2013). Interestingly, this scenario
would imply that after transfer of the cytokinin genes from
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the plastid genome to the nuclear genome, they would have
been adapted to regulate development and function of their
place of origin, the plastids. Currently, however, the evolutionary path of the cytokinin genes has not been traced back
unequivocally to a cyanobacterial origin (Frébort et al., 2011;
Spíchal, 2012; Gruhn et al., 2014). The earliest traces appear
before the landfall of plants and the complexity of the cytokinin system has increased during the development of modern land plants (Gruhn and Heyl, 2013).
Chloroplasts, sites for cytokinin
biosynthesis
The cytokinin metabolism and signalling system is distributed to a number of subcellular compartments, including
the plastids. Chloroplasts of A. thaliana contain four of the
seven IPT enzymes (IPT1, IPT3, IPT5, IPT8) catalysing the
first and rate-limiting step of the isopentenyl adenine (iP)
and trans-zeatin (tZ)-type cytokinins, while other enzymes
of this cytokinin synthesis pathway are located in the mitochondria (IPT7) or in the cytosol (IPT4) (Kasahara et al.,
2004). The plastid localization of these IPTs is linked to the
use of dimethylallyl diphosphate, which is produced through
the methylerythritol phosphate pathway in the plastids themselves as a substrate for the biosynthesis of tZ and iP within
the chloroplasts. The localization of IPT4 in the cytosol is
related to the mevalonate-derived prenyl-group of the cZ-type
cytokinin. However, while tZ and iP are known as the most
active cytokinins and their functions have been described,
the cis-zeatin (cZ) biosynthetic pathway has not yet been
completely elucidated and the hormonal functions of cZ are
not well understood (Gajdošová et al., 2011; Spíchal, 2012).
Noteworthy, an Agrobacterium T-DNA-encoded IPT enzyme
is also localized to plastids (Sakakibara et al., 2006), further
corroborating the relevance of this organelle for cytokinin
synthesis. Interestingly, Galichet et al. (2008) showed that
the subcellular localization of IPT3 depends on its modification by farnesylation. Wild-type IPT3 is localized in plastids
and preferentially synthesizes tZ-type cytokinin, while mutation of the farnesylation site causes cytosolic localization of
the enzyme and accumulation of iP-type cytokinins. Taken
together, the presence of different sets of IPT proteins in the
plastids and other cellular locations suggests a specific function for the plastid pathway in maintaining cellular cytokinin homeostasis. This is further supported by the finding
that chloroplasts contain a specific set of cytokinin metabolites, including free bases, ribosides, and ribotides, as well
as N-glucosides (Benková et al., 1999). However, neither the
specific functional significance of plastid-derived cytokinin
nor the occurrence of cytokinin within plastids is presently
known.
Chloroplasts, targets for cytokinin action
The initial study describing a role of cytokinin during chloroplast-related processes was published by Richmond and Lang
(1957) and some years later confirmed by Mothes (1960). The
observation of chlorophyll retention in cytokinin-treated
leaves made in these early reports is due to the now well-documented anti-senescence effect of cytokinin, which will not
be discussed in this review (for further reading: Zwack and
Rashotte, 2013).
Later studies on the role for cytokinin during chloroplast
development have mostly focused on its influence during deetiolation and described changes in chloroplast ultrastructure and chlorophyll biosynthesis. De-etiolation is the switch
of the developmental programme of dark-grown seedlings,
named skotomorphogenesis, to photomorphogenesis upon
perception of light. Photomorphogenesis causes inter alia
development of a shorter hypocotyl as well as unfolding
and greening of cotyledons. This de-etiolation response was
employed as an easy accessible experimental system to study
cytokinin-induced chloroplast development because strong
changes are induced (see further details below). However,
cytokinin also has an important role in the light for chloroplast function because it modulates the photosynthetic performance. Because of lack of specific cytokinin inhibitors
and the absence of well-defined cytokinin mutants, most of
the earlier studies reported the consequences of exogenous
addition of cytokinin, of the increase of the endogenous
cytokinin content upon introduction of an IPT gene, and/
or the correlation of phenotypic changes with changes in the
endogenous cytokinin content. The outcome of these studies
has given very valuable hints on the functions of cytokinin;
however, the results have been mostly preliminary as no clear
conclusions on the true physiological relevance of cytokinin
in the analysed processes could be drawn. We will, therefore,
pay particular attention to the question of whether these earlier observations have been confirmed by analysis of loss-offunction mutants, which were mainly obtained in Arabidopsis.
Cytokinin influences chloroplast ultrastructure
One of the first reports describing a stimulating influence
of cytokinin on chloroplast development was by Stetler and
Laetsch (1965), who reported induction of chloroplast differentiation from proplastids by the artificial cytokinin, kinetin.
Numerous studies in the 1970s, mostly done in Lupinus luteus
(lupine), Hordeum vulgare (barley), or Nicotiana tabacum
(tobacco), confirmed that during dark-to-light transitions
cytokinin can accelerate the differentiation of the prolamellar body into the typical lamellar structure, and that this is
accompanied by an increase in chlorophyll biosynthesis rate
(for review: Parthier, 1979). In 1994, Chory and co-workers
compared the plastid morphology of etiolated A. thaliana
seedlings grown in the absence or presence of cytokinin
(Chory et al., 1994). Seedlings grown without cytokinin had
small etioplasts containing a prolamellar body whereas the
plastids of seedlings grown in the presence of cytokinin were
larger, lens-shaped, lacked a prolamellar body, and contained
some bi-thylakoid membrane structures. Kusnetsov et al.
