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RESEARCH ARTICLE
GTL1 and DF1 regulate root hair growth through
transcriptionalrepression of ROOT HAIR DEFECTIVE 6-LIKE 4 in
ArabidopsisMichitaro Shibata1,¶, Christian Breuer1,*,¶, Ayako
Kawamura1, Natalie M. Clark2,3, Bart Rymen1,Luke Braidwood1,‡,
Kengo Morohashi4, Wolfgang Busch5,§, Philip N. Benfey6, Rosangela
Sozzani2,3 andKeiko Sugimoto1,**
ABSTRACTHow plants determine the final size of growing cells is
an important,yet unresolved, issue. Root hairs provide an excellent
model systemwith which to study this as their final cell size is
remarkably constantunder constant environmental conditions.
Previous studies havedemonstrated that a basic helix-loop helix
transcription factor ROOTHAIR DEFECTIVE 6-LIKE 4 (RSL4) promotes
root hair growth, buthow hair growth is terminated is not known. In
this study, wedemonstrate that a trihelix transcription factor
GT-2-LIKE1 (GTL1)and its homolog DF1 repress root hair growth in
Arabidopsis. Ourtranscriptional data, combined with genome-wide
chromatin-bindingdata, show that GTL1 and DF1 directly bind the
RSL4 promoter andregulate its expression to repress root hair
growth. Our data furthershow that GTL1 and RSL4 regulate each
other, as well as a set ofcommon downstream genes, many of which
have previously beenimplicated in root hair growth. This study
therefore uncovers a coreregulatory module that fine-tunes the
extent of root hair growth by theorchestrated actions of opposing
transcription factors.
KEY WORDS: Root hair, Cell growth, Trihelix transcription
factor,Gene regulatory network
INTRODUCTIONPlant cells often undergo extensive post-mitotic
cell expansion andcan reach up to several hundred-fold their
original size (Sugimoto-Shirasu and Roberts, 2003). Controlling the
final size of post-mitotically growing cells is of fundamental
importance, as failure inthis control can result in severe defects
in plant organ growth anddevelopment (Braidwood et al., 2014;
Breuer et al., 2010). Growing
to an optimal size is also physiologically relevant for
somespecialized cell types such as root hairs, which must increase
theirsurface area to permit better uptake of nutrients and water
from thesurrounding environment (Grierson et al., 2014). Root hairs
grow ina polarized fashion through a localized deposition of cell
wallmaterials at the root hair apex. This is enabled by highly
directionalmembrane trafficking towards the growing tip of cells
andsubsequent exocytosis of vesicles that contain cell
wallpolysaccharides and cell wall proteins that need to be
incorporatedinto newly developing cell walls (Grierson et al.,
2014). As formany other cell types in plants, root hair growth is
oftenaccompanied by an increase in nuclear DNA content, or
ploidy,through successive rounds of endoreduplication (Breuer et
al., 2007;Sugimoto-Shirasu et al., 2005, 2002). It is also known,
however,that root hair growth is ploidy independent to some degree
as roothairs can elongate without changing their ploidy levels (Yi
et al.,2010).
Root hairs in Arabidopsis thaliana (Arabidopsis) have served
asan excellent model system for studying cell size control in
plants,and molecular genetic studies over the past few decades
haveuncovered key regulatory mechanisms that control root hair
growth.Given that root hairs are formed in a specific pattern of
cell files inArabidopsis roots, initiation of root hair outgrowth
is regulated by agenetic program that determines cell fate
(Grierson et al., 2014;Salazar-Henao et al., 2016). Key regulators
that translate thesedevelopmental cues into hair initiation are the
basic helix-loop-helix(bHLH) transcription factor ROOT HAIR
DEFECTIVE 6 (RHD6)and its close homolog RHD6-LIKE 1 (RSL1) (Masucci
andSchiefelbein, 1994; Menand et al., 2007). RHD6, together
withRSL1, induces the expression of another RHD6 homolog ROOTHAIR
DEFECTIVE 6-LIKE 4 (RSL4), leading to the accumulationof RSL4
proteins prior to the initiation of hair outgrowth (Dattaet al.,
2015; Yi et al., 2010). Remarkably, constitutiveoverexpression of
RSL4 by the Cauliflower Mosaic Virus 35Spromoter is able to
maintain root hair elongation until the hair cellsdie (Yi et al.,
2010), indicating that RSL4 is sufficient to promoteroot hair
growth. Several recent studies have identified 132 genesthat are
regulated by RSL4 and have shown that RSL4 promotes theexpression
of these target genes by binding the root hair specific cis-element
(RHE) in their promoter sequences (Hwang et al., 2017;Kim et al.,
2006; Vijayakumar et al., 2016; Won et al., 2009; Yiet al., 2010).
