Charles University in Prague, Faculty of Science
Department of Biochemistry
Summary of the Ph.D. Thesis
Analyzing the complexity of auxin-related processes
and their regulation
Sibu Simon
Supervisor: Assoc. Prof. RNDr. Eva Zažímalová, Ph.D.
Co-Supervisor: Jan Petrásek, Ph.D.
Supervisor
Assoc. Prof. RNDr. Eva Zažímalová, Ph.D.
Institute of Experimental Botany ASCR, Prague, Czech republic
Department of Experimental Plant Biology, Faculty of Science, Charles University
in Prague
Czech Republic
Co-supervisor
Jan Petrásek, Ph.D.
Institute of Experimental Botany ASCR, Prague, Czech republic
Department of Experimental Plant Biology, Faculty of Science, Charles University
in Prague
Czech Republic
1 Introduction
1
1.1 Phytohormones
1
1.2 Auxin
2
1.2.1 Auxin in plant development
2
1.2.2 Auxin transport
3
1.2.3 Auxin signal transduction
4
1.2.4 Chemical biology in auxin research
6
2. Aim of the study
7
3. Results and discussion
7
3.1 Charecterization of different auxin analogues and using them in various
auxin-related processes
7
3.2 5-F-IAA as a tool to study inhibition of endocytosis by auxin
10
3.3 Effect of FM dyes on transient re-localization of memebrane proteins
10
3.4 Auxin-cytokinin cross talk in root meristem activity
11
3.5 Endogenous auxins: A collective and detailed review focusing on endogenous
Auxins
11
4 Conclusions
5 References
6 Curriculam Vitae
12
13
20
1 Introduction
Hormones are modulators of biological activity and play a crucial role in growth and
development of all multi cellular organisms. Hormones are generally classified based on their
structure and biological action and in animals they are peptide hormones, steroid hormones and
amine-derived hormones. Cells respond to a hormone when the corresponding receptor of
hormone is binding the hormone and formation of the hormone-receptor complex initiates the
specific signaling cascade. In animals dedicated tissue called ‘glands’ are responsible for the
production of hormones and they are transported to its region of action through blood stream.
Plant hormones are often called as phytohormones and sometimes as plant growth substances or
plant growth regulators. They differ significantly from classical animal hormones in various
ways such as 1) Plants produce very few hormones compared to animals. 2) Mostly two or more
plant hormones work together for the regulation of particular biological effect and their function
- in contrast to animals - is not so strictly defined. Plant hormones function is usually pleiotropic.
3) In plants all meristematic cells are highly active in production of hormones and also most of
the plant cells can produce certain level of any plant hormone, while in animals it is the duty of
the ‘glands’.
1.1 Phytohormones
Because of the sessile life style and autotrophic nature, plants demand special adaptation
mechanism to respond to various environmental stress/developmental stimulations. These
adaptation mechanisms are mainly mediated by endogenous signaling molecules such as
phytohormones which regulate various physiological processes in plants. Phytohormones consist
of various classes of molecules, from the first identified auxin to recently identified
strigolactones (Davies, 2004; Gomez-Roldan et al., 2008; Umehara et al., 2008). Mostly,
different phytohormones have functional distinctions; in general, auxin, cytokinins (CK),
gibberellins (GA), brassinosteroids (BR) and ethylene mainly have their role predominantly in
plant growth and developmental events, whereas abscisic acid and jasmonic acid act as the
abiotic and biotic stress response molecules. In addition to them, recently found strigolactones
function as a branching inhibitor to maintain plant apical dominance (Davies, 2004; GomezRoldan et al., 2008; Umehara et al., 2008).
1
1.2 Auxin
In 1880, Darwins discovered the existence of movable cue, which was identified as a
transporting signal molecule which responded to light (Darwin and Darwin, 1880). This concept
was experimentally proved by Frits Went (Went, 1928) and the substance was identified as
indole-3-acetic acid (IAA) (Kogl et al., 1934). IAA is the major endogenous auxin in plants apart
from few others like 4-chloroindole-3-acetic acid (4-Cl-IAA), phenyl acetic acid (PAA), indole3-butyric acid (IBA) There are few chemically more stable synthetic auxins such as 2,4dichlorophenoxy acetic acid (2,4-D) and naphthalene-1-acetic acid (NAA) that are widely used
in agriculture and micropropagation techniques (Thimann, 1977). Auxin proved to have very
important role in regulating various plant growth and developmental processes. Last few decades
witnessed very intensive research aiming to elucidate the complexity of auxin-related processes,
and more information was added to elucidate the complex regulation of auxin-mediated plant
growth and development.