(1998) concluded from work in lupine that the stimulatory
effect of cytokinin on chloroplast development was dependent on an intact prolamellar body and a cytokinin-dependent increased stability of POR enzymes. Taken together,
most studies show consistently that cytokinin supports the
Cytokinin function in chloroplasts | 5003
formation of the thylakoid membranes. This is illustrated
in Fig. 3, which shows an overview of cytokinin-induced
changes in chloroplast ultrastructure at different time points
after transferring dark-grown Arabidopsis seedlings into the
light. Indeed, even in the dark cytokinin [1 µM BA (6-benzyladenine)] causes formation of prothylakoid membranes,
whereas these membranes are only visible in the wild-type
seedlings after about 6 h light treatment. The acceleration of
the etioplast-to-chloroplast transition is apparent from the
earlier formation of mature thylakoid membranes.
Most reports describe the effect of cytokinin on chloroplast
ultrastructure during de-etiolation, but the hormone also causes
alterations in light-grown plants. Farineau and Rousseaux
(1975) reported abnormal features of the plastids of cytokinintreated light-grown Cucumis sativus (cucumber) leaves, such as
an increase in the amount of thylakoids per granum.
Hyperstacking of grana and increased starch content in
response to cytokinin were also noted years later (Quanten
et al., 2007; Criado et al., 2009). Abnormal structures, such
as large crystalloids and globular bodies within the chloroplasts, were reported as a consequence of increased endogenous cytokinin content by Synková et al. (2006); however, it
could be that these anomalies are a pleiotropic effect and not
specifically due to cytokinin action. By contrast, chloroplasts
in 35S:CKX1 tobacco plants with a lower endogenous cytokinin content have only few starch grains and show reduced
grana stacking (Werner et al., 2008). This indicates that in
light-grown plants a normal level of cytokinin is required to
assure the development of proper chloroplasts.
Besides their effect on chloroplast ultrastructure, cytokinins
also influence chloroplast number. Boasson and Laetsch
(1969) noted that cytokinin treatment increased the chloroplast number per cell in etiolated tobacco leaf discs incubated
in light. The addition of cytokinin or expression of an IPT
gene complements a Physcomitrella patens mutant defective
in chloroplast division (Reutter et al., 1998). In Arabidopsis,
it was shown that cytokinin regulates plastid division by acting on the level of PLASTID DIVISION2 (PDV2) as will be
detailed below (Okazaki et al., 2009).
Cytokinin regulates chlorophyll biosynthesis
Much attention has been paid to the role of cytokinin in regulating chlorophyll biosynthesis (Fig. 1), which is often seen
as an indicator for chloroplast biogenesis. Sugiura (1963)
described the stimulating effect of kinetin on chlorophyll biosynthesis in greening of detached primary leaves of Phaseolus
vulgaris. Stimulation of chlorophyll accumulation during the
etioplast–chloroplast transition has been described for various species and could be linked to two important steps within
the chlorophyll biosynthesis pathway: ALA synthesis and
the photoconversion of accumulated protochlorophyllide to
chlorophyllide (for review: Parthier, 1979). Fletcher and coworkers (Fletcher et al., 1973) reported the absence of the
normally occurring lag phase in chlorophyll biosynthesis during the de-etiolation process upon pre-treatment of cucumber
cotyledons with kinetin, due to stimulation of the formation
of the ALA-synthesizing system, resulting in an immediate
Fig. 3. Effect of cytokinin on chloroplast ultrastructure during de-etiolation.
Electron microscopic pictures of 3-day-old etiolated Arabidopsis thaliana
seedlings grown in dark on full MS medium (Murashige and Skoog,
1962) without sucrose in the presence or absence of cytokinin (1 µM
BA). Scale bars represent 1200 nm. After 3 days growth in the dark, the
etioplasts contain large prolemellar bodies (p). In presence of cytokinin,
the prolamellar body is still present in the etioplast but it also contains
prethylakoids (pt). After 6 hours illumination, the prolamellar bodies start
to disintegrate and prethylakoid membranes are formed. With cytokinin,
no prolamellar body is visible anymore at this time point and the thylakoid
(th) membranes are fully developed. After 12 hours illumination, a fully
functional chloroplast is formed with thylakoid membranes and grana
stacking (g). A similar observation is made in the presence of cytokinin but
starch (st) has already started to accumulate.
chlorophyll biosynthesis after light exposure. Later, Masuda
et al. (1992, 1994, 1995) found in etiolated cucumber cotyledons that cytokinin increases ALA synthesis through stimulation of glutamyl-tRNA reductase activity and of the activity
of glutamyl-tRNA synthase or GSA-AT. The contribution of
cytokinin to regulating the expression and activities of the
genes and enzymes of the entire tetrapyrrole biosynthetic
pathway was investigated by Yaronskaya et al. (2006) in barley seedlings. Also in this case, glutamyl-tRNA reductase and
GSA-AT were elevated in dark-grown cytokinin-treated barley seedlings. However, the rate of ALA synthesis was only
significantly increased when plants were exposed to light.
The synthesis of protochlorophyllide and the photoreduction of protochlorophyllide a to chlorophyllide a by POR
enzymes (see Fig. 1) are also cytokinin-responsive. An early
report stated that cytokinin enhances protochlorophyllide
formation (Banerji and Laloraya, 1967), which has been connected to increased ALA synthesis (Lew and Tsuij, 1982).