As expected, RSL4 target genes include those involvedin cell wall
biosynthesis and remodeling, vesicle trafficking, cellularsignaling
and metabolism, thus highlighting how the RSL4-mediated
transcriptional program orchestrates various subcellularprocesses
required for root hair growth.
Root hair growth is also fine-tuned by various hormonal
andenvironmental cues, and several studies have shown that some
ofthis regulation involves transcriptional upregulation of RSL4
andReceived 1 October 2017; Accepted 9 January 2018
1RIKEN Center for Sustainable Resource Science, Yokohama
230-0045, Japan.2Department of Plant and Microbial Biology, North
Carolina State University,Raleigh, NC 27708, USA. 3Biomathematics
Graduate Program, North Carolina StateUniversity, Raleigh, NC
27695, USA. 4Department of Applied Biological Science,Faculty of
Science and Technology, Tokyo University of Science, Noda
278-8510,Japan. 5Gregor Mendel Institute (GMI), Austrian Academy of
Sciences, ViennaBiocenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna,
Austria. 6Department of Biology,Howard Hughes Medical Institute,
Duke University, Durham, NC 27695, USA.*Present address: PtJ,
Division Bioeconomy BIO7, Forschungszentrum Jülich,52425 Jülich,
Germany. ‡Present address: Department of Plant Sciences,University
of Cambridge, Cambridge CB2 3EA, UK. §Present address:
PlantMolecular and Cellular Biology Laboratory, Salk Institute for
Biological Studies, LaJolla, CA 92037, USA.¶These authors
contributed equally to this work
**Author for correspondence ([email protected])
M.S., 0000-0002-7008-8437; B.R., 0000-0003-3651-9579; L.B.,
0000-0002-0233-3605; K.S., 0000-0002-9209-8230
This is an Open Access article distributed under the terms of
the Creative Commons AttributionLicense
(http://creativecommons.org/licenses/by/3.0), which permits
unrestricted use,distribution and reproduction in any medium
provided that the original work is properly attributed.
1
© 2018. Published by The Company of Biologists Ltd | Development
(2018) 145, dev159707. doi:10.1242/dev.159707
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subsequent activation of its downstream pathway (Franciosini et
al.,2017; Marzol et al., 2017; Yi et al., 2010). Among various
planthormones, auxin is well known to enhance root hair growth
(Knoxet al., 2003; Lee and Cho, 2013; Pitts et al., 1998). A recent
studydemonstrated that several AUXIN RESPONSE FACTORs (ARFs),which
are central transcriptional regulators of auxin signaling, bindthe
RSL4 promoter and directly activate its expression (Manganoet al.,
2017), providing the first molecular link between auxinsignaling
and transcriptional control of root hair development(Zhang et al.,
2016). Exogenous application of ethylene alsopromotes root hair
growth (Pitts et al., 1998) and this physiologicalresponse is
accompanied by increased RSL4 expression. Limitedphosphate
availability is another trigger for extended root hairgrowth, and
this regulation also involves upregulation of RSL4expression (Datta
et al., 2015; Yi et al., 2010).Accumulating genetic evidence
suggests that plants are also
equipped with a regulatory system to actively repress root
hairgrowth. For example, double mutants in the bHLH
transcriptionfactors Lotus japonicas ROOTHAIRLESS LIKE 4 (LRL4)
andLRL5 produce longer root hairs compared with wild-type
plants(Breuninger et al., 2016), demonstrating that root hair
growth isnegatively regulated by LRL4- and LRL5-dependent
mechanisms.It has also been reported that ROOTHAIR SPECIFIC 1
(RHS1) andRHS10, which encode a calcium-binding protein and a
receptor-likekinase, respectively, repress root hair growth, as
mutating eithergene results in extended hair growth (Hwang et al.,
2016;Won et al.,2009). These observations thus suggest that there
are multiple levelsof regulation by which root hair growth can be
blocked, although theexact molecular details of this control remain
unknown.We have previously reported a transcriptional mechanism
that
terminates cell growth in Arabidopsis leaf trichomes, another
celltype that undergoes extensive post-mitotic cell expansion
(Breueret al., 2009). Loss-of-function mutants in the trihelix
transcriptionfactor GT-2-LIKE 1 (GTL1) develop larger trichomes
than wildtype, and this phenotype is associated with an increase in
nuclearDNA content (Breuer et al., 2009). We showed that
GTL1terminates cell growth in a ploidy-dependent manner by
repressing the expression of CELL CYCLE SWITCH PROTEIN52 A1
(CCS52A1), a key driver of plant endoreduplication (Breueret al.,
2012). Whether GTL1 acts as a general repressor of cellgrowth
remains an unresolved issue as gtl1 single mutants do notdisplay
obvious growth defects beyond trichomes (Breuer et al.,2009). We
reasoned that there may be other transcription factorsacting
redundantly with GTL1. In support of this notion, anothertrihelix
protein, called DF1, has twin trihelix binding domains thatshow 70%
amino acid sequence identity with GTL1 (Breuer et al.,2009). In
this study, we have characterized gtl1 df1 double mutantsand
tissue-specific overexpression lines of GTL1 and DF1 todemonstrate
that both GTL1 and DF1 negatively regulate root hairgrowth. Our
data from transcriptional and chromatinimmunoprecipitation (ChIP)
studies suggest that GTL1 and DF1directly repress RSL4 as well as a
set of genes previously implicatedin root hair growth. We further
used gene regulatory network (GRN)inference and mathematical
modeling to show that GTL1 and RSL4likely form a negative-feedback
loop to cooperatively control roothair growth.
