ERECTA Family Genes Regulate Auxin Transport in the Shoot Apical Meristem and Forming Leaf

42 Pages·2013·2.08 MB·English

Plant Physiology Preview. Published on July 2, 2013, as DOI:10.1104/pp.113.218198

Running head: ERECTA Family Genes Regulate Auxin Transport
Keywords: ERECTA, auxin, PIN1, leaf primordium, shoot apical meristem

Correspondence to:
Elena Shpak, Department of Biochemistry, Cellular and Molecular Biology, M407 Walters Life
Sciences, 1414 Cumberland Avenue, University of Tennessee, Knoxville, TN, 37996, USA
865-974-8383 (phone); 865-974-6306 (fax)
e-mail:[email protected]

Research category: Genes, Development and Evolution

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Copyright 2013 by the American Society of Plant Biologists
ERECTA Family Genes Regulate Auxin Transport in the Shoot Apical Meristem and Forming Leaf
Primordia.
Ming-Kun Chena, Rebecca L. Wilsona, Klaus Palmeb, Franck Anicet Ditengoub, and Elena D. Shpaka

Author Affiliations:
aDepartment of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville,
TN, 37996, USA
bInstitute of Biology II/Botany, Faculty of Biology, Albert-Ludwigs-University of Freiburg; Centre of
Biological Systems Analysis, Freiburg Institute of Advanced Sciences (FRIAS); Centre for Biological
Signalling Studies (bioss), Albert-Ludwigs-University of Freiburg, 79104 Freiburg, Germany

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Funding:
This work was supported by the National Science Foundation [IOS-0843340 to E.S], the German
Research Council (EXC 294), Bundesministerium für Forschung und Technik (BMBF), and the
Deutsches Zentrum für Luft und Raumfahrt.

Correspondence to:
Elena Shpak
e-mail:
[email protected]

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Abstract:
Leaves are produced postembryonically at the flanks of the shoot apical meristem. Their initiation
is induced by a positive feedback loop between auxin and its transporter PIN1. The expression
and polarity of PIN1 in the shoot apical meristem is thought to be regulated primarily by auxin
concentration and flow. The formation of an auxin maximum in the L1 layer of the meristem is the
first sign of leaf initiation and is promptly followed by auxin flow into the inner tissues, formation
of the midvein, and appearance of the primordium bulge. The ERECTA family genes (ERfs) encode
leucine-rich repeat receptor-like kinases and in Arabidopsis this gene family consists of ERECTA
(ER), ERECTA-LIKE 1 (ERL1) and ERL2. Here we show that ERfs regulate auxin transport during leaf
initiation. The shoot apical meristem of the er erl1 erl2 triple mutant produces leaf primordia at a
significantly reduced rate and with altered phyllotaxy. This phenotype is likely to be due to
deficiencies in auxin transport in the shoot apex, as judged by altered expression of PIN1, the
auxin reporter DR5rev::GFP, and the auxin inducible genes MP, IAA1, and IAA19. In er erl1 erl2
auxin presumably accumulates in the L1 layer of the meristem, unable to flow into the
vasculature of a hypocotyl. Our data demonstrate that ERfs are essential for PIN1 expression in
the forming midvein of future leaf primordia and in the vasculature of emerging leaves.

