Bimodular Auxin Response Controls Organogenesis in Arabidopsis

Abstract

Like animals, the mature plant body develops via successive sets of instructions that determine cell fate, patterning, and organogenesis. In the coordination of various developmental programs, several plant hormones play decisive roles, among which auxin is the best-documented hormonal signal. Despite the broad range of processes influenced by auxin, how such a single signaling molecule can be translated into a multitude of distinct responses remains unclear. In Arabidopsis thaliana, lateral root development is a classic example of a developmental process that is controlled by auxin at multiple stages. Therefore, we used lateral root formation as a model system to gain insight into the multifunctionality of auxin. We were able to demonstrate the complementary and sequential action of two discrete auxin response modules, the previously described Solitary Root/indole-3-Acetic Acid (IAA)14-Auxin Response Factor (ARF)7-ARF19-dependent lateral root initiation module and the successive Bodenlos/IAA12-Monopteros/ARF5-dependent module, both of which are required for proper organogenesis. The genetic framework in which two successive auxin response modules control early steps of a developmental process adds an extra dimension to the complexity of auxin's action.

Full text

Bimodular auxin response controls organogenesis
in Arabidopsis
Ive De Smet
a,b,c,d,1
, Steffen Lau
c,d,2
, Ute Voß
d,e,2
, Steffen Vanneste
a,b
, René Benjamins
f,3
, Eike H. Rademacher
g
,
Alexandra Schlereth
d,4
, Bert De Rybel
a,b
, Valya Vassileva
a,b,5
, Wim Grunewald
a,b
, Mirande Naudts
a,b
,
Mitchell P. Levesque
h,6
, Jasmin S. Ehrismann
d,7
, Dirk Inzé
a,b
, Christian Luschnig
f
, Philip N. Benfey
h
, Dolf Weijers
d,g
,
Marc C. E. Van Montagu
a,b,1
, Malcolm J. Bennett
e
, Gerd Jürgens
c,d,8
, and Tom Beeckman
a,b,8
a
Department of Plant Systems Biology, Flanders Institute for Biotechnology, B-9052 Ghent, Belgium;
b
Department of Plant Biotechnology and Genetics, Ghent
University, B-9052 Ghent, Belgium;
c
Department of Cell Biology, Max Planck Institute for Developmental Biology, D-72076 Tübingen, Germany;
d
Center for
Plant Molecular Biology, University of Tübingen, D-72076 Tübingen, Germany;
e
Plant Sciences Division and Centre for Plant Integrative Biology, School of
Biosciences, University of Nottingham, Loughborough LE12 5RD, United Kingdom;
f
Department for Applied Genetics and Cell Biology, University of Natural
Resources and Applied Life Sciences, A-1190 Vienna, Austria;
g
Laboratory of Biochemistry, Wageningen University and Research Centre, 6703 HA
Wageningen, The Netherlands; and
h
Department of Biology and IGSP Center for Systems Biology, Duke University, Durham, NC 27708
Contributed by Marc C. E. Van Montagu, December 30, 2009 (sent for review September 17, 2009)
Like animals, the mature plant body develops via successive sets of
instructions that determine cell fate, patterning, and organogene-
sis. In the coordination of various developmental programs, several
plant hormones play decisive roles, among which auxin is the best-
documented hormonal signal. Despite the broad range of processes
influenced by auxin, how such a single signaling molecule can be
translated into a multitude of distinct responses remains unclear. In
Arabidopsis thaliana, lateral root development is a classic example
of a developmental process that is controlled by auxin at multiple
stages. Therefore, we used lateral root formation as a model sys-
tem to gain insight into the multifunctionality of auxin. We were
able to demonstrate the complementary and sequential action of
two discrete auxin response modules, the previously described SOLI-
TARY ROOT/INDOLE-3-ACETIC ACID (IAA)14-AUXIN REPONSE FAC-
TOR (ARF)7-ARF19–dependent lateral root initiation module and
the successive BODENLOS/IAA12-MONOPTEROS/ARF5–dependent
module, both of which are required for proper organogenesis. The
genetic framework in which two successive auxin response modules
control early steps of a developmental process adds an extra dimen-
sion to the complexity of auxin’s action.