RESULTSGTL1 and DF1 repress root hair growth through a
ploidy-independent mechanismTo explore the functional redundancy
between GTL1 and DF1, weisolated an Arabidopsis T-DNA insertion
mutant for DF1 thatresulted in a null allele (Fig. S1A). As shown
in Fig. 1A, root hairgrowth of 7-day-old gtl1-1 and df1-1 single
mutants isindistinguishable from wild type. In contrast, we found
that roothairs in gtl1-1 df1-1 double mutants are significantly
longercompared with wild type, and their final hair length is, on
average,more than 200 μm longer than wild type (Fig. 1A,B). Long
roothairs in gtl1-1 df1-1 could be caused by either faster growth
and/oran extended period of their growth. Our time-lapse analysis
showedthat the rate of root hair growth is comparable between wild
type andgtl1-1 df1-1 but gtl1-1 df1-1 root hairs continue to grow
after wild-type root hairs halt their growth (Fig. 1C). To confirm
that thisgrowth phenotype is caused by the lack of GTL1 and DF1,
we
Fig. 1. GTL1 and DF1 repress roothair growth in Arabidopsis. (A)
Roothair phenotypes of wild-type, gtl1-1,df1-1, gtl1-1 df1-1,
pEXP7:GTL1-GFPand pEXP7:DF1-GFP seedlings. Scalebar: 500 μm. (B)
Quantitative analysisof root hair length. Data are mean±s.e.m.
(n=120). Different letters indicatemeans that differ significantly
(Tukey-Kramer test, P
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transformed gtl1-1 df1-1 plants with pGTL1:GTL1-GFP or
pDF1:DF1-GFP constructs. Introduction of either of these constructs
fullyrescues the hair growth phenotype, indicating that both GTL1
andDF1 contribute to the termination of root hair growth (Fig.
S1B,C).Confocal microscopy revealed that both GTL1-GFP and DF1-
GFP proteins are broadly expressed along the longitudinal axis
ofroots, and we detected clear GFP expression in expanding root
hairs(Fig. S2). To test whether overexpression of GTL1 and DF1
issufficient to inhibit root hair growth, we ectopically
expressedGTL1-GFP andDF1-GFP under the root-hair specific
EXPANSIN7(EXP7) promoter (Cho and Cosgrove, 2002). The resulting
pEXP7:GTL1-GFP and pEXP7:DF1-GFP plants showed an increase in
theexpression of GTL1-GFP and DF1-GFP by 15-fold and
30-fold,respectively (Fig. S1D). Importantly, these plants had
significantlyshort, often undetectable, root hairs (Fig. 1A,B),
demonstrating thatectopically expressed GTL1 and DF1 can repress
root hair growth.Next, we tested whether the root hair phenotypes
in gtl1-1 df1-1
mutants are accompanied by changes in ploidy levels by
examining4′6-diamidino-2-phenylindole (DAPI)-stained nuclei in
fully grownroot hairs. We observed that the size of DAPI-stained
nuclei iscomparable between wild-type and gtl1-1 df1-1 root hairs,
andquantitative analysis of DAPI-stained nuclei confirmed that
thenuclear DNA content is not different between wild-type and
gtl1-1df1-1 root hairs (Fig. S3). Consistently, unlike in
trichomes, theccs52a1 mutation does not rescue the hair growth
phenotype ofgtl1-1 df1-1 (Fig. S3C,D), indicating that CCS52A1 does
not actdownstream of GTL1 and DF1 in root hair development.
Theseobservations suggest that GTL1 and DF1 control root hair
growthploidy independently.