Cytokinin also increases the levels of POR mRNA and
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the corresponding enzyme in etiolated cucumber seedlings
(Kuroda et al., 1996, 2001) and lupine (Kusnetsov et al.,
1998). The latter publication additionally demonstrated that
the light-sensitive POR enzyme is stabilized in the presence of
cytokinin. These results clearly indicate that cytokinin regulates POR activity.
The studies cited above mostly focused on the cytokinin
effect on pigment and protein levels and enzyme activities.
Later studies revealed that cytokinin alters transcript levels of
specific genes of the tetrapyrrole biosynthesis pathway during de-etiolation. Cytokinin increases the steady state mRNA
levels of the HEMA1, CHLH, and CHL27 genes in etiolated
Arabidopsis seedlings (Tanaka et al., 2011). The regulation
of these and additional chlorophyll synthesis genes (GSA1,
GUN4, CHLM) by cytokinin was shown to be reduced in ahk2
ahk3 cytokinin receptor mutants (Cortleven et al., unpublished). This coincided with a decreased greening rate and
lack of increased GluTR protein levels in response to cytokinin treatment, which indicated that the stimulating effect of
cytokinin on chlorophyll biosynthesis during de-etiolation is
mediated by the AHK2 and AHK3 receptors. Similar as for
the ahk2 ahk3 mutant, the cytokinin-dependent induction of
some chlorophyll biosynthesis genes was lowered in the arr1
arr12 mutant suggesting participation of ARR1 and ARR12
in regulating these genes (Cortleven et al., unpublished).
Most of the observations described above were made during de-etiolation, but it has been repeatedly demonstrated
that the endogenous cytokinin content of light-grown plants
is positively correlated with the chlorophyll content as well.
For example, in Pssu-ipt transgenic tobacco plants that
express the IPT gene of Agrobacterium tumefaciens, under
control of the light-inducible promoter of the small subunit of Rubisco of Pisum sativum, the chlorophyll content
was 50% higher as compared to wild type (Cortleven and
Valcke, 2012), while cytokinin-deficient 35S:CKX1 transgenic tobacco plants showed an ~35% reduction in chlorophyll content (Werner et al., 2008). In these 35S:CKX1
plants, the ALA synthesizing capacity was four times lower
as compared to wild type. A fully functional cytokinin signalling system is also important to reach a normal chlorophyll content. The chlorophyll content was reduced to 75%
of wild-type levels in ahk3 and cre1 ahk3 mutants and to
only about 60% in ahk2 ahk3 mutants (Riefler et al., 2006).
This indicates that the AHK3 receptor, in part together
with AHK2, plays a central role in mediating the effect of
cytokinin on regulating the chlorophyll content. Similarly,
in the triple B-type ARR mutant arr1 arr10 arr12 the chlorophyll content was reduced to almost 50% of wild-type
levels, suggesting that the corresponding transcription factors ARR1, ARR10, and ARR12 regulate the expression
of specific target genes required for optimal chlorophyll
biosynthesis (Argyros et al., 2008). It is important to note
that a basal level of chlorophyll is formed independent of
cytokinin because triple cytokinin receptor mutants are still
green (Higuchi et al., 2004; Nishimura et al., 2004; Riefler
et al., 2006), indicating that cytokinin is not absolutely necessary for chlorophyll biosynthesis but rather modulates its
concentration.
Cytokinin affects photosynthesis
The question arose whether cytokinin would also directly
influence the most relevant process taking place in chloroplasts, namely photosynthesis. In 1977, Buschmann
and Lichtenthaler concluded that cytokinin promotes the
light-induced formation of the electron transport chain in
Raphanus by increasing the Hill activity (oxygen evolution),
P700 concentration (reaction-centre chlorophyll of PSI), and
the level of plastoquinone (electron acceptor associated with
PSII). However, this was not confirmed in Sinapis (Zerbe and
Wild, 1980) where neither an effect on Hill activity nor on
the non-cyclic phosphorylation was observed after exogenous
application of cytokinin. Evaluation of photosynthetic activity in transgenic plants with elevated endogenous cytokinin
content showed no or an only slightly improved photosynthetic activity (Šiffel et al., 1992; Čatský et al., 1993). When
the endogenous cytokinin level exceeded a certain threshold,
a decline in several photosynthetic parameters even occurred
(Ondrej et al., 1990; Čatský et al., 1993). A more detailed
analysis of photosynthesis in Pssu-IPT transgenic tobacco
plants showed that the partial reactions of the electron transport chain were differently influenced by an elevated cytokinin level (Synková et al., 1999). The activity of the reaction
centre of PSII was almost not affected whereas PSI activity and the electron transport chain were inhibited by 70%.
Similar observations were made by Cortleven and Valcke
(2012), who investigated photosynthetic activity in tobacco
plants with increased or lowered endogenous cytokinin content. Cytokinin had no effect on the initial responses of the
photosynthetic apparatus on the dark-light transition but the
further kinetic behaviour was affected, which led to the suggestion that cytokinin can induce structural changes in different parts of the electron transport chain. Taken together,
it appears that cytokinin influences photosynthesis, but a
more profound analysis particularly of cytokinin mutants is
required to elucidate the specific changes that are caused.
A relevant role of cytokinin with respect to photosynthesis that is only rarely considered is its protective role for the
photosynthetic apparatus under light stress. It is known, for
example, that cytokinin prolongs antioxidant-based protection in chloroplasts resulting in an extension of their life span
(Procházková et al., 2008). A detailed study on the protective
function of cytokinin during high light stress reported that
plants with a lower cytokinin status show increased photoinhibition (Cortleven et al., 2014). This was caused by a higher
degree of photodamage, a partly impaired D1 repair cycle,
and reduced efficiency of the photoprotective scavenging system. The protective function of cytokinin during light stress
was dependent on the AHK2 and AHK3 receptors and the
transcription factors ARR1 and ARR12.