1.2.1 Auxin in plant development
Auxin plays a major role in almost every aspect of plant growth and development. Its
span of actions ranges throughout the life of a plant. The apical and basal polarity in plants are
triggered during embryogenesis and auxin is involved in specifying the root meristem founder
cell called hypophysis and maintains root meristem (Friml et al., 2003; Aida et al., 2004; Blilou
et al., 2005; Weijers et al., 2006). It also maintains the shoot apical meristem size and function in
co-operation with another plant hormone cytokinin (Zhao et al., 2010). It has very important role
in initiation of different organs and their growth (Laskowski et al., 1995; Reinhardt et al., 2000,
2003; Benková et al., 2003; Heisler et al., 2005). It is involved in vascular tissue formation
(Sachs, 1991; Mattsson et al., 1999), regulating the tropic response of plants towards light
(phototrophism) and gravity (gravitropism) (Li et al., 1991; Friml et al., 2002; Swarup et al.,
2005; Esmon et al., 2006), controlling the apical dominance and apical hook formation in
Arabidopsis (Leyser, 2005; Zádníková et al., 2010; Vandenbussche et al., 2010) etc. These are
some of the plant growth and developmental functions of auxin and the action spectrum of auxin
actions is still expanding.
2
1.2.2 Auxin transport
In animals ‘morphogens’ form a group of signaling molecules which regulate body
patterning of multi-cellular organisms. In general morphogen forms a concentration gradient at
its point of action and it acts in a concentration-dependent manner directly in a target cell
(Bhalerao and Bennett, 2003; Benková et al., 2009). In plants, from the available knowledge,
plant hormone auxin is the only molecule which at least partially fits into the term of
‘morphogen’. There are many studies that suggest the localized concentration maxima are
necessary for the initiation of many plant developmental events such as wood formation (Uggla
et al., 1996) and organ formation (Benková et al., 2003) etc. These developmentally important
auxin gradients are formed mainly by the machinery of polar auxin transport, which consists of
different class of auxin transport proteins.
Polar auxin transport is based on the chemiosmotic polar diffusion model which was
suggested by Raven (1974) and Rubery and Sheldrake (1974) and generalized by Mary Helen
Goldsmith in 1977. According to this model, non-dissociated lipophilic form of IAA can enter
cells from slightly acidic extracellular space (pH 5.5) through passive diffusion, and inside the
cell, majority of IAA molecules dissociate to form hydrophilic anion (IAA-) because of
cytoplasmic neutral environment (pH 7). This dissociated auxin molecule (auxin anion) requires
the assistance of carrier proteins to be exported out from the cell. In extracellular space auxin
exists in both dissociated and non-dissociated (lipophilic) forms and dissociated form enters the
cell through auxin influx carriers. Thus, the polar auxin transport machinery consists of both
influx and efflux carriers which have functionally opposite roles.
The first auxin influx carrier was discovered based on a mutant screen where the mutant
showed agravitropic phenotype. The auxin resistant mutant 1 (aux1) mutant was resistant
towards 2,4-D which was considered as a substrate for the active influx, whereas it was sensitive
to membrane permeable auxin NAA (Maher and Martindale, 1980; Yamamoto and Yamamoto,
1998). This led to the discovery of AUX1 as the auxin influx carrier. The AUX1 protein is
similar to amino acid permeases which form a group of proton-gradient-driven secondary
transporters (Bennett et al., 1996). Later three other influx carriers called LIKE-AUX1 (LAX1-3)
were also identified (Parry et al., 2001). The role of influx carriers in plant development will be
discussed later in this thesis.