GTL1 and DF1 directly repress RSL4 expression in rootsIt has
been previously shown that RSL4 promotes root hair growthin a
ploidy-independent manner and without affecting theelongation rate
(Yi et al., 2010). As the extended root hair growthin the gtl1 df1
loss-of-function line resembles that of the 35S:RSL4line, we asked
whether the level of RSL4 expression is increased ingtl1-1 df1-1
mutants. As shown in Fig. 2A, our RT-qPCR analysisrevealed that
RSL4 expression is significantly upregulated in gtl1-1df1-1 double
mutants, suggesting that GTL1 and DF1 are requiredto repress RSL4
expression. We also found that RSL4 expression isdownregulated in
pEXP7:GTL1-GFP roots (Fig. 2A), indicatingthat ectopic
overexpression of GTL1 is sufficient to block RSL4expression.
Intriguingly, overexpression of DF1 does not causesignificant
downregulation of RSL4 in pEXP7:DF1-GFP roots(Fig. 2A), implying
that GTL1 has a stronger impact on RSL4expression. In addition, our
RT-qPCR analysis revealed that, amongthe RHD6/RSL homologs, RHD6
expression is elevated in the gtl1-1df1-1 doublemutants but its
expression is not significantly changed inpEXP7:GTL1-GFP and
pEXP7:DF1-GFP roots (Fig. 2A).Conversely, we did not detect
significant changes in RSL1, RSL2and RSL3 expression in the gtl1-1
df1-1 double mutant, but we didobserve that RSL2 and RSL3 are
downregulated in pEXP7:GTL1-GFP and pEXP7:DF1-GFP roots (Fig. 2A).
These results suggestthat the primary target of GTL1 and DF1 in
root hair development isRSL4, because among the RHD6/RSL homologs,
RSL4 shows asignificant transcriptional response in both GTL1/DF1
gain-of-function and loss-of-function lines.To investigate whether
transcriptional activation of RSL4 leads to
the increased levels of RSL4 proteins in root hairs, we
introducedthe pRSL4:GFP-RSL4 construct (Yi et al., 2010) into
gtl1-1 df1-1double mutants and examined the GFP-RSL4 accumulation
byconfocal microscopy. As previously reported, GFP-RSL4
proteins
accumulate in trichoblast cells prior to root hair initiation
and theiraccumulation sharply declines when hair cells complete
theiroutgrowth in thewild-type background (Yi et al., 2010 and Fig.
2B).In sharp contrast, we found that GFP-RSL4 detection persists
muchlonger in maturing root hairs in the gtl1-1 df1-1
background.Accordingly, our quantitative analysis showed that the
number ofRSL4-GFP-expressing cells in trichoblast cell files is
significantlyincreased in gtl1-1 df1-1 roots (Fig. 2B,C). In order
to test whetherRSL4 upregulation is responsible for the extended
hair growth ingtl1-1 df1-1, we introduced the rsl4-1mutation into
the gtl1-1 df1-1mutant background. As shown in Fig. 3A,B,
introduction of rsl4-1into gtl1-1 df1-1 rescues the root hair
phenotype in gtl1-1 df1-1,further substantiating that RSL4 is a key
regulator of root hairgrowth acting downstream of GTL1 and DF1. We
subsequentlyinvestigated whether GTL1 and DF1 directly bind the
RSL4promoter by immunoprecipitating GTL1-GFP and DF1-GFPproteins
from 7-day-old pGTL1:GTL1-GFP and pDF1:DF1-GFProots. Our chromatin
immunoprecipitation (ChIP) followed byquantitative PCR (ChIP-qPCR)
analysis showed an enrichment ofboth GTL1-GFP and DF1-GFP around
500-1000 bp upstream of theRSL4 start codon containing two binding
motifs for GTL1 (Breueret al., 2012) (Fig. 3C). To further support
these results, we co-bombarded the p35S:GTL1 construct and
pRSL4:LUC promoterinto ArabidopsisMM2D culture cells and tested
whether GTL1 canrepress the RSL4 promoter activity. As shown in
Fig. S4, applicationof p35S:GTL1 significantly suppresses the
expression of pRSL4:LUC compared with the vector control. These
results thus suggestthat GTL1 and DF1 directly bind the promoter
region of RSL4 torepress its expression.
GTL1 and DF1 act in a parallel pathway with auxin signalingto
regulate root hair developmentAuxin is known to promote root hair
growth by activating theexpression of RSL4 (Mangano et al., 2017;
Yi et al., 2010). Toinvestigate whether GTL1 and DF1 are also
involved in auxin-induced root hair growth, we first studied the
auxin response in gtl1-1 df1-1 mutants, as well as in GTL1 and DF1
overexpression lines.As previously reported (Grierson et al.,
2014), wild-type plantsgrown in the presence of 10 nM
indole-3-acetic acid (IAA) displaysignificantly longer root hairs
compared with control plants(Fig. 4A,B). In contrast, root hair
growth is comparable betweencontrol and IAA-treated gtl1-1 df1-1
mutants (Fig. 4A,B). We nexttested whether IAA can rescue the
compromised hair growth inpEXP7:GTL1-GFP and pEXP7:DF1-GFP plants
but found thatIAA has little to no impact on root hair growth in
theseoverexpression lines (Fig. 4A,B). Conversely, the application
ofan auxin inhibitor, auxinole (Hayashi et al., 2012),
completelysuppresses root hair growth in wild type, gtl1-1 df1-1
and GTL1 orDF1 overexpression lines (Fig. 4A,B). These results
togethersuggest that GTL1 and DF1 likely function independently
fromauxin signaling in controlling root hair growth.