Cytokinin acts on plastids through the
regulation of gene expression
Part of the plastid responses to cytokinin are due to the
control of expression of nuclear genes encoding plastid or
plastid-related proteins; some of the transcription factors
Cytokinin function in chloroplasts | 5005
involved in this regulation have been identified. In addition,
cytokinin also regulates the expression of plastid genes, as
will be detailed below.
RBCS and CAB mRNA. This activity was confirmed by
work in lupine (Kusnetsov et al., 1994) but not addressed specifically in later research.
Cytokinin regulates nuclear genes encoding plastid
and plastid-related proteins
Specific transcription factors mediate cytokinin effects
on plastids
A large number of studies documenting the influence of
cytokinin on plastid-related nuclear genes were cited in an
earlier review describing cytokinin-regulated gene expression (Schmülling et al., 1997). For example, CAB, encoding chlorophyll a binding proteins of photosystem II, and
genes encoding both the small and large subunit of RubisCO
(RBCS/RBCL) were identified in several species (tobacco,
cucumber, Arabidopsis) to be strongly upregulated by cytokinin (e.g. Lerbs et al., 1984; Ohya and Suzuki, 1991; Chory
et al., 1994).
Genome-wide transcript analyses documenting the influence of cytokinin on gene expression, which have been performed primarily in Arabidopsis but also in Oryza sativa and
other species such as Medicago trunculata, Brassica oleracea,
and Populus (e.g. Hirose et al., 2007; Ramirez-Carvajal et al.,
2009; Liu et al., 2013), identified additional cytokinin-regulated chloroplast-related genes. For example, in Arabidopsis
>100 cytokinin-regulated genes related to photosynthesis were
identified, extending, among others, the list of the above-mentioned nuclear-encoded cytokinin-regulated chlorophyll biosynthesis and chloroplast genes (Brenner et al., 2005; Brenner
and Schmülling, 2012). One other example is the induction
of a gene encoding oligogalactosyldiacylglycerol synthase, an
enzyme involved in the synthesis of galactolipids, more specifically of monogalactosyldiacylglycerol and digalactosyldiacylglycerol, key components of the thylakoid membranes.
A connection between cytokinin and the formation of these
galactolipids had been made also by Yamaryo et al. (2003)
in cucumber. An interesting aspect is that cytokinin treatment shifts root gene expression towards a more shoot-like
pattern. Separate analysis of the transcriptomic responses of
roots and shoots to cytokinin revealed a particularly strong
induction of nuclear genes encoding chloroplast proteins in
the root. This was interpreted as a cytokinin-induced identity
shift from root to shoot prior to the occurrence of morphological changes (Brenner and Schmülling, 2012).
To identify a core set of cytokinin-responsive genes, metaanalyses of transcriptomic studies have been performed
(Brenner et al., 2012; Bhargava et al., 2013, Brenner and
Schmülling, 2015). Two transcription factor genes previously described as being involved in chloroplast development and division, CGA1 and CRF2 (Okazaki et al., 2009;
Chiang et al., 2012), have been listed among the top cytokinin-regulated genes (Brenner et al., 2012; Bhargava et al.,
2013). Both genes will be discussed in more detail in the next
section. Although the outcome of transcriptomic studies is
most often interpreted as reflecting the influence of cytokinin
on gene transcription, it could also be that the hormone has
posttranscriptional effects on the stability of mRNAs. This
has previously been suggested by Flores and Tobin (1986),
who showed that cytokinin slows down the degradation of
Which transcription factor(s) mediates cytokinin action
on plastids? The pale green phenotype of arr1 arr10 arr12
mutants (Argyros et al., 2008) suggests that the corresponding B-type response regulators operate downstream of AHK3
and AHK2 receptors to link their action to the regulation of
specific target genes. However, direct interaction between
these transcription factors and genes involved in chlorophyll
biosynthesis remains to be shown. Several additional transcription factors that could mediate cytokinin action on chloroplast development have been identified as will be described
in the following sections. An overview of transcription factors acting downstream of the cytokinin signalling pathway
can be found in Fig. 3.
Two transcription factor genes linking cytokinin and plastid development are cytokinin-responsive GATA factor 1
[CGA1, also known as GNL (GNC-like) or GATA22], and its
paralog GNC (also known as GATA, nitrate-inducible, carbon metabolism involved, or GATA21) (Naito et al., 2007).