AUXIN/INDOLE-3-ACETIC ACID
|
AUXIN RESPONSE FACTOR
|
cell cycle
|
lateral root
Unlike animals, plants produce new organs primarily post-
embryonically. The formation of these new structures fol-
lows a precise pattern that guarantees an optimal spacing of
plant organs and that contributes to their functionality. To
investigate how a general signal, such as auxin, can be translated
into various distinct responses (1), we used the postembryonic
development of lateral roots in Arabidopsis thaliana as a model
process. Lateral roots originate from a few asymmetrically
dividing pericycle cells and develop according to a highly regular
pattern (2–4). Each step in this process is controlled predom-
inantly by the phytohormone auxin, and a number of AUXIN
RESPONSE FACTOR (ARF) (transcriptional regulators) and
AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) (inhibitors of
ARF) proteins are implicated in lateral root development (3–5).
Most prominently, the SOLITARY ROOT (SLR)/IAA14-
ARF7-ARF19–mediated auxin response module is required for
cell cycle activation and controls the initial, asymmetric pericycle
cell divisions (4, 6, 7). Whereas genetic stimulation of the basic
cell cycle machinery is capable of bypassing the SLR/IAA14-
mediated control on cell division, it is insufficient for de novo
lateral root organogenesis (7). Previous analyses of auxin
response markers (8–10) have suggested that additional AUX/
IAA-ARF–mediated signaling might be required for lateral root
development, however.
Here we demonstrate that the capacity of pericycle cells to
form new lateral roots in response to auxin is enhanced con-
siderably on genetic stimulation of the basic cell cycle machinery,
arguing for the existence of an additional auxin response module
during lateral root initiation. We show that in dividing pericycle
cells, the BODENLOS (BDL)/IAA12-MONOPTEROS (MP)/
ARF5–mediated auxin response guarantees organized lateral
root patterning downstream of SLR/IAA14.
Results and Discussion
Cell Cycle Activation Sensitizes Pericycle Cells for Auxin-Induced
Lateral Root Initiation. To explore whether during lateral root
formation other auxin response modules are active after or
besides the SLR/IAA14-dependent signaling, we released cell
cycle regulation in the pericycle from its SLR/IAA14-dependent
repression via overexpression of core cell cycle regulators. WT
roots with enhanced cell cycle activity caused by 35S-driven
overexpression (OE) of the heterodimeric G1-S E2Fa/DPa
transcription factor (E2Fa/DPa
OE
) displayed stretches with small
pericycle cells that clearly deviated from the larger WT pericycle
cells or the typical lateral root initiation pattern (Fig. 1 A–C).
Tight control of patterned pericycle cell division has been shown
to be required for de novo lateral root formation (7, 11, 12).
Consequently, the lateral root density of the E2Fa/DPa
OE
seedlings—in which the cell cycle is not inhibited in dividing
pericycle cells—was significantly reduced (by >60%) and, inter-
estingly, significantly higher in the presence of auxin compared
with WT seedlings (Fig. 1D). A similar significant increase in
lateral root density was observed in auxin-treated plants with
35S-driven overexpression of the G1-S–related D-type cyclin
Author contributions: I.D.S., S.L., D.I., P.N.B., M.J.B., G.J., and T.B. designed research; I.D.S.,
S.L., U.V., S.V., A.S., B.D.R., V.V., W.G., M.N., M.P.L., and J.S.E. performed research; R.B., E.
H.R., A.S., J.S.E., C.L., and D.W. contributed new reagents/analytic tools; I.D.S., S.L., U.V., S.
V., M.P.L., P.N.B., D.W., M.C.E.V.M., M.J.B., G.J., and T.B. analyzed data; and I.D.S. and T.B.
wrote the paper.
The authors declare no conflict of interest.
1
To whom correspondence should be addressed. E-mail: ive.desmet@tuebingen.mpg.de
or marc.vanmontagu@ugent.be.
2
S.L and U.V. contributed equally to this work.
3
Present address: Department of Molecular Genetics, Faculty of Bio logy, University of
Utrecht, 3584 CH Utrecht, The Netherlands.
4
Present address: Syngenta Crop Protection, CH-4332 Stein, Switzerland.
5
Present address: M. Popov Institute of Plant Physiology, Bulgarian Academy of Sciences,
1113 Sofia, Bulgaria.
6
Present address: Department of Genetics and Genomics, Max Planck Institute for Devel-
opmental Biology, D-72076 Tübingen, Germany.