It has been demonstrated previously that the application
ofanother synthetic auxin, 1-naphthaleneacetic acid
(NAA),upregulates RSL4 expression (Yi et al., 2010). Consistently,
ourRT-qPCR analysis revealed that IAA promotes RSL4 expressionand
that auxinole strongly suppresses its expression in both wild-type
and gtl1-1 df1-1 roots (Fig. 4C). Our RT-qPCR data, inaddition,
showed that overexpression of GTL1 and DF1 counteractsthis
auxin-induced RSL4 upregulation as the level of RSL4expression is
strongly reduced in pEXP7:GTL1-GFP and pEXP7:DF1-GFP plants,
regardless of treatment with IAA or auxinole(Fig. 4C). Unlike RSL4,
the expression of GTL1 and DF1 does not
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RESEARCH ARTICLE Development (2018) 145, dev159707.
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seem to be affected by IAA, although their expression tends to
belower in plants treated with auxinole (Fig. 4D). These
results,therefore, support the hypothesis that GTL1 and DF1 control
roothair growth via regulation of RSL4 in a parallel pathway with
auxin.In addition to auxin, phosphate (Pi) availability is well
known toaffect root hair growth (Datta et al., 2015; Yi et al.,
2010). Similar toauxin treatment, however, our RT-qPCR data showed
thatphosphate conditions do not change the expression of GTL1
andDF1 (Fig. 4E), suggesting that GTL1 and DF1 do not act via
thephosphate-mediated control of root hair growth.
GTL1 and DF1 directly regulate a subset of RSL4
targetgenesRecent studies have identified 132 genes that might be
directlyactivated by RSL4 in Arabidopsis roots (Hwang et al.,
2017;Vijayakumar et al., 2016; Won et al., 2009; Yi et al., 2010).
Havinguncovered that GTL1 and DF1 repress RSL4 expression, we
sought
to determine how much the GTL1/DF1-regulated gene
regulatorynetwork overlaps with that of RSL4. To identify genes
regulated byGTL1 and DF1 in root hairs, we collected root hair
cellsoverexpressing GTL1-GFP and DF1-GFP, respectively,
frompEXP7:GTL1-GFP and pEXP7:DF1-GFP plants by
fluorescence-activated cell sorting. We also collected root hair
cells from gtl1-1df1-1 plant expressing the root hair specific
pEXP7:NLS-GFPmarker (Ikeuchi et al., 2015) and compared their
transcriptionalprofile using the Affymetrix ATH1 microarray. We
defined GTL1or DF1 response genes as those that show more than a
twofoldchange of gene expression in gtl1-1 df1-1 plants compared
withpEXP7:GTL1-GFP or pEXP7:DF1-GFP plants (P
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genes are repressed by GTL1 (Fig. S5A and Table 1),
stronglysuggesting that GTL1 and RSL4 share many common targets
butthey act in opposing manners. For DF1, we identified 755
activatedand 918 repressed genes, but only 7 (5.3%) of the
repressed genesare RSL4 response genes (Fig. S5A, Table S1). Given
that all sevenof these RSL4 response genes are also repressed by
GTL1(Fig. S5B), we focused our remaining analysis on the 36
genescommonly regulated by GTL1 and RSL4.The 36 GTL1-RSL4 common
downstream genes include those
that encode proteins associated with cell wall biosynthesis
andremodeling (e.g. EXT12, XTH26 and LRX2), those likely acting
incellular signaling (e.g. RHS1, RLK genes and ACA12), and
thosewith functions in membrane trafficking or membrane
transport(e.g. ABCB3, ABCB5, IRT2 and COW1) (Table 1), suggesting
thatGTL1 and RSL4 regulate a wide range of subcellular
processesunderlying root hair growth. Having uncovered a
substantialoverlap between RSL4-activated genes and
GTL1/DF1-repressedgenes, we wanted to test whether GTL1 and DF1
also directly bindany of these genes. To achieve this, we performed
awhole-genomeChIP-chip analysis using pGTL1:GTL1-GFP and
pDF1:DF1-GFP roots. We identified 9200 putative direct targets for
GTL1and 7803 putative targets for DF1, 5208 (66.7% of DF1 targets)
ofwhich are common between GTL1 and DF1 (Z-score>1.5) (Fig.S6A
and Table S2). Using these ChIP-chip data, we found that 12(33.3%)
of the 36 targets shared between GTL1 and RSL4 aredirectly bound by
GTL1 (Table 1). We also found that DF1directly binds 11 (30.6%) of
the 36 genes but only four of these 11(36.4%) are transcriptionally
repressed by DF1 (Table 1),implying that the physical binding of
DF1 to the target promotersequence does not necessarily cause
downstream transcriptionalrepression. These data demonstrate that
GTL1, DF1 and RSL4control root hair growth by regulating partially
overlappingtranscriptional pathways.