They belong to the 29 GATA transcription factors, plant type
IV zinc finger proteins, encoded by the Arabidopsis genome
(Riechmann et al., 2000). Both genes are the sole members in
an expression clade showing the strongest difference among
the GATA factors between light- and dark-grown seedlings
(Manfield et al., 2007). CGA1 is rapidly induced by cytokinin in an AHK2/AHK3-dependent manner, whereas GNC
appears to be much less induced (Naito et al., 2007; Chiang
et al., 2012). Cytokinin-treated phyA phyB mutants showed
an induction of CGA1, indicating that red light and cytokinin act in parallel to regulate its expression. CGA1 expression strongly correlates with the expression of genes involved
in light responses, including HYH and HY5 (Monte et al.,
2004). Both cga1 and gnc mutants have defects in greening (Bi
et al., 2005; Richter et al., 2010) but only the single gnc and
double gnc cga1 mutant have strongly reduced chlorophyll
levels in the shoots, indicating that GNC plays a dominant
role in regulating chlorophyll biosynthesis (Chiang et al.,
2012). Overexpression of GNC and CGA1 promotes the differentiation of proplastids to etioplasts in the dark and to
chloroplasts in the light (Chiang et al., 2012) and the ectopic
production of chloroplasts in roots (Richter et al., 2010;
Köllmer et al., 2011; Chiang et al., 2012). Altering CGA1 and
GNC expression modulates the expression of important chlorophyll biosynthesis genes, including GUN4 and HEMA1
(Hudson et al., 2011), indicating that these transcription factors might be responsible for the cytokinin-dependent regulation of these genes (Cortleven et al., unpublished). In sum,
GNC and CGA1 appear to be key regulators of chloroplast
development, integrating both light and cytokinin pathways. Lastly, CGA1 and GNC transcription factors are also
involved in gibberellin (GA) and auxin signalling (Richter
et al., 2010; Richter et al., 2013) and could form a point of
5006 | Cortleven and Schmülling
convergence between cytokinin and these other hormones
during de-etiolation as is discussed below.
Another family of transcription factors that is a positive
regulator of chloroplast development and co-regulate the
expression of nuclear photosynthetic and chlorophyll biosynthesis genes is GOLDEN2-LIKE (GLK) (Fitter et al., 2002;
Waters et al., 2009). glk1 glk2 Arabidopsis double mutants are
pale green and contain small chloroplasts with sparse thylakoid membranes unable to form grana. GLK2 expression is
strongly induced by cytokinin in roots and inhibited by auxin.
Overexpression of GLK2 results, just like CGA1 and GNC
overexpression, in root greening (Kobayashi et al., 2012).
The same study revealed that the combination of HY5 and
GLKs, functioning downstream of light and auxin/cytokinin
signalling pathways, is responsible for coordinated expression
of key genes in chloroplast biogenesis in the root. However,
while there is an absolute requirement for HY5 to respond
to cytokinin it was apparent that GLK1 and GLK2 are not
essential transcription factors for cytokinin-dependent root
greening and that the hormone can regulate chlorophyll biosynthesis in the root in a manner at least partially independent of GLKs (Kobayashi et al., 2012).
CRF2, a member of the cytokinin response factor family,
which belongs to the APETALA2/ETHYLENE RESPONSE
FACTOR (AP2/ERF) class of transcription factors (Rashotte
et al., 2006), has been implicated in controlling chloroplast division. Overexpression of CRF2 or cytokinin treatment causes
an increased level of PDV2 protein and increased chloroplast
division (Okazaki et al., 2009). PDV2 is a nuclear-encoded
protein that is required for plastid division and determines,
together with its paralog PDV1, the rate of plastid division
(Okazaki et al., 2009). The results suggest the possibility that
cytokinin regulates chloroplast division by acting through
CRF2 and subsequent induction of PDV2 expression.
Cytokinin regulates the expression of plastid genes
The expression of plastid genes is influenced by cytokinin
and an early report noted a long-term influence of the hormone on steady-state mRNA levels of plastid genes (Lerbs
et al., 1984). In their Arabidopsis microarray study, Brenner
et al. (2005) identified seven plastid genes rapidly (15–
120 min) induced by cytokinin (PETA, PSBG, YCF10, YCF5,
MATK, PSBA, and PSBI), coding for different functions
that included carbon uptake into chloroplasts, RNA splicing,
and photosystem II components. Zubo et al. (2008, 2009)
used a run-on transcription assay to explore the influence of
cytokinin on chloroplast transcription in barley leaves. The
transcription of a number of chloroplast genes (e.g. PETD,
ATPA, RRN16) was highly responsive to cytokinin treatment
in an age- and light-dependent manner, while others were
not responsive (e.g. PSBA). At first glance this is a surprising result because cytokinin signalling is not known to occur
in chloroplasts. To explain the regulation of plastid genes
by cytokinin, the involvement of sigma factors, which are
nuclear-encoded and fine-tune plastid RNA polymerases, has
been investigated. It was shown that the induction of chloroplast genes by cytokinin was dependent on specific sigma
factors (SIG1, SIG2, and SIG6), because the corresponding
single mutants displayed a reduced response of chloroplast
genes to cytokinin (Borsellino, 2011). In addition, these sigma
factors, and especially SIG2, are linked to chlorophyll biosynthesis (Kanamaru and Tanaka, 2004). However, there is as yet
no direct proof that regulation of sigma factors is responsible
for the cytokinin response of chloroplast transcription.
Cytokinin acts on plastids through changes
in the proteome
To date, few studies have investigated the effect of cytokinin
on the plants’ proteome. Lochmanová et al. (2008) studied the
proteomic changes during cytokinin-induced photomorphogenesis in dark-grown Arabidopsis. They reported that even
a modest increase in the endogenous cytokinin content (<2fold) resulted in the upregulation of 37 proteins identified by
MALDI-TOF MS and/or LC-MS/MS. Chloroplast proteins
involved in photosynthetic processes or chloroplast biogenesis
represented a major fraction (26%) of the cytokinin-induced
proteins. These proteins included the gamma-subunit of ATP
synthase, glyceraldehyde-3-phosphate dehydrogenase, ATPdependent Clp proteolytic subunit, and ribosomal protein
L21.There was only a small overlap with those proteins identified previously to be induced during photomorphogenesis.