3
Intensive mutant screening for the purpose of identifying potent auxin efflux carrier(s) led
to the identification of pin-formed 1 (pin1) which has a needle like inflorescence stem and lack
of flowers, an almost identical phenotype observed upon chemical inhibition of auxin transport
(Okada et al., 1991). PIN1 encodes a transmembrane protein with two transmembrane regions
separated by a hydrophilic loop (Galweiler et al., 1998). Further investigations identified that
PIN is a gene family consisting of eight members in Arabidopsis (PIN1-8). The PIN1, PIN2,
PIN3, PIN4 and PIN7 proteins have longer hydrophilic loop and they predominantly localize to
plasma membrane. In contrast, the PIN5, PIN6 and PIN8 have much shorter hydrophilic loop
and they localize to endomembranes and or on endoplasmic reticulum (ER) (Vieten et al., 2005;
Petrášek et al., 2006; Zazímalová et al., 2007; Mravec et al., 2009; Petrášek and Friml, 2009;
Křeček et al. 2009; Zazímalová et al., 2010; Grunewald and Friml, 2010). The PIN proteins also
have their distinct tissue localization in various organs of plants. Another class of proteins which
are responsible for auxin transport are phospho-glycoproteins (PGPs) forming the ABCB subgroup
of
the
ATP-binding
cassette
(ABC)
transporters
of
the
multidrug
resistance/phosphoglycoproteins (ABCB/MDR/PGP) (Noh et al., 2001). The best characterized
transporters in this class are ABCB1, ABCB4 and ABCB19 carriers in Arabidopsis. ABCBs are
considered to be involved in mediating long distance auxin transport (Noh et al., 2001). ABCB1
and ABCB19 are structurally very similar and their auxin efflux function is very well
documented (Titapiwatanakun and Murphy, 2009). ABCB4 appears to behave differently than
other two in a way that it may function as both auxin influx and efflux carrier (Terasaka et al.,
2005; Cho et al., 2007). Another subgroup of ABC transporters, namely, PLEOTROPIC DRUG
RESISTANCE/ABCG (PDR/ABCG) are also reported to be active in auxin transport. So far
only two members of this protein class are characterized as auxin transporters. ABCG37/PDR9 is
reported to be a 2,4-D effluxer as well as transporter for the endogenous auxin IBA (Ito and
Gray, 2006; Strader et al., 2008; Ruzicka et al., 2010). Another member of this subfamily,
ABCG36/PDR8 is also involved in IBA efflux (Strader and Bartel, 2010).
1.2.3 Auxin signal transduction
Last few decades explored very crucial components of auxin signaling. Identification of
Transport Inhibitor Response 1 (TIR1) as auxin receptor was an important discovery in auxin
signaling field (Dharmasiri et al., 2005a; Kepinski and Leyser, 2005). This F-box protein is a
4
subunit of S-phase kinase associated protein 1//Cullin/F-box (SCF) ubiquitin ligase (E3)
complex (Dharmasiri and Estelle, 2002). This ‘genomic’ auxin signaling is then regulated by two
families of transcription factors; the auxin/indole-3-acetic acid (Aux/IAA) and the auxin
response factors (ARFs). Auxin responsive gene’s promoter region carries the auxin responsive
element (ARE) which is the binding site for ARFs and this binding either activates or inhibits
transcription of the gene depending on the type of particular ARF (Guilfoyle and Hagen, 2007).
These ARFs are under the repression by Aux/IAA repressors through their common domain III
and IV thus repressing the auxin-regulated transcription. In presence of auxin TIR1 auxin
receptor binds auxin directly and this binding favors the interaction of TIR1 with Aux/IAA
repressors. Structural studies of the TIR1 protein revealed a hydrophobic pocket on the surface
of the leucine-rich repeat domain of TIR1. Auxin binds to the base of this pocket forming
‘molecular glue’ between TIR1 and Aux/IAA repressor (Tan et al., 2007). Interaction of TIR1
and Aux/IAA repressor in presence of auxin promotes the ubiquitination and then degradation of
Aux/IAA repressors and so it releases ARFs from repression to act on auxin response gene
promoter and regulate its expression (Mockaitis and Estelle, 2008). In addition to TIR1,
Arabidopsis genome contains five more F-box proteins (AFB1-5) which have varying degree of
sequence similarity and most of them were also identified as native auxin receptors (Dharmasiri
et al., 2005b; Walsh et al., 2006; Parry et al., 2009). AFB5 is an exclusive receptor for synthetic
auxin picloram (Walsh et al., 2006) and a very recent study reported the role of the AFB4 auxin
receptor as a negative regulator of auxin signaling (Greenham et al., 2011).