GTL1 and RSL4 control root hair growth through a
negative-feedback loopOur transcriptome and ChIP-chip data suggest
that GTL1 and DF1directly regulate RSL4 and a subset of its
downstream targets. Tofurther investigate the regulatory roles of
GTL1 and DF1, weemployed gene network inference with ensemble of
tree 3(GENIE3) (Huynh-Thu et al., 2010) and inferred a
generegulatory network (GRN) among GTL1, DF1, RSL4 and their36
common target genes using our expression data from GTL1 andDF1
mutants (gtl1-1, df1-1, gtl1-1 df1-1) and from GTL1 and
DF1overexpression lines ( pEXP7:GTL1-GFP and pEXP7:DF1-GFP).We also
obtained the sign (positive/negative) of regulation
usingtime-course RNA-seq data from 3-, 4-, 5-, 6- and 7-day-old
wild-type roots (de Luis Balaguer et al., 2017). Our GRN predicts
thatGTL1 regulates 30 (83%) of the 36 downstream targets, 28 of
which(93%) are also predicted to be regulated by RSL4 (Fig. 5A).
Thesedata further substantiate that GTL1 and RSL4 co-regulate many
ofthese root hair genes to control root hair growth. Of the 30
targetsGTL1 is predicted to regulate, our sign algorithm predicts
that 29 ofthose (97%) are repressions (Fig. 6A). In contrast, our
signalgorithm predicts that of the 28 genes regulated by RSL4,
21(75%) are activated by RSL4 (Fig. 5A). In agreement with DF1
notplaying a central role in transcriptional repression of root
hair genes,we observed that DF1 has only one predicted downstream
target,XTH26, which is not predicted to be regulated by RSL4 (Fig.
5A).
Our GRN, in addition, predicted that RSL4 and GTL1 form
anegative-feedback loop (Fig. 5A). As positive regulation of GTL1by
RSL4 has also been reported by a previous study using
RSL4-inducible lines (Vijayakumar et al., 2016), we hypothesized
that thismutual regulation is important for establishing proper
root hairgrowth. To explore this reciprocal regulation further, we
developeda mathematical model between RSL4 and GTL1, and tested
whethertheir protein levels can reach a steady state. In addition
to the
Fig. 3. GTL1 and DF1 directly bind the RSL4 promoterand regulate
its expression. (A) Root hair phenotypes ofwild-type, gtl1-1 df1-1,
rsl4-1 and gtl1-1 df1-1 rsl4-1seedlings. Scale bar: 500 μm. (B)
Quantitative analysis ofroot hair length. Data are mean±s.e.m.
(n=120). Differentletters indicate means that differ significantly
(Tukey-Kramertest, P
-
negative-feedback loop between GTL1 and RSL4, our ChIP-chipdata
and transient promoter-LUC data suggested that GTL1 is ableto
repress itself (Table S2 and Fig. S4). Thus, we incorporated
theauto-repression of GTL1 into the model. We incorporated
severalparameters, such as production and degradation rates of GTL1
andRSL4, to estimate the levels of GTL1 and RSL4 over time.
Ourmodel predicted that for certain parameter values, there exists
asteady state for GTL1 and RSL4, likely representing the
wild-typesituation where root hairs grow to a constant size (Fig.
5B andFig. S7A). We subsequently used a sensitivity analysis to
identifythe most important parameters in the model that influence
the steady
states of GTL1 and RSL4. Using Sobol decomposition, we foundthat
the production rate of RSL4, k1, and the production rate ofGTL1,
k2, are the two most important parameters in the model(Fig. S7B and
Table S4).