Černý et al. (2011) described a rapid cytokinin impact
on the proteome and the phosphoproteome in 7-day-old
Arabidopsis seedlings. Only 15 min after cytokinin treatment,
significant changes were seen in 53 spots of the proteome
and 31 spots of the phosphoproteome. These changes were
mainly in the range of 1.3- to 2-fold, which is much smaller
than those seen on the transcript level, but they were consistently obtained with different cytokinins and a large part
was absent or reduced in cytokinin receptor mutants. The
highest number of phosphoproteins was regulated by AHK3,
followed by CRE1/AHK4 and AHK2. A remarkably high
percentage (45%) of the differently expressed proteins and
phosphoproteins are involved in various processes of chloroplast biogenesis and function. The authors proposed that
phosphorylation induced by cytokinin might mainly regulate
protein–protein and protein–ligand/substrate interactions. As
yet, it is unclear by which mechanism the abundance or phosphorylation state of a large number of specific chloroplastrelated proteins is rapidly changed by cytokinin. Elucidating
the functional relevance of these changes will be an excellent
contribution to understanding cytokinin action in plastids.
Cortleven et al. (2011) studied the effects of constitutively
altered endogenous cytokinin content on the proteome of
two chloroplast sub-fractions (stroma and thylakoids) in
tobacco. Few quantitative differences in protein abundance
were found in the stroma fraction and concerned proteins of
the Calvin-Benson cycle and photoprotective mechanisms.
In agreement with Lochmanova et al. (2008), the level of an
ATP-dependent Clp protease was identified to be regulated
by cytokinin. However, the low overall number of changes
contrasts with the numerous and rapid changes found shortly
after cytokinin application (Lochmanova et al. 2008; Černý
Cytokinin function in chloroplasts | 5007
et al., 2011) and indicates that plastids are able to adapt to an
altered cytokinin status.
Recently, Zd’árská et al. (2012) analysed the proteome of
A. thaliana under the same settings as in the transcriptome
analysis by Brenner et al. (2005). They found only limited
overlap of regulated genes and proteins, which might be due
in part to the delay caused by translation of target genes, the
limited coverage of proteomic analysis, and/or to a more relevant action of cytokinin on the post-transcriptional level for
some genes. Overall the abundance of only a few chloroplastrelated proteins was found to be cytokinin-regulated, among
them RubisCO and two enzymes involved in chlorophyll biosynthesis (Mg-chelatase and GSA-AT).
Together, the few studies reporting the effect of cytokinin
on the plant’s proteome has given valuable hints about the
functioning of cytokinin. An important outcome is that the
proportion of the rapidly cytokinin-induced changes in the
plastid-related proteome appears in most studies to be larger
than the changes noted in the plastid-related transcriptome.
Thus, post-transcription and/or post-translation regulatory
mechanisms might be more relevant than mechanisms acting
on transcription.
Crosstalk between cytokinin and light
Light and cytokinin cause in part similar effects on plant
growth and development. Miller (1956) showed that, similar to red light, cytokinin induces expansion of leaf discs
and elongation of epicotyl segments in etiolated Phaseolus
vulgaris seedlings, and induces germination in light-sensitive Lactuca sativa seeds. Particularly well studied is the
interaction between cytokinin and light during de-etiolation, which includes the development of chloroplasts from
etioplasts. Phytochromes and cryptochromes, photoreceptors that detect red/far-red and blue light respectively,
mediate the effect of light in this process. A series of genetic
screens to uncover regulators of light-dependent seedling
development revealed constitutively photomorphogenic
(cop), de-etiolated (det), and fusca (fus) mutants that all
resemble light-grown seedlings when grown in darkness
(for review: Kim et al., 2002). The COP/DET/FUS proteins are central repressors of photomorphogenesis, and
members of the ubiquitin system that targets photomorphogenesis-promoting transcription factors such as HY5
for degradation in the dark. Light signals mediated by the
above-mentioned photoreceptors repress the activity of
the COP/DET/FUS complex, leading to the accumulation
of said transcription factors and thus promoting photomorphogenesis. Besides the COP/DET/FUS proteins, a
group of basic helix-loop-helix transcription factors called
phytochrome interacting factors (PIFs) are also essential
for the repression of photomorphogenesis in darkness (for
review: Leivar and Monte, 2014).
The phenotype of Arabidopsis seedlings germinated in
the dark on cytokinin-containing medium resembled the
phenotype of cop/det mutants and indicated that cytokinin
promotes photomorphogenesis, even in the absence of light
(Chory et al., 1994). Later it was shown that this activity
depends on the AHK2 and AHK3 receptors (Riefler et al.,
2006). A mutant with increased endogenous cytokinin levels, amp1, also shows a de-etiolated phenotype (Chin-Atkins
et al., 1996) lending support for a role of the hormone in
de-etiolation. Further, the cin4/cop10 mutation renders
Arabidopsis insensitive to the induction of the ethylenemediated triple response by cytokinin in the dark (Vogel
et al., 1998). This links cytokinin action to light signalling
because COP10 has a central role in repressing photomorphogenesis (Lau and Deng, 2012), although it is currently
not known whether COP10 is a target of cytokinin to promote de-etiolation. Together, these reports all support a
role for cytokinin as a positive regulator of de-etiolation.
However, it should be noted that in studies on de-etiolation,
which focus mainly on hypocotyl elongation, chloroplast
development has often not been specifically addressed.
Therefore, it is not guaranteed that the results on crosstalk
between cytokinin and other effectors during the de-etiolation process also apply entirely for their action on chloroplast development.