ABP1 has been identified much earlier than any other auxin receptor by analyzing its
ability to bind radiolabelled auxin (Hertel et al., 1972). This protein is a small glycoprotein,
major portion of its molecules resides in ER and a small portion is distributed on the plasma
membrane (Napier et al., 2002). The ABP1-mediated auxin signaling seems to play a role mainly
in performing fast auxin responses where immediate gene induction is not necessary. One of the
best characterized fast auxin responses involving ABP1 was the activation of H+-ATPase and the
inward flow of K+ ions resulting in cell expansion (Hager et al., 1991). In addition to this there
are many more plant developmental events where ABP1 has been reported to play a significant
role, such as cell division, vesicle trafficking, induction of early auxin-regulated genes,
Arabidopsis root growth etc. (Tromas et al., 2009; Tromas et al., 2010; Effendi et al., 2011).
5
1.2.4 Chemical biology in auxin research
The complexity of auxin biology is studied, among other things, also by the use of
different synthetic chemicals which are structurally analogous to endogenous plant compounds.
Chemically more stable synthetic auxins 2,4-D and NAA are used widely as exogenous auxin to
study various auxin-related processes. Using analogous structures helps to explore important
information about various aspects of auxin behavior in detail. One of the best examples depicting
this kind of study is using 2,4-D as a marker for auxin influx carriers activity whereas NAA can
serve as a marker for auxin efflux carrier activity (Delbarre et al., 1996). This differential
property of these compounds helps to study auxin influx and efflux part of the active auxin
transport separately. There are more chemicals used to study the auxin transport machinery, such
as 1-naphthylphthalmic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA) which have been used
widely as inhibitors of carrier-mediated auxin efflux (Katekar and Geissler, 1980; Morris, 2000).
There are several inhibitors that are in use to characterize also the auxin influx machinery, such
as 1-naphthoxyacetic acid (1-NOA), 2-naphthoxyacetic acid (2-NOA) and 3-chloro-4hydroxyphenylacetic acid (CHPAA) (Parry et al., 2001; Lankova et al., 2010). Another auxin
transport inhibitor identified was gravacin, reported to inhibit the ABCB-mediated auxin
transport (Rojas-Pierce et al., 2007). After the characterization of the auxin signaling
components, there are few synthetic chemicals designed, which specifically targets the auxin
signaling pathway, such as yokonolide B (Ykb) (Hayashi et al., 2003), terfestatin A (TrfA)
(Yamazoe et al., 2005), toyocamycin (Hayashi et al., 2009) and juglone (Dharmasiri et al., 2003).
Fungal toxin brefeldin A (BFA) has been a very useful tool for elucidating the constitutive
cycling of the PIN proteins and, generally, it is an inhibitor of vesicle transport (Nebenfuhr et al.,
2002; Geldner et al., 2001; Geldner et al., 2003). There are also various styryl FM dyes used
frequently to study membrane trafficking in plants, including the auxin-related phenomena
(Bolte et al., 2004; Aniento and Robinson, 2005).
Altogether, the chemical biology approach proved to be a very useful tool in the auxin
research, even if not fully exploited yet. That is why, we have concentrated predominantly on
using this type of approach to characterize specificity aspects of various auxin-related
physiological processes, and - possibly - to uncover compound(s) with very distinct behavior
towards distinct auxin-related processes, so that they could be used to separate these processes
and to characterize them individually.
6
2 Aim of the study
In our study we used a group of compounds structurally related to major endogenous
auxin indole-3-acetic acid, as well as synthetic auxins 2,4-dichlorophenoxy acetic acid (2,4-D)
and naphthalene-1-acetic acid (NAA). We aimed to characterize the auxin specificity of
developmentally important processes such as carrier-mediated auxin transport, and
‘transcriptional’ and ‘non-transcriptional’ auxin signaling. In addition to the characterization of
these compounds we also hoped to get an insight into the complex regulatory mechanism of
auxin-related processes and to possibly find a particular compound with distinct behavior
towards particular processes. By making use of such compounds and other molecular tools we
aimed to analyze the mechanism of ‘non-genomic’ auxin signaling. In this study we also tried to
understand the mode of action of FM (Fei Mao) styryl dyes on the dynamics of membranelocalized auxin transporters, and to study the involvement of other phytohormones like cytokinin
in regulation of auxin levels to mediate particular physiological action.