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Leaves are formed during postembryonic development by the shoot apical meristem
(SAM), a dome-shaped organ with a stem cell reservoir at the top and with leaf initiation taking
place slightly below in the peripheral zone. The initiation of leaf primordia depends on the
establishment of auxin maxima at the site of initiation (Braybrook and Kuhlemeier, 2010). Auxin is
polarly transported through the epidermal layer of the meristem to the incipient primordium
initiation site (Heisler et al., 2005), and then moves inward where it promotes formation of a
vascular strand (Scarpella et al., 2006; Bayer et al., 2009). The developing vascular tissue acts as
an auxin sink, depleting auxin in the epidermal layer (Scarpella et al., 2006). PIN1, an auxin efflux
protein, is a central player in formation of auxin maxima and it is involved in the transport of
auxin in both the epidermis and the forming vascular strand during leaf initiation (Benková et al.,
2003; Reinhardt et al., 2003). PIN1 is the earliest marker for midvein formation (Scarpella et al.,
2006), which starts to form before a leaf primordium bulges out of the meristem. The
mechanisms determining PIN1 expression and polar localization in the SAM are central to
understanding leaf initiation. In the L1 layer of the SAM PIN1 is polar localized in the plasma
membrane towards cells with higher auxin concentration (Jönsson et al., 2006; Smith et al., 2006).
Formation of the vein is explained by the canalization hypothesis, in which high auxin flux
reinforces PIN1 expression (Kramer 2008). Of all plasma membrane-localized PIN family
transporters, only PIN1 has been detected in the vegetative SAM and linked with initiation of
rosette leaves (Guenot et al., 2012). At the same time, rosette leaves are positioned nonrandomly
in pin1 mutants suggesting that additional PIN1-independent mechanisms also have a role in
regulating leaf initiation (Guenot et al., 2012).
Here we investigate the role of ERECTA family receptor-like kinases during leaf initiation.
Previously ERfs have been shown to be involved in the regulation of epidermis development and
of plant growth along the apical-basal/proximodistal axis in aboveground organs (Torii et al.,
1996; Shpak et al., 2004, 2005). Triple er erl1 erl2 mutants form a rosette with small, round leaves
that lack petiole elongation. During the reproductive stage, the main inflorescence stem exhibits
striking elongation defects and reduced apical dominance. ER has been implicated in vascular
development, with the er mutation causing radial expansion of xylem (Ragni et al., 2011) and
premature differentiation of vascular bundles (Douglas and Riggs, 2005). Recently, the dwarfism
of described mutants was attributed to the function of ERf genes in the phloem where they
perceive signals from the endodermis (Uchida et al., 2012a). In the epidermis all three genes
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inhibit the initial decision of protodermal cells to become meristemoid mother cells (Shpak et al.,
2005). In addition, ERL1 and to a lesser extent ERL2 are important for maintaining cell
proliferative activity in stomata lineage cells and for preventing terminal differentiation of
meristemoids into guard mother cells. The activity of ERf receptors in the epidermis is regulated
by a different set of peptides than in the phloem. EPF1 and EPF2 are expressed in stomatal
precursor cells. They inhibit development of new stomata in the vicinity of a forming stoma (Hara
et al., 2007, 2009; Hunt and Gray, 2009). EPFL9/stomagen is expressed in the mesophyll and in
contrast it promotes development of stomata (Kondo et al., 2010; Sugano et al., 2010). EPFL4 and
EPFL6/CHALLAH are expressed in the endodermis and their perception by phloem-localized ERfs is
critical for stem elongation (Uchida et al., 2012a).
While ERfs are very strongly expressed in the vegetative SAM and in forming leaf
primordia, only recently has it became clear that these genes are involved in the regulation of
meristem size and leaf initiation (Uchida et al., 2012b, 2013). It was suggested that ERfs regulate
stem cell homeostasis in the SAM via buffering its cytokinin responsiveness by an unknown
mechanism (Uchida et al., 2013). Here we further investigate involvement of ERfs in the control of
leaf initiation and phyllotaxy. Our data suggest that ERfs are essential for PIN1 expression in the
vasculature of forming leaf primordia. Based on analysis of the DR5rev::GFP reporter, auxin may
accumulate in the L1 layer of the SAM in the mutant but is not able to move into the vasculature,
consistent with drastically reduced PIN1pro:PIN1-GFP expression there. These data suggest that
the convergence of PIN1 expression in the inner tissues of the SAM during leaf initiation is a
complex process involving intercellular communications enabled by ERfs. The importance of ERfs
for efficient auxin transport is further supported by reduced phototropic response in the er erl1
erl2 mutant.