Vandenbussche and co-workers (2007) revealed that the
interaction between light and cytokinin is mediated by
common signalling intermediates. HY5, a bZIP transcription factor and positive regulator of photomorphogenesis
that acts downstream of cryptochrome and phytochrome,
has been described as a point of convergence between
blue light-dependent cryptochrome and cytokinin signalling, both triggering anthocyanin accumulation. Mutation
of HY5 caused a reduced response in blue light to cytokinin, similar to mutation of CRY1. Moreover, cytokinin
increased HY5 stability similar to blue light. Since no stimulation of anthocyanin accumulation by either blue light or
cytokinin was observed in cop1-5 mutants, it was concluded
that both blue light and cytokinin act together to prevent
degradation of HY5 by COP1. However, it should be noted
that cytokinin and blue light act through separate pathways
on hypocotyl elongation and that development of chloroplasts as part of photomorphogenesis was not specifically
addressed in this study.
Other mechanisms of cytokinin-light crosstalk have
been suggested. Kusnetsov et al. (1999) demonstrated that
the expression of ATPC, encoding the gamma-subunit of
the chloroplast ATP synthase, is stimulated by both cytokinin and light via the same CAAT-box cis-acting element
located in the promoter of ATPC. Examining the binding of CAAT-box binding factor to this CAAT-box, they
could not distinguish between a common or independent
signalling pathway of light and cytokinin. Yet another
mechanism for crosstalk was deduced from the finding that
ARR4, a type-A ARR, interacts with the N-terminus of the
red light photoreceptor PhyB (Sweere et al., 2001) and thus
stabilizes the Pfr-form of PhyB. Overexpression of ARR4
in Arabidopsis causes increased sensitivity of hypocotyl
growth to red light. Mira-Rodado et al. (2007) showed that
ARR4 activity depends on the phosphorylation of a specific Asp-residue within ARR4. These findings support the
idea that ARR4 acts as a direct molecular switch integrating red light and cytokinin signals.
5008 | Cortleven and Schmülling
Crosstalk of cytokinin with other plant
hormones in photomorphogenesis
The involvement of other hormones in regulating chloroplast
development is less well documented than it is for cytokinin.
Early reports with exogenous application of other phytohormones often did not unambiguously support a direct connection to chloroplast development. However, in recent years,
more reports appeared pointing to a role for abscisic acid
(ABA), GA, auxin, and ethylene during photomorphogenesis and greening. The action of these hormones and their
crosstalk with cytokinin will be addressed in the following
paragraphs. An overview is given in Fig. 4. Please note that,
similar to the interaction between light and cytokinin, chloroplast development and function has not been addressed specifically in several of the studies reported below.
Abscisic acid
An inhibitory effect of ABA on plastid biogenesis during deetiolation has been known for many years (Khokhlova et al.,
1978). Several points of convergence for the antagonistic
actions of ABA and cytokinin in controlling greening and
thus chloroplast development are known. One is the opposite
action of both hormones on regulating the amount of POR
enzyme. While cytokinin-treated lupine cotyledons green
faster with a higher level and slower degradation of the lightsensitive POR enzyme, ABA treatment has the opposite effect
(Kusnetsov et al., 1998). The inhibitory effect of ABA on
greening is mediated by ABSCISIC ACID INSENSITIVE5
(ABI5), a bZIP transcription factor (Finkelstein and Lynch,
2000; Lopez-Molina and Chua, 2000) which is itself possibly
regulated by HY5 (Chen and Xiong, 2008). Cytokinin antagonizes the ABA-mediated inhibition of cotyledon greening by
inducing the degradation of ABI5 (Guan at al., 2014) (Fig. 4).
This action is dependent on several proteins of the cytokinin
signalling pathway, namely the receptor CRE1/AHK4; the
phosphotransmitter proteins AHP2, AHP3, and AHP5; and
the B-type transcription factor ARR12 (Guan at al., 2014).
Gibberellins
GA inhibits photomorphogenesis in the dark through
DELLA proteins, which act as repressors of GA signalling
by blocking different transcription factors, including the PIFs
that are repressors of photomorphogenesis (de Lucas et al.,
2008; Feng et al., 2008). In light, GA levels are reduced by the
catabolic enzyme GA2ox2 (Ait-Ali et al., 1999), thus promoting DELLA accumulation and thereby abrogating negative
control by DELLA targets such as PIFs. The expression of
the GA2ox2 gene has been shown in Pisum sativum to be regulated by LONG1, an ortholog of HY5 (Weller et al., 2009).
This indicates that, similar to ABA, HY5 also has a central
role in mediating GA hormonal activity (Fig. 4). In addition,
GA reduces HY5 stability and, accordingly, mutants defective
in GA production and response have a photomorphogenic
development corresponding to high HY5 levels (Alabadi
et al., 2008). Recently, it was shown that GNC and CGA1 (see
above) are important targets downstream of GA, DELLA,
and PIFs, regulating germination, greening, flowering, and
hypocotyl elongation (Richter et al., 2010). This suggests that
these transcription factors are another point of convergence
for GA and cytokinin signalling.