3 Results and Discussion
This PhD thesis is based on published articles and a submitted manuscript where the author has
significant contributions. All publications are included under ‘Publication’ section of this thesis.
This Result and Discussion part is aimed to combine and highlight the notable findings from all
publications together and to discuss them. Detailed documentation of results and discussion has
been done in individual publications. At the end of each paragraph related to particular paper, my
contribution is depicted (in italics).
3.1 Characterization of different auxin analogues and using them in studies on various
auxin-related processes
The submitted manuscript by Simon et al., entitled ‘Examining auxin biology: a study using
auxin analogues’ targets to characterize a group of compounds which are structurally analogous
to the major endogenous auxin IAA and synthetic auxins 2,4-D and NAA. We assessed their
physiological auxin-like action using well-established assays for determination of the auxin
activity. Thus, we have measured the ability of selected compounds to inhibit Arabidopsis
primary root growth, to promote lateral root growth and to promote cell division in suspension7
grown tobacco BY-2 cells. We have also analyzed the ability of these compounds to undergo the
active influx to and efflux from cells as well as their involvement in TIR1/AFB-mediated auxin
signaling. Finally, we measured their role in inhibiting endocytosis of PIN proteins. In addition,
to characterize these compounds we have also aimed to get an insight in to the complex
regulatory mechanism of the above mentioned auxin related processes. The compounds analyzed
in our study are IAA and its analogues halogenated on various positions on the indole ring , 5fluoroindole-3-acetic aid (5-F-IAA), 4-Cl-IAA, 5-chloroindole-3-acetic acid (5-Cl-IAA), 6chloroindole-3-acetic acid (6-Cl-IAA), 5-bromoindole-3-acetic acid (5-Br-IAA), indole-3propionic acid (IPA), IBA, indole-3-lactic acid (ILA), indole-3-acetyl-L-alanine (IAAla), indole
itself, then naphthalene and its carboxy-derivatives NAA, naphthalene-2-acetic acid (2-NAA),
and
phenyl
and
phenoxy
acetic
acids,
phenylacetic
acid
(PAA),
2,4-D,
2,4,5-
trichlorophenoxyacetic acid (2,4,5-T, ) 2-(2,4-dichlorophenoxy) propionic acid (and 2,4-DP). For
all experiments, benzoic acid (BA) was used as negative control.
In Arabidopsis primary root growth inhibition assay, most of the halogenated derivatives of IAA
were very efficient and 6-Cl-IAA was the most active compound, and it was even more active
than endogenous auxin IAA. 5-Cl-IAA and 5-Br-IAA were less efficient among halogenated
IAA group. In contrast to this, in lateral root promotion, 5-Cl-IAA was the most active
compound. In general, all halogenated derivatives of IAA were promoting lateral root formation
efficiently but in a varying degree. In the case of BY-2 cell division assay, endogenous auxin
IAA was not active at all whereas halogenated forms of IAA, especially 5-Cl-IAA and 5-F-IAA,
were very efficient at higher concentrations. All these data show that halogenation on different
positions of IAA often have a beneficial effect for the auxinic property of the molecule. This
effect, however, is not uniform for different assays. Other IAA analogues such as IPA, ILA,
IAAla, or indole had no or weak effects but the activity of IBA in BY-2 cell division assay was
surprisingly high. IBA is an endogenous compound and it has been believed to be a precursor of
IAA. So, our data contradict this notion and suggest a possibility of the IAA-independent mode
of action of this particular compound. Another compound whose effect should be especially
noted is 2-NAA; it is considered to be an inactive analogue of synthetic auxin NAA. However, in
our study 2-NAA was moderately active in Arabidopsis root growth assay. 2,4-D structural
analogues were highly active especially in BY-2 cell division but 2,4,5-T was comparatively less
effective than 2,4-D and 2,4-DP in Arabidopsis root growth assays.