7
Present address: Lehrstuhl für Medizinische Genetik, Universität Tübingen, D-72076 Tü-
bingen, Germany.
8
G.J. and T.B. contributed equally to this work.
This article conta ins supporting in formation online at www.p nas.org/cgi/c ontent/full/
0915001107/DCSupplemental.
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CYCD3;1 (CYCD3;1
OE
), which exhibited ectopic cell divisions in
the shoot (13) but no initial decrease in lateral root density in the
absence of auxin (Fig. 1D). The increase in the number of pri-
mordia on auxin treatment was greater for E2Fa/DPa
OE
than for
CYCD3;1
OE
at both concentrations used, possibly reflecting the
greater cell division competence obtained by overexpression of
the heterodimeric E2F-DP transcription factors. We conclude
that although genetic stimulation of the basic cell cycle machi-
nery is not sufficient for de novo lateral root formation, it
enhances the capacity of pericycle cells to divide and to form new
lateral roots in response to auxin.
Other Auxin Response Modules After SLR/IAA14-Dependent Cell Cycle
Regulation. In the gain-of-function mutant slr-1, lateral root for-
mation cannot be induced by auxin treatment or by cell cycle stim-
ulation, although the latter results in stretches of small cells (6, 7)
(Fig. 2 A–D). The absence of lateralroots is supported by the absent
expression of ARABIDOPSIS CRINKLY4 (ACR4) (a receptor-like
kinase marking the first divisions of the lateral root initiation site)
(2) and the lack of increased expression of PLETHORA3 (PLT3)(a
highly auxin-responsive AP2-domain transcription factor involved
in primary root meristem formation) (7, 14) (Fig. 2 G–Iand K).
However,when slr-1xCYCD3;1
OE
seedlingswere treated with auxin,
ACR4 was expressed in thestretches of dividing pericycle cells (Fig.
2J), and PLT3 expression was induced (Fig. 2K). This reactivation
of markers augured the formation of lateral root organs in auxin-
treated slr-1xCYCD3;1
OE
(Fig. 2E)andslr-1xE2Fa/DPa
OE
(Fig. 2F).
These observations imply that in the slr-1 mutants as well, genetic
stimulation of the basic cell cycle machinery enhances the capacity
of pericycle cells to divide and to form new lateral roots in response
to auxin. But endogenous auxin is not sufficient to induce lateral
roots in slr-1xCYCD3;1
OE
and slr-1xE2Fa/DPa
OE
,mostlikely
because in slr-1, PIN-dependent auxin redistribution—which is
required for lateral root development—is affected as well (7, 15),
and this is largely overcome by exogenous auxin application. Very
little is known about how differential cell cycle activity and auxin
regulate specific division patterns during lateral root initiation. Our
findings suggest that along with the SLR/IAA14-dependent cell
cycle regulation, other auxin response modules might work to
coordinate organogenesis and to prevent proliferative division.
BDL-MP–Dependent Auxin Response Module Acts in Lateral Root
Initiation. To identify additional auxin response modules, we
analyzed the expression profiles of other AUX/IAA and ARF
genes in a highly specific transcript data set from pericycle cells
undergoing lateral root initiation (2). Of the retrieved AUX/IAA
and ARF genes, which showed significant transcriptional induc-
tion during lateral root initiation and for which no role in lateral
root development has been documented until now (3, 16), the
most intriguing were BDL/IAA12, its paralog IAA13, and its
interaction partner MP/ARF5. Because the extensively studied
role of the BDL/IAA12–MP/ARF5 pair in embryogenesis
implies a function in cell fate determination and asymmetric cell
division (5, 17–21), we investigated the functional involvement of
these two proteins in postembryonic lateral root development.