Auxin
Auxin is involved in regulating almost all aspects of plant growth
and development and it also has a role in photomorphogenesis,
CK(4)
SAM
ACC
ARR1
(1)
AHK4
CK(2,3)
AHK4
light
AHP2/3/5
ethylene
AHK2
AHK3
COP
ABA
ARR12
ETR
GA
CTR
EIN2
DELLA
EIN3/EIL1
PIFs
GA2ox2
HY5
ABI5
IAA
Photomorphogenesis
IAA14/SLR
AXR2/IAA7
Auxin
response
genes
Pchlide
GNC CGA1
ARF7/
ARF19
ARF2
B-type ARRs
AHP
AHK
CK(5)
Fig. 4. Crosstalk of cytokinin with other hormonal signalling pathways during photomorphogenesis. Cytokinin (CK) action is highlighted in red. Positive
and negative interactions are represented by arrows or inhibition lines, respectively. Numbers refer to the following studies: (1) Hansen et al. (2009); (2)
Vandenbussche et al. (2007); (3) Riefler et al. (2006); (4) Guan et al. (2014); (5) Chiang et al. (2012). For further information, see text. IAA, auxin; SAM,
S-adenosyl-1-methionine; Pchlide, protochlorophyllide (this figure is available in colour at JXB online).
Cytokinin function in chloroplasts | 5009
as is suggested by the promotion of dark-induced hypocotyl elongation in auxin-response mutants. Numerous auxin
responses are mediated by auxin response factors (ARFs),
which bind to promoters of auxin responsive genes to regulate
transcription, and auxin/indole-3-acetic acid inducible (Aux/
IAA) proteins that repress ARF function. The suppressor of
hy2 (shy) mutant is defective in a member of the auxin-induced
Aux/IAA family and shows a partially de-etiolated phenotype
in darkness (Kim et al., 1996). HY5 and its homolog HYH
play a central role in suppressing auxin signalling (Cluis et al.,
2004; Sibout et al., 2006), linking auxin action to the cytokinin
signalling pathway in regulating photomorphogenesis (Fig. 4).
HY5 suppresses auxin signalling through the activation of
AUXIN RESISTANT2 (AXR2)/IAA7 and SOLITARY ROOT
(SLR)/IAA14 genes (Cluis et al., 2004).
Auxin derived from the shoot negatively regulates
root greening through SLR/IAA14, ARF7, and ARF19
(Kobayashi et al., 2012). Richter et al. (2013) noted that mutations of the auxin repressors ARF2 and SLR/IAA14 cause
some phenotypes similar to the ones of GNC and CGA1 overexpressors, including root greening. They revealed that ARF2
can bind to the promoters of GNC and CGA1 genes, resulting
in their repression, which is abolished in arf2. Because root
greening can also be achieved by cytokinin presumably acting
through GNC and CGA1 (Köllmer et al., 2011; Kobayashi
et al., 2012), it could be that GNC and CGA1 integrate auxin
and cytokinin signals in the regulation of root greening.
Notably, ARF2 and SLR/IAA14 are putative targets of HY5
(Cluis et al., 2004), presumably placing the auxin/cytokinin
crosstalk downstream of HY5.
Ethylene
Ethylene induces the triple response in dark-grown seedlings
and has been reported to enhance seedling development and
cotyledon greening under high salinity or excess glucose
(Zhou et al., 1998; Cao et al., 2007). A recent study revealed
that ethylene represses dark-induced protochlorophyllide
accumulation and that EIN3/EIL1, a transcriptional activator initiating transcriptional reprogramming in response to
ethylene, can directly bind to specific response elements present in PORA/PORB promoters, causing induction of POR
gene expression. It has been suggested that ethylene signalling
via EIN3/EIL1 presents a new pathway to decrease the accumulation of protochlorophyllide in darkness and to facilitate
protochlorophyllide reduction to synthesize chlorophyll upon
light exposure (Zhong et al., 2009; 2010) in cooperation with
PIF1. In addition, PIF3 is involved in mediating this ethylene-induced protochlorophyllide suppression by EIN3/EIL1
(Zhong et al., 2014). Besides this, PIF3 is directly activated by
EIN3/EIL1 and is responsible for both the ethylene-induced
reduction and promotion of hypocotyl elongation in the dark
and the light, respectively, owing to an interaction with ERF1
(Zhong et al., 2012).
Ethylene is derived from methionine and is produced in
three steps involving S-adenylmethionine synthetase, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, and
ACC oxidase (Yang and Hoffman, 1984). Cytokinin induces
ethylene biosynthesis in etiolated seedlings (Woeste et al.,
1999) by increasing the stability of ACC synthase (Hansen
et al., 2009). In addition, these authors showed that CRE1/
AHK4 and B-type ARR1 are necessary for cytokinin-induced
ethylene production. It could be that cytokinin achieves part
of its influence on photomorphogenesis by stimulating the
ethylene pathway (Fig. 4).
Conclusions
In this review, we have given a comprehensive overview of
the findings connecting cytokinin with chloroplast development and function. Knowledge derived from earlier gain-offunction studies has been largely confirmed and extended by
work done with plants displaying a lower cytokinin status.
Key steps in chlorophyll biosynthesis regulated by cytokinin
are ALA synthesis and the photoreduction of protochlorophyllide. However, cytokinin is not absolutely required but
rather a modulator of chlorophyll synthesis and chloroplast
function. The molecular mechanisms underlying its function
are only partly known and numerous details are still missing. However, a number of transcriptional regulators mediating cytokinin action on chloroplasts have been identified;
refined analysis of transcriptomic data and more targeted
genetic approaches will certainly contribute in the future to
a more detailed understanding of this long-known cytokinin
function.
Acknowledgements
This work was supported by a grant of the Deutsche Forschungsgemeinschaft
to TS. Special thanks to Beatrix Fauler and Thorsten Mielke (MPI of
Molecular Genetics, Berlin) for technical support with electron microscopy.
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