8
The active transport potential of experimental compounds used was analyzed by assessing their
ability to compete with 3H-2,4-D for the active influx into cells and with 3H-NAA for efflux
from cells (Delbarre et al. 1996; Petrášek et al., 2006). All halogenated forms of IAA appeared to
be substrates for efflux machinery in tobacco BY-2 cells but IAA itself was a poor substrate.
This lesser ability of IAA to be transported from cells probably results from the fact that the
major portion of IAA is metabolized inside cells, but this is not the case for its halogenated
forms. Surprisingly, 2-NAA, an inactive analogue of NAA, was also a substrate for efflux
carriers in tobacco cells, though slightly less active than NAA. The efflux activity of these
compounds in Arabidopsis suspension showed more or less same trend as in tobacco BY-2 cells
with slight variation among individual compounds.
At the influx level IAA was the most active compound, and its halogenated derivatives also
showed considerable influx activity. The active influx machinery in both BY-2 and Arabidopsis
cells showed high affinity to 2-NAA whereas NAA was only a weak substrate. Notably, IBA
slightly increased the accumulation of 2,4-D in BY-2 cells but not in Arabidopsis. This increase
is probably due to common transporter for both 2,4-D and IBA from the PDR group of ABC
transporters which is acting as an effluxer for both compounds (Ito and Gray, 2006; Strader et al
2008). The absence of this effect in Arabidopsis cells may result from the fact that the
Arabidopsis cell suspension we used was derived from stem explants while the PDR transporters
are mostly expressed in roots.
The involvement of these compounds in ‘transcriptional’ auxin signaling was analyzed by
tracking their ability to promote expression of the reporter gene (GFP) driven by the auxinresponsive promoter DR5 (Ulmasov et al. 1997; Benková et al. 2003) and also by measuring the
primary root growth resistance of tir1 knockout mutant seedlings to these compounds. IAA and
all its halogenated derivatives induced the DR-5-driven gene expression. Again, synthetic
compounds 2-NAA and 2,4,5-T showed a remarkable activity at higher concentration. In contrast
to this, IBA expressed weak DR-5 induction. In tir1 mutant seedlings, root growth inhibition by
2,4-D and its structural analogues 2,4,5-T and 2,4-DP was less compared to control suggesting
higher affinity of TIR1 (and possibly also other members of AFB family due to functional
redundancy) to these compounds. The effect of 2,4,5-T was very interesting because it is a weak
activator of DR-5 auxin reporter. So it is reasonable to assume that the auxinic effect of 2,4,5-T
is mainly attributed by TIR1 other than other members of F-box proteins. Very less activity of
9
IPA in this assay in a physiologically active concentration shows that the auxinic nature of IPA is
mainly attributed by F-box proteins other than TIR1.
The determination of ability to inhibit endocytosis of PIN efflux carriers by these compounds
provided interesting data. All halogenated derivatives of IAA inhibited endocytosis efficiently
but the important exception was 5-F-IAA which did not show any effect in this assay.
This paper showed that the auxin-related processes in plants share very similar structure-activity
relationships; however, there are few exceptions of synthetic compounds (5-F-IAA, 2-NAA,
IPA) pointing at differential establishment of structural requirements of these processes during
evolution in the absence of a selection pressure from these synthetic compounds. These
particular compounds can be used to distinguish between various auxin-related processes and to
characterize them. The example is given in the next chapter.
Contribution of Sibu Simon to this paper:
I am first author of the manuscript. I have designed the experiments in consultation with my PhD
supervisor and co-supervisor and performed most of the experiments. Analyzed data and
prepared the first draft of the manuscript.
3.2 5-F-IAA as a tool to study inhibition of endocytosis by auxin
In the article Robert and Kleine-Vehn et al.: ABP1 Mediates Auxin Inhibition of ClathrinDependent Endocytosis in Arabidopsis (Cell 143, 111–121, 2010) we have dealt with
elucidation of the mechanism underlying the inhibition of endocytosis by auxin. This study used
5-F-IAA as one of the tools to study this mechanism because of its dual effect as being a
physiologically active auxin-like compound but inactive in inhibition of endocytosis.