Using promoter fusions with GFP and β-glucuronidase (GUS),
ProBDL:n3xGFP and ProMP:GUS, we found that BDL (Fig. 3A)
and MP (Fig. 3B) were indeed expressed in the lateral root ini-
tiation site. Because homozygous gain-of-function bdl mutants
lack a main root (18), we analyzed the lateral root phenotype of
hemizygous ProBDL:bdl:GUS plants (ProBDL:bdl
hemi
) (19). At 11
days after germination, the density of emerged lateral roots was
significantly lower in the mutants compared with WT seedlings
(Fig. 3C). A detailed microscopic analysis revealed fused pri-
mordia (Fig. 3F) and stretches with a two- or three-layer pericycle
(Fig. 3 Dand E) that were not present in WT roots, which had
single-layer pericycle (Fig. 1B), and clearly differed from normal
lateral root initiation sites (Fig. 1A), where only a limited number
of cells divide periclinally. Emerging ProBDL:bdl
hemi
lateral roots
did not display the regular longitudinal positioning seen in the WT
root primordia (Fig. 3G) but rather were grouped in clusters (Fig.
3H). Similarly, the weak loss-of-function mp
S319
mutant (22)
exhibited zones of ectopic pericycle cell division, although they
were less pronounced (Fig. 3I). Although the 35S-driven over-
expression of MP (MP
OE
) did not result in a significant increase in
the number of emerged lateral roots (Fig. 3J), closely positioned
lateral root initiation sites and aberrantly spaced lateral root pri-
mordia were occasionally seen in the MP
OE
roots (Fig. 3 Kand L).
Together, these findings demonstrate that BDL and MP are
involved in lateral root organogenesis.
The BDL-MP–Dependent Auxin Response Module Follows the SLR-
ARF7-ARF19–Dependent Auxin Response Module. Both gain-of-
function bdl and loss-of-function mp mutants displayed different
defects arising at later stages than those of the lateral rootless
phenotypes of slr-1 and arf7arf19 (3–7, 23, 24). Using a system of
synchronized lateral root induction after gravistimulation (25), we
found that an increase in expression of at least ARF19, which
together with ARF7 has been proposed to interact with SLR (26),
precedes an increase in MP expression (Fig. 4A). In addition, we
found a decrease in MP expression in the absence of lateral root
initiation in slr-1 and arf7arf19 roots (Fig. 4B). Therefore, it is
Fig. 1. Auxin response module after cell cycle activation. (A) WT (Col-0)
lateral root initiation. Arrowheads indicate adjacent (red) and short cell
boundaries (blue). (Band C) Pericycle cell boundaries (black arrowheads) in a
Col-0 (B) and E2Fa/DPa
OE
(C) root regions. The yellow arrowhead indicates
single pericycle layer. DIC, differential interference contrast (Nomarski)
imaging. (Scale bars: 50 μM.) (D) Number of lateral roots/cm in WT (Col-0 and
Ler), CYCD3,1
OE
, and E2Fa/DPa
OE
(mean ±SEM) at different NAA concen-
trations. Seedlings 5 days after germination were transferred to NAA for 5
days. *Statistically significant differences for values compared with WT as
determined by Student's ttest (P<0.05).
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reasonable to postulate that BDL and MP act later than SLR,
ARF7, and ARF19 during lateral root organogenesis.
To test this hypothesis, we used 35S-driven overexpression of
MP in the slr-1 mutant. The number of emerged lateral roots in
slr-1xMP
OE
increased significantly (Fig. 4C), and occasionally
those lateral roots were fused or irregularly spaced, or both (Fig.
4D), in contrast to the complete absence of lateral roots in the
slr-1 mutant (Figs. 2Aand 4C). Such a strong rescue of the slr-1
lateral rootless phenotype could not be obtained even through
overexpression of ARF19 (slr-1xARF19
OE
) and ARF7 (slr-
1xARF7
OE
) (Fig. 4C); however, in slr-1xARF19
OE
and slr-1xAR-
F7
OE
, the ARF19 and ARF7 overexpression levels, respectively,
were not as high as the MP overexpression level in slr-1xMP
OE
.In
addition, we specifically overexpressed MP in the xylem pole
pericycle of slr-1 plants with a GAL4-based transactivation
expression approach using the J0121 driver line (Pro
J0121
>>MP)
(27). Although the slr-1xJ0121 control did not display any
emerged lateral roots, the slr-1xPro
J0121
>>MP lines exhibited
Fig. 2. Auxin response module after SLR-dependent cell
cycle activation. Absence of lateral roots (A–C), lack of
expression of the marker ProACR4:H2B:YFP (G–I), and lack
of increased expression of PLT3 (K)inslr-1 (A,G, and K), slr-
1grown on NAA (B,H, and K), and slr-1 with increased cell
cycle activity (slr-1xCYCD3;1
OE
)(C,I, and K), but presence of
stretches of short cells in slr-1xCYCD3;1
OE
(D). Induction of
lateral roots (Eand F) and/or turning on of ProACR4:H2B:
YFP (J) and PLT3 (K)inslr-1xCYCD3;1
OE
(E,J, and K) and slr-
1xE2Fa/DPa
OE
(F) by the combination of NAA and increased
cell cycle activity. Insets (G, and I) show the presence of
ProACR4:H2B:YFP in the root tip. Seedlings 5 days after
germination were transferred to 10 μM NAA for 96 h (B,E,
and F) and 30 h (Hand J). Arrowheads indicate the peri-
cycle (yellow) (Dand J), which consists of one layer (D)orof
one layer starting to undergo periclinal divisions (J)and
pericycle cell boundaries (black) (D). DIC, differential
interference contrast (Nomarski) imaging. (K) Quantitative
RT-PCR expression analysis of PLT3. Seedlings 5 days after
germination were treated with 10 μM NAA for 24 h.
Fig. 3. Role of BDL and MP in lateral root development. (A
and B) Expression of ProBDL:n3xGFP (A) and ProMP:GUS (B)
in the lateral root initiation site. Arrowheads indicate adja-
cent (red) and short cell boundaries (blue). (C–L)bdl gain of
function [hemizygous ProBDL:bdl:GUS (ProBDL:bdl
hemi
)] (C–F
and H), mp partial loss of function (mp
S319
)(I), and MP
overexpression (MP
OE
)(J–L) leading to defects in pericycle
cell division, lateral root initiation, and positioning compared
with WT (Col-0) (C,G, and J). (Cand J) Emerged lateral root
density at 10 (J) and 11 (C) days after germination. (Gand H)
Lateral root positioning in Col-0 WT (G) and ProBDL:bdl
hemi
(H) at 15 days after germination. The red dotted line indi-
cates separation between two pericycle cell layers, and the
yellow dashed line indicates outer boundaries of the two- or
three-layer pericycle (D). Arrowheads indicate two- (green)
and three-layer pericycle (white) (E), small pericycle cell
boundaries (black) (Dand I), and pericycle (yellow), which is
undergoing periclinal division (I) or consists of one layer (K).
Red asterisks indicate lateral root primordia (Fand L)and
sites that strongly resemble a lateral root initiation site (K).
Data are mean ±SEM. *Statistically significant differences
for values compared with WT as determined by Student's t
test (P<0.01).
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Fig. 4. BDL-MP–dependent auxin response module after SLR/IAA14-ARF7-ARF19 action. (Aand B) Quantitative RT-PCR expression analysis of ARF7,ARF19,
and MP during synchronized lateral root initiation (A) and of MP in slr-1 (5 days after germination) and arf7arf19 roots (10 days after germination) compared
with WT expression level (dashed line) (B), indicating two successive modules. Just prior to stage I in (A) indicates at which time point CYCB1;1 expression is
induced, which coincides with the onset of lateral root initiation. (C–H) Induction of lateral roots in slr-1 by expression in whole roots MP
OE
(Cand D)and
pericycle-specific expression (Pro
J0121
>>MP)ofMP (Cand E–H). (C) Emerged lateral root density at 12 days (slr-1,slr-1xMP
OE
,slr-1xARF7
OE
, and slr-1xARF19
OE
)
and 15 days (slr-1xJ0121 and slr-1xPro
J0121
>>MP) after germination. Data are mean ±SEM. *Statistically significant differences for values compared with
control or as indicated with a black line as determined by Student’sttest (P<0.01). (D–H) Red asterisks indicate emerged lateral roots (Dand F) and sites that
strongly resemble a lateral root initiation site (H)inslr-1xMP
OE
(D) and in slr-1xPro
J0121
>>MP (Fand H) compared with the control slr-1xJ0121 (Eand G).
Arrowheads indicate pericycle cell boundaries (black) (Gand H) and a pericycle consisting of a single layer (yellow) (Gand H). DIC, differential interference
contrast (Nomarski) imaging.
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numerous emerged lateral roots (Fig. 4 C,E, and F). This
increase in emerged lateral roots was also reflected in a sig-
nificant increase in xylem pole pericycle cell divisions, resembling
lateral root initiation sites that were absent in the slr-1xJ0121
control (Fig. 4 Gand H). These lateral root initiation sites gave
rise to closely spaced emerged lateral roots (Fig. 4F).
Although the possibility that the difference in rescue between
slr-1xMP
OE
and slr-1xARF19
OE
or slr-1xARF7
OE
is due to
unequal expression levels cannot be ruled out, we know of no
evidence indicating that MP also activates direct targets of ARF7
and ARF19 in the root. For example, LATERAL ORGAN
BOUNDARIES-DOMAIN16 (LBD16) and LBD29 (28) showed
no clear difference in expression in slr-1xPro
J0121
>>MP and slr-
1xMP
OE
compared with control (Fig. S1).
These data strongly suggest the existence of a second auxin
response module that is sufficient to activate lateral root ini-
tiation, that is controlled by BDL/IAA12-MP/ARF5, and that
acts after the well-known SLR/IAA14-ARF7-ARF19–dependent
control. However, based on the gain-of-function bdl and loss-of-
function mp mutants, it appears that along with its role in acti-
vating lateral root initiation, the BDL/IAA12-MP/ARF5 module
also might play a role in inhibiting lateral root formation, similar
to what was observed for the receptor-like kinase ACR4 (2).
Conclusions
In multicellular organisms, growth and development, including
proper pattern formation and organogenesis, must be tightly
regulated. In plants, the phytohormone auxin plays a prominent
role in controlling nearly every step in growth and development
(1). Just how one generic signal, such as auxin, can be translated
into so many diverse developmental responses is unclear, how-
ever. Our results demonstrate that the control exerted by auxin
on lateral root initiation is at least bimodal and consists of the
crucial early SLR/IAA14-ARF7-ARF19–dependent auxin
response module (3–7, 23, 24), followed by a second BDL/
IAA12-MP/ARF5-dependent module. Most likely, more mod-
ules are involved, because other ARFs have been implicated in
adventitious and lateral root development (3, 29–34). We pro-
pose that discrete auxin response modules successively coor-
dinate distinct developmental processes, most probably through
the regulation of unique targets, comparable to the spatially
distinct, bipartite auxin response during hypophysis specification
(19). Thus, a genetic framework for many diverse functions is
provided for a single molecule, such as auxin. We hypothesize
that such bimodular or multimodular response mechanisms
might represent a general principle for auxin signaling in plants.
Materials and Methods
Plant Material and Growth Conditions. We analyzed A. thaliana (L.) Heynh.
ecotypes Columbia (Col-0) and Landsberg erecta (Ler), the mutants slr-1 (6) and
mp
S319
(22), and the transgenic lines CYCD3;1
OE
(13), slr-1xCYCD3;1
OE
(7), slr-
1xE2Fa/DPa
OE
(7), E2Fa/DPa
OE
(35), slr-1xProACR4:H2B:YFP (2), slr-1xCYC-
D3;1
OE
xProACR4:H2B:YFP (2), ProMP:GUS,ProBDL:bdl:GUS (19), UAS::MP:HA
(19), MP
OE
,arf7arf19 (24, 36), ARF7
OE
(37), ARF19
OE
(23), slr-1xJ0121 (7),and J0121
(27). For slr-1xMP
OE
,slr-1xARF7
OE
slr-1xARF19
OE
,andslr-1xPro
J0121
>>MP
OE
,the
F1 seedling roots were analyzed. For analysis of expression patterns and lateral
root densities, seedlings were grown on normal or half-strength Murashige and
Skoog (MS) medium as described previously (38). For the auxin treatment, 5-day-
old seedlings were transferred for 5 days from MS medium to MS medium
supplemented with various concentrations of 1-naphthalene acetic acid (NAA)
foranalysisoflateralrootdensitiesasdescribed previously (11), or, alternatively,
4- to 5-day-old seedlings were incubated for 24 h in liquid half-strength MS
medium with or without 1% sucrose supplemented with NAA for quantitative
RT-PCR analyses as described previously (15). Seedlings were incubated in a
growth chamber under continuous light at 22 °C or under 16-h light/8-h dark
conditions at 24 °C.
Histochemical, Histological, and Microscopic Analyses. The GUS assays were
conducted as described previously (39). Microscopic analyses we performed
as described previously (2).
Quantitative RT-PCR. RNA extraction and quantification by qRT-PCRwas done in
triplicate as described previously (15). The following primers were used to
quantify the gene expression levels: PLT3,5′-CGGCGAATGCAGCTTCTGACTC-3′/
5′-GGTGCCATAAGTCCCATTGTTCCC-3′;ARF7,5′-GCTCATATGCATGCTCCACA-3′/
5′-GCAATGCATCTCTGTCATATTTG-3′;ARF19,5′-CACCGATCACGAAAACGATA-
3′/5′-TGTTCTGCACGCAGTTCAC-3′;andMP,5′-CGAGCTTTGTGGGTGAGTTAG-
TAG-3′/5′-ACAAGCT TTAAAGACTACGAGGAGCTA-3′.
New Constructs and Transgenic Lines. ProMP:GUS was generated as follows.
The primers 5′-GGGTTCTAGATTGGAGATCCTTTGATTCAAATAT-3′/5′-CACCT-
CTAGAGAAGTAATACACTAAGCTCCCAA-3′were used to amplify a 2.5-kb
genomic fragment consisting of ≈2kbofMP promoter region and further
extending into exon 2 of the MP-coding region, which was inserted via XbaI
into pPZP-GUS (40). pGreenIIKanProBDL:NLS:3xEGFP::nost (ProBDL:n3xGFP)
was constructed by inserting ProBDL into pGreenIIKanNLS:3xEGFP::nost (41)
via the EcoRI and BamHI restriction sites. ProBDL was amplified as two frag-
ments which were fused via the PstI restriction site; those fragments were
amplified with primers 5′-CCGAATTCCATGTGGTAGTGTCGAG-3′/5′-GGCTG-
CAGACAACAAGAAGAGAAAGAG-3′and 5′-GGCTGCAGCTCCATTCTCTCTGTG-
3′/5′-CCGGATCCGTCAATAACAAAACCC-3′, respectively. 35S::MP (MP
OE
)was
generated by fusing the MP cDNA and a sequence encoding an HA epitope-tag
(36) to the double-enhanced CaMV 35S promoter. Constructs were used for
floral dip transformation of Col-0 plants (42). Transformants were selected on
kanamycin and subsequently confirmed for a 3:1 segregation. For MP
OE
, lines
were selected that had elevated MP mRNA levels in the qRT-PCR, namely an
increase of ≥40-fold compared with WT.
ACKNOWLEDGMENTS. We thank J. Murray, H. Fukaki, T. Berleth, and A.
Theologis for sharing materials; M. Cook (Comprehensive Cancer Facility,
Duke University) for providing expert assistance with cell sorting; and D.
Slane, M. Bayer, and M. De Cock for critically reading the manuscript. This
work was funded by grants from the Interuniversity Attraction Poles
Programme (P6/33 and P5/13), initiated by the Belgian State Science Policy
Office (BELSPO); the National Science Foundation AT2010 program (to P.N.
B.); the Deutsche Forschungsgemeinschaft (SFB 446, to G.J.); the Austrian
Science Fund (to C.L. and R.B.), Research Foundation-Flanders (travel grant,
to I.D.S.), and the Netherlands Organization for Scientific Research (ALW-
VIDI 864-06.012, to D.W.). Financial support was provided by the University
of Nottingham , Biotechnology and Biological Sciences Research Council
(BBSRC), and Engineering Physics Scientific Research Council (EPSRC) award
to the Centre for Plant Integrative Biology (to U.V. and M.J.B.), a fellowship
for non–European Union researchers from BELSPO (to V.V), fellowships from
the Institute for the Promotion of Innovation by Science and Technology in
Flanders (to I.D.S. and S.V.), the Bijzondere Onderzoeksfonds of Ghent Uni-
versity (B.D.R. and W.G.), the European Molecular Biology Organization
(ALTF 108-2006, to I.D.S.; ALTF 142-2007, to S.V.), and the Marie Curie
Intra-European Fellowship scheme (FP6 MEIF-CT-2007–041375, to I.D.S).
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www.pnas.org/cgi/doi/10.1073/pnas.0915001107 De Smet et al.