RESULTS
ERfs are Critical for Initiation of Leaf Primordia and Establishment of Phyllotaxy
ERf genes are expressed in the vegetative SAM and in leaf primordia (Yokoyama et al.,
1998; Uchida et al., 2013). Previously, a QTL analysis suggested that ER regulates total leaf
number (El Lithy et al., 2004, 2010) and based on analysis of scanning electron microscope images
it was proposed that the number of formed leaves is decreased in the er erl1 erl2 mutant (Uchida
et al., 2012b). In addition, down-regulation of the ERf signaling pathway in tomatoes by a
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dominant negative version of ER resulted in reduced leaf formation (Villagarcia et al., 2012). To
explore ERf function in leaf initiation in more detail and to obtain quantitative data we analyzed
single, double and triple mutants of ERf genes under long and short day conditions. Under long
day conditions no change in the leaf formation rate in erl1, erl2, erecta, and erl1 erl2 mutants was
detected; however, er erl1 and especially er erl2 formed leaves at a slower rate (Fig. 1A). The
smaller number of visible leaves in the mutants could be due to a reduction in primordia initiation
or to a decreased growth rate of formed primordia, or both. To address the underlying cause of
the reduced visible leaf number, we analyzed the structure of SAMs during early seedling
development in er erl2 and er erl1 erl2 mutants. This experiment detected a very small but
statistically significant decrease in leaf initiation in the er erl2 mutant at day five (p<0.01; based
on Student t-test here and below) and at day seven (p<0.0001) post germination (Fig. 1B). In
addition, in er erl2 we observed decreased longitudinal primordia growth from day one to day
three (Supplemental Fig. S1). While at day one there was no significant difference between the
size of first true leaves in the wild type and er erl2, from day one to day three these leaves
increased in size 3.17 times on average in the wild type versus 2.67 times in er erl2. Thus, the
reduced number of visible leaves in the er erl2 mutant is likely caused by both a slight reduction in
leaf primordia initiation and a reduced rate of leaf elongation. The change in leaf primordia
initiation was much more dramatic in the er erl1 erl2 mutant (Fig. 1B and 2A). While all of the wild
type and er erl2 seedlings had formed at least two leaf primordia by day one post germination,
most of the er erl1 erl2 seedlings (72%) did not have a single primordium and 28% had only one
tiny primordium. On day three a majority of the wild type and er erl2 seedlings had four primordia
whereas 54% of er erl1 erl2 seedlings did not have any, 36% had one, and 10% had two.
Since changes in the rate of leaf formation are easier to detect when plants are grown in
short days, we also observed our mutants under those conditions. We did not detect a novel
phenotype in the single erl1 and erl2 mutants, but er mutants formed leaves at a slightly slower
rate (Fig. 1C). To check whether this phenotype could be rescued by the ERpro:ERECTA construct
(Godiard et al., 2003), we analyzed the rate of leaf formation in short days in segregated T2
families derived from 4 independent transgenic lines. All 4 transgenic lines contained a single
ERpro:ERECTA insertion in the er background. Plants containing the construct were easily
identified at maturity based on the length of pedicels and siliques and on plant height. In all four
lines we observed that the rate of leaf formation in er plants with the ERpro:ERECTA construct
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increased to the wild type levels (Fig. 1D). Analysis of double mutants in short days demonstrated
that the addition of the erl2 mutation to er further decreased the rate of leaf formation, while the
addition of erl1 did not (Fig. 1C). The phenotype was most severe in the triple er erl1 erl2 mutant,
with leaves appearing ~2.4 times slower compared to the wild type. Interestingly, erl1 erl2
mutants formed leaves at a faster rate than the wild type. Based on these mutant phenotypes, we
conclude that all three receptors help to control leaf formation in short days with ER being more
effective than ERL1 and ERL2. The increased leaf formation rate in the erl1 erl2 mutant may be
due to improved efficiency of ER in the absence of ERL1 and ERL2, as it does not need to compete
with the other receptors for ligands or other components of the signaling pathway.
To determine whether ERf genes are important for establishment of phyllotaxy we attempted to
measure leaf divergence angles in er erl1 erl2 seedlings grown in soil. However, due to very short
petioles and altered shape of leaf blades in the mutant this approach did not provide reliable
data. We tehn examined leaf phyllotaxy directly in shoot apices using scanning electron
microscopy (SEM). This experiment demonstrated that leaf primordia divergence angles in er erl1
erl2 mutants strongly deviated from the 137o found in the wild type plants (Fig. 2,A-D). While in
the wild type new leaf primordia appeared consecutively and as far as possible from already-
formed primordia in er erl1 erl2 primordia often formed almost simultaneously immediately
adjacent to each other. Measurement of divergent angles between successive siliques along the
main inflorescence stem also demonstrated differences in phyllotactic patterning between the
wild type and the er erl1 erl2 mutant (Fig 2, E and F). In er erl1 erl2 we observed decreased
stability of the phyllotactic pattern with an increased number of flower primordia forming at
angles significantly different from 137o. In the mutant only 31% of flower primordia formed at the
angle between 120 o and 150o while in the wild type 64% did. The average divergence angle of
flower primordia were 138.0±3.1o (±SE; n=126) in wild type and 159.0±5.5o (±SE; n=102) in the
mutant. Thus, ERf receptors are essential for establishment of leaf phyllotaxy and they contribute
strongly to phyllotactic patterning of flower primordia.