Contribution of Sibu Simon to this paper:
Most of the experiments for this article were performed by Jiri Friml’s lab. I have selected a
group of auxin-related compounds and 5-F-IAA was one among them. The ability to induce DR5
auxin reporter by 5-F-IAA was performed by me and initial experiments to assess the ability to
inhibit endocytosis was also performed.
3.3 Effect of FM dyes on transient re-localization of membrane proteins
The study by Jelínková and Malínská et al.: Probing plant membranes with FM dyes:
tracking, dragging or blocking? (Plant Journal 61: 883–892, 2010) is reporting an unexpected
10
effect of FM dyes. FM dyes are fluorescent membrane markers which are widely used in cell
biology mainly to track membrane trafficking in eukaryotes.
This study is an important reminder for cell biologists using FM-dyes in their experiments as the
results demonstrated an unexpected effect of these widely used probes, and pointed to possible
misinterpretation of observed effects.
Contribution of Sibu Simon to this paper:
I have performed auxin accumulation assay in BY-2 cells after application of FM-dyes to see
how these dyes affect the function of membrane proteins. Transient internalization effect of
membrane protein in Arabidopsis seedlings was also performed by me.
3.4 Auxin-cytokinin cross talk in root meristem activity
The article by Růžička et al.: Cytokinin regulates root meristem activity via modulation of
the polar auxin transport (Proc. Natl. Acad. Sci. U. S. A. 106: 4284-4289, 2009) is mainly
focusing on auxin-cytokinin crosstalk in control of root meristem activity. This study revealed
the molecular mechanism behind the auxin-cytokinin interaction in root growth. Cytokinin (CK)
modulated the polar transport of auxin by controlling the transcription of PIN proteins.
Contribution of Sibu Simon to this paper:
Eva Benkova’s lab was the major contributor to this work. My contribution to this work was the
preparation of PIN1:RFP construct.
3.5 Endogenous auxins: A collective and detailed review focusing on endogenous auxins
The review article by Simon and Petrášek: Why plants need more than one type of auxin
(Plant Science 18: 454-460, 2011) is a focused review on endogenous auxins. The term auxin is
mainly referred to the major endogenous auxin IAA and auxin action is mainly meant as the
physiological actions mediated by IAA itself. In addition to IAA, plant produces few more
endogenous compounds such as 4-chloroindole-3-acetic acid (4-Cl-IAA), indole-3-butyric acid
(IBA) and phenylacetic acid (PAA), which are also considered to be native auxins. In this review
we are trying to analyze the term auxin and auxin action, and to what extent the endogenous
auxins other than IAA are fitting into the conventional definition of auxin and auxin action.
After the identification of the F-box proteins TIR1 and AFBs, auxin biologists started defining
auxins as molecules which are able to bind to TIR1/AFB to initiate ubiquitin-mediated
11
degradation of the Aux/IAA repressors. However, the right definition for ‘auxin action’ is
difficult because auxin is involved in a wide array of plant growth and developmental events and
sometimes these physiological processes are mediated in co-operation with other plant
hormones.
Contribution of Sibu Simon to this paper:
This review wrote based on the invitation received by Jan Petrasek from ‘Plant Science’ journal.
Review topic was selected in consultation with Jan Petrasek and first draft of the review is
prepared by me.
4 Conclusions
Using various analogues of both native and synthetic auxins, it was shown that generally (and
not surprisisngly) the structure-activity relationships in auxin biology share similar requirements
for the auxin-like behavior of various compounds. Nevertheless, this ‘chemical biology’
approach revealed non-canonical behavior of few synthetic substances, namely 5-F-IAA, IBA, 2NAA 2,4,5-T, IPAetc. These particular compounds represent potentially very useful tools as they
can be used to characterize particular auxin-related processes separately.
Thus, usage of 5-F-IAA that was inactive in inhibition of endocytosis but it was an active
compound in induction of ‘genomic’ auxin signaling, contributed to identify a putative auxin
receptor ABP1 as the mediator of inhibition of endocytosis by auxin and to understand its
involvement in ‘non-genomic‘ auxin signaling.
The detailed study using auxin analogues also revealed the potential of 2,4,5-T and IPA to
explore more about receptor mediated auxin signaling.
Last but not least, the observation of distinct behavior of native auxin-like compound IBA in
BY-2 cell division bio-assays pointed to possible IBA-specific mode of action, independent of
major native auxin IAA.
Some of FM-dyes that do not interfere with the internalization of auxin transporters do interfere
with the overall transport active of auxin inside and outside of the cell. This observation is of
particular importance as these dyes are routinely and widely used as markers of endocytosis.
The construct PIN1:RFP was very useful in identifying cytokinin’s effect on PIN protein
expression regulation.
12
Based on careful evaluation of data known about endogenous auxin’s activities, the need of
redefining the term ‘endogenous auxin’ was suggested, and detailed characterization of
properties of
endogenous auxins other than IAA and namely their involvement in auxin
signaling and transport pathways was proposed.
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19
Curriculum Vitae
Name and Surname: Sibu Simon
Date of Birth:
26.05.1979
Nationality:
Indian
Employe:
Institute of Experimental Botany
Address:
Rozvojová 263, 165 02 Prague 6 – Lysolaje
E-mail:
[email protected]
Phone:
+420 225 106 425
Education:
2006-Present
-
PhD student, Department of Biochemistry, Charles University, Prague, Czech
Republic
2001-2003
- MSc. in Biotechnology, Allahabad Agricultural Institute, India
1997-2000
- BSc. in Biology, Andhra Loyola College Vijayawada, India
Research Experience:




2006 March-Present: Working as a PhD student researcher at the Institute of Experimental Botany,
Prague, under the supervision of Assoc. Prof. Eva Zažímalová. Title of the thesis: ‘Mechanism of
auxin transport – biochemical characterisation of auxin efflux from cells’.
2005 July-February: Worked as a pre-graduate researcher at the Molecular farm laboratory of
Institute of Experimental Botany, Prague, under Dr. Karel Angelis. Title of the project: ‘Cloning and
over expression of various commercially important proteins in tobacco for commercial harvesting’
2002 May-2003 January: Worked for master thesis at the Banaras Hindu University, India under the
supervision of Prof. Mercy J Raman. Title of the thesis: ‘Status of DNA Double Strand Break repair
efficiency in rat testicular extract collected from high natural radiation area’
2001 May-July: Worked as a summer student at the Centre for Cellular and Molecular Biology, India
under the supervision of Dr. Kshitish Majumdar. Title of the project: ‘Fish mitochondrial DNA
isolation and sequencing’
Languages Known:
English, Hindi, Malayalam, Tamil and Telugu
20
List of publications
Examining auxin biology: A study using auxin analogues
(Submitted to Biochemical journal)
Sibu Simon, Martin Kubeš, Stéphanie Robert, Pawel Baster, Petre Ivanov Dobrev, Jiří Friml,
Jan Petrášek, Eva Zažímalová
ABP1 Mediates Auxin Inhibition of Clathrin-Dependent Endocytosis in Arabidopsis
Cell 143: 111-121, 2010
Stephanie Robert, Jü rgen Kleine-Vehn, Elke Barbez, Michael Sauer, Tomasz Paciorek,
Pawel Baster, Steffen Vanneste Jing Zhang, Sibu Simon, Milada Covanova´, Kenichiro
Hayashi, Pankaj Dhonukshe, Zhenbiao Yang, Sebastian Y. Bednarek, Alan M. Jones,
Christian Luschnig, Fernando Aniento, Eva Zažímalová, Jiří Friml
Probing plant membranes with FM dyes: tracking, dragging or blocking?
The Plant Journa 61: 883-892, 2010
Adriana Jelínkova´, Kateřina Malínska´, Sibu Simon, Jü rgen Kleine-Vehn, Markéta
Pařezova´, Přemysl Pejchar, Martin Kubeš, Jan Martinec, Jiří Friml, Eva Zažímalová, Jan
Petrášek
Cytokinin regulates root meristem activity via modulation of the polar auxin transport
PNAS 106: 4284-4289, 2009
Kamil Růžička, Mária Simášková, Jerome Duclercqa, Jan Petrášek, Eva Zažímalová, Sibu
Simon, Jiří Friml, Marc C. E. Van Montagua, Eva Benková
Why plants need more than one type of auxin
Plant Science 180: 454-460, 2011
Sibu Simon, Jan Petrášek
21