To test whether RSL4 and GTL1 alone are able to account forthe
observed root hair phenotypes, we subsequently modified thevalues
of their production rates (k1 and k2, respectively) andexamined the
changes in the steady state. First, to simulate GTL1overexpression,
we increased the production rate of GTL1 (k2)until the steady state
value of GTL1 was 15-fold higher than inwild type, as estimated by
our RT-qPCR data on pEXP7:GTL1-GFP plants (Fig. S1D). Our model
predicts that the steady stateof RSL4 is 16% of the value in wild
type (Fig. 5B, Fig. S7A),suggesting that, as GTL1 increases, RSL4
decreases and causesshorter root hairs, which is the phenotype we
observed inpEXP7:GTL1-GFP plants (Fig. 1A). Next, to simulate the
gtl1-1df1-1 mutant, we set the production rate of GTL1 (k2) to 0,
as ourRT-qPCR data show that GTL1 expression is effectively zero
ingtl1-1 df1-1 (Fig. S1D). Our model shows that in this scenariothe
steady state of RSL4 is 1.5-fold higher than in wild type,which is
supported by our RT-qPCR data (Figs 2A and 5B,Fig. S6A). These
results thus suggest how the expression levelsof GTL1 and RSL4 can
be altered to determine the final lengthof root hairs.
DISCUSSIONThe GTL1-RSL4 module in the GRN of root hair growthIn
this study we demonstrate that the final size of root hair cells
isregulated by coordinated action of GTL1 and RSL4, which serve asa
repressor and activator, respectively, of root hair growth.
Previousstudies have identified several negative regulators of root
hairgrowth (Breuninger et al., 2016; Hwang et al., 2016), but how
theysuppress root hair growth is not known. Our data uncover
aneffective strategy to fine-tune root hair growth that relies on
thenegative regulation of RSL4, which is one of the central hubs in
thetranscriptional network of root hair growth (Datta et al.,
2015;Marzol et al., 2017; Yi et al., 2010). It is interesting that
GTL1 andDF1 control only the duration of root hair growth and not
the rate ofhair growth. These observations are consistent with
previous datashowing that the ectopic RSL4 expression does not
change the rateof hair growth and only prolongs hair growth (Yi et
al., 2010). Theseresults thus suggest that these two parameters of
root hair growth canbe mechanistically uncoupled and, although the
GTL1-RSL4module controls the extent of root hair growth, other as
yetunknown pathways control the rate of root hair growth. As
gtl1-1df1-1 double mutants, but not their single mutants, display
root hairphenotypes (Fig. 1A,B), we hypothesized that GTL1 and DF1
workredundantly on root hair development. In agreement with this,
thereis a large overlap between GTL1- and DF1-response genes(Fig.
S5B and Table S1). DF1, however, has less of an impact onRSL4
expression than GTL1 (Fig. 2A), and our GRN predicts thatthe
contribution of DF1 to regulating known root hair genes is muchless
than GTL1 (Fig. 5A). These results thus suggest that DF1
mayfunction as a backup system for GTL1.
Our data show that GTL1 and RSL4 regulate a set of
commondownstream genes (Figs 5 and 6), which allows robust control
oftarget gene expression. Another important aspect of our root
hairGRN is that GTL1 and RSL4 form a negative-feedback loop
thatresults in steady-state levels of their expression, thus
permittingconsistent hair growth in wild-type plants (Fig. 5). In
addition toRSL4, our RT-qPCR analysis shows that RHD6 is also
upregulatedin gtl1-1 df1-1 mutants (Fig. 2A), suggesting that RHD6
is another
Fig. 4. GTL1 and DF1 regulate root hair growth in a parallel
pathway toauxin signaling. (A) Root hair phenotypes of wild-type,
gtl1-1 df1-1, pEXP7:GTL1-GFP and pEXP7:DF1-GFP seedlings treated
with 10 nM IAA or 10 μMauxinole. Scale bars: 300 μm. (B)
Quantitative analysis of root hair length. Dataare mean±s.e.m.
(n=60). Different letters indicate means that differsignificantly
(Tukey-Kramer test, P
-
potential target of GTL1 and/or DF1. Indeed, our ChIP-chip
datasupport this notion as both GTL1 and DF1 bind the
promotersequence of RHD6 (Table S2). Given that RHD6 directly
bindsRSL4 (Yi et al., 2010), this suggests a feed-forward loop
betweenGTL1, RHD6 and RSL4. Interestingly, expression levels
ofLotus japonicus ROOTHAIRLESS1-LIKE1 (LRL1), whichencodes a bHLH
transcription factor that acts as an activator ofroot hair growth
(Karas et al., 2009; Lin et al., 2015), is alsocorrelated with GTL1
and DF1 expression based on ourtranscriptome data (Table S1). Thus,
the GTL1-RSL4 GRN weunveiled in this study may be expanded in
future studies toinclude RHD6, LRL1 and perhaps other root hair
regulators tofurther understand the core transcriptional modules
that controlroot hair growth.
Physiological roles of GTL1 in root hair growthWe found that the
majority of the 36 GTL1-RSL4 common targetsare genes related to
cell wall biosynthesis and remodeling (Table 1and Fig. 6). We
believe this is reasonable as cell-wall construction isdirectly
involved in cell expansion. In addition, RHS1, one of theGTL1-RSL4
common downstream genes (Table 1 and Fig. 6), is acalmodulin-like
protein that is thought to regulate root hair growththrough Ca2+
signaling (Won et al., 2009). Another GTL1-RSL4target, CAN OF WORMS
1 (COW1), is implicated in membranetrafficking and required for
root hair tip growth, as its loss-of-function mutation causes
shorter root hairs (Böhme et al., 2004;Grierson et al., 1997).
GTL1-RSL4 common targets also includegenes associated with several
other physiological functions. IRONREGULATED TRANSPORTER 2 (IRT2),
for example, regulates
Table 1. Genes commonly regulated by GTL1, DF1 and RSL4
AGIdesignation
Genesymbol Molecular and biological function
Transcriptionalregulation
Promoterbinding
ReferenceGTL1 DF1 GTL1 DF1
At1g05990 RHS1 EF hand calcium-binding protein family −2.04 − +
+ 3At1g12950 RHS2 MATE efflux family protein −1.00 −1.15 + ++
3At1g27740 RSL4 bHLH transcription factor −1.08 − + + 1At1g30850
RHS4 A hypothetical protein of unknown function −1.59 − − −
1At1g31750 Proline-rich family protein −1.60 − − − 1At1g34330
Pseudogene, putative peroxidase −2.83 − NA NA 1At1g34510 Peroxidase
superfamily protein −5.99 − − − 1At1g34540 CYP94D1 Cytochrome P450,
involved in oxidation-reduction process −5.14 − − − 1At1g59850 ARM
repeat superfamily protein −4.57 − + − 1At1g61080
Hydroxyproline-rich glycoprotein family protein −5.02 −3.97 − −
1At1g62440 LRX2 Leucine-rich repeat/extensin, involved in cell wall
organization −1.41 − − − 3At1g63600 Receptor-like protein
kinase-related family protein −3.60 −1.17 − + 1At2g02990 RNS1
Ribonuclease T2 family −1.96 − − − 1At2g03980 GDSL-like
lipase/acylhydrolase superfamily protein, involved in lipid
catabolic process−6.19 −1.46 − − 1
At2g20520 FLA6 FASCICLIN-like arabinogalactan 6, plasma membrane
protein −1.01 − ++ − 1At2g25240 CCP3 Serine protease inhibitor
(SERPIN) family protein −3.01 − + − 1At3g60280 UCC3 Blue
copper-binding protein III, involved in the oxidation-reduction
process −2.27 − NA NA 1At3g63380 ACA12 ATPase E1-E2 type family
protein/haloacid dehalogenase-like hydrolase
family protein−1.25 − − − 1
At4g01820 ABCB3 ATP-BINDING CASSETTE B3, involved in basipetal
auxin transport −1.02 − − − 1At4g01830 ABCB5 ATP-BINDING CASSETTE
B5, involved in basipetal auxin transport −1.02 − − − 1At4g02270
RHS13 Required for optimal hair growth −2.06 − − − 1At4g13390 EXT12
Proline-rich extensin-like family protein, involved in cell wall
organization −1.55 − − ++ 1At4g19680 IRT2 Iron and zinc transporter
−1.96 − ++ + 1At4g22080 RHS14 Involved in pectin catabolic process
−2.75 − − − 1At4g22090 Involved in pectin catabolic process −2.75 −
++ − 1At4g25220 RHS15 Involved in carbohydrate transport −5.68
−1.82 − − 1At4g25790 CAP (cysteine-rich secretory proteins, antigen
5 and pathogenesis-related
1 protein) superfamily protein−5.10 −1.92 − + 1
At4g28850 XTH26 Xyloglucan endotransglucosylase/hydrolase,
involved in cell wallbiogenesis and organization
−1.42 − ++ ++ 1
At4g34580 COW1 A phosphatidylinositol transfer protein, involved
in cell tip growth −1.85 − − + 1At5g19800 HRGP2 A
hydroxyproline-rich glycoprotein family protein −1.01 − − −
1At5g22410 RHS18 Involved in hydrogen peroxide catabolic process
−1.11 − − − 1At5g24880 Chromodomain cec-like protein −1.01 − + −
1At5g26080 Proline-rich family protein −2.84 1.11 − − 1At5g43580
UPI Serine-type endopeptidase inhibitor, functions in resistance
to
necrotrophic fungi and insect herbivory−5.05 −2.37 + + 1
At5g58375 Methyltransferase-related protein −1.21 − NA NA
2At5g61650 CYCP4 P-type cyclins, involved in the cell cycle −1.15 −
++ ++ 1
Root hair-specific microarray analysis identifies 36 genes
regulated by both GTL1 and RSL4. Among them, seven genes are
co-regulated by DF1. Transcriptionalregulation by GTL1 and DF1 are
shown as log2(pEXP7:GTL1-GFP/gtl1-1