ERf Genes Regulate Size of the SAM
Formation of a leaf primordium requires a sufficient number of founder cells, and
reduced leaf initiation could be related to decreased meristem size. However, this is not the case
for the er erl1 erl2 mutant. Recent work suggests that at day nine er erl1 erl2 mutants have flatter
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and broader meristems (Uchida et al., 2013). To obtain quantitative data at earlier developmental
stages we analyzed meristem size at day one and day five post germination. In both cases we
observed that the meristem in er erl1 erl2 is approximately two times broader compared to either
the wild type or er erl2 (Fig. 3, A and B). This result is consistent with the timing of ERf expression
in the shoot meristematic region. Analysis of ER, ERL1, and ERL2 promoter-GUS fusions
demonstrated that ERf genes are not expressed in mature dry seeds or in seeds imbibed in water
before germination (Supplemental Fig. S2, A-C), but their expression becomes noticeable during
testa rupture in shoot and root meristematic regions (Supplemental Fig. S2, D-F). During the next
24 hours the expression of all three ERf genes dramatically increases in the SAM and in forming
leaf primordia (Supplemental Fig. S2, G-I and M-R). This upregulation of ERf gene expression
during germination is not dependent on light, as a similar pattern of expression was detected in
etiolated seedlings (Supplemental Fig. S2, J-L). The increased size of SAMs in er erl1 erl2 is linked
with raised expression of WUSCHEL (WUS) and SHOOT MERISTEMLESS (STM) (Fig. 4A), key
regulators of meristem development (Ha et al., 2010). At the same time, we observed decreased
expression of ASYMMETRIC LEAVES1 (AS1) (Fig. 4B), a MYB transcription factor involved in
specification of cotyledons and leaves (Byrne et al., 2000) and of AINTEGUMENTA (ANT), a gene
expressed in the incipient leaf primordia (Long and Barton, 2000). A closer look at the meristemic
region in er erl1 erl2 mutants demonstrated that cells in the L1 and L2 layers were significantly
wider (Fig. 3, C and D) and thus, while er erl1 erl2 meristems were twice as broad, the number of
cells in the L1 and L2 layers was only moderately increased. Therefore, the increase of the er erl1
erl2 SAM size cannot be caused solely by increased cell proliferation due to WUS overexpression
or by decreased incorporation of cells into leaf primordia.

YODA and a GSK3-like Kinase Function Downstream of ERfs in the SAM
A MAP kinase cascade consisting of YODA, MKK4, MKK5, MPK3, and MPK6 functions
downstream of ERf receptors, regulating both stomata development and growth along the
proximal/distal axis (Bergmann et al., 2004; Wang et al., 2007; Meng et al., 2012). To investigate
whether ERfs use the same signaling cascade in the meristem during leaf initiation we analyzed
the ability of constitutively active YODA (CA-YODA) (Lukowitz et al., 2004) to rescue the SAM
defects of the er erl1 erl2 mutant. Expression of CA-YODA in the mutant increased leaf primordia
initiation and their growth rate (Fig. 5, A and B) and decreased the size of the meristem (Fig. 5C).
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Recently it was proposed that a GSK3-like kinase can module the ERf signaling pathway
(Gudesblat et al., 2012; Kim et al., 2012). One of the experiments supporting this conclusion
demonstrated the ability of bikinin, a highly specific inhibitor of GSK3-like kinases, to rescue the
stomata clustering phenotype of the er erl1 erl2 mutant (Kim et al., 2012). We found that
treatment of er erl1 erl2 with bikinin can also partially rescue leaf initiation (Fig. 5D).
Unfortunately it was not possible to measure meristem size as bikinin treatment changed the
meristem shape from dome to concave in both wild type seedlings and the mutant, and we were
not able to clearly define the meristematic zone. Together, these experiments suggest that in the
SAM the signal from ERfs is transduced by a similar mechanism as during epidermis development
or plant elongation along the proximal/distal axes.

Auxin Distribution is Abnormal in er erl1 erl2 Seedlings
Formation of an auxin maximum in the meristem is the first sign of new leaf primordium
initiation. To determine the distribution of auxin in the meristematic region of er erl1 erl2 mutants
we observed the expression of DR5rev::GFP, where the eGFP protein is targeted to the
endoplasmic reticulum (Friml et al., 2003). In the wild type vegetative SAM the DR5rev::GFP signal
was observed to form maxima in the L1 layer and directly below in the internal tissues (Fig. 6, A
and B). In er erl1 erl2 we observed very high expression of DR5rev::GFP in the L1 layer of the
meristem but no maxima formation. A spotted expression of DR5rev::GFP was sometimes noticed
in the internal tissues of the SAM, but it did not form well-defined stripes as in the wild type. In
addition, DR5rev::GFP expression differed in formed leaf primordia. In the wild type DR5rev::GFP
was expressed at the tips of incipient primordia in a very limited region of the L1 layer (Fig. 6, A
and B) while in the mutant the expression at the tips was much broader (Fig. 6, C and D). But the
most dramatic difference of DR5rev::GFP expression was observed in the vasculature of
hypocotyls where this construct was very highly expressed in the wild type but not in the mutant
(compare Fig. 6E with 6F). Since formation of auxin maxima in the meristem and auxin flow onto
the vasculature are critical for leaf initiation (Braybrook and Kuhlemeier, 2010), the observed
abnormalities in auxin distribution in er erl1 erl2 should be unfavorable for efficient leaf initiation.
The changed distribution of auxin in er erl1 erl2 correlates with drastically reduced
expression of the auxin-inducible genes MONOPTEROS (MP), IAA1, and IAA19 (Fig. 4B). However,
we were unable to detect substantial changes in expression of enzymes involved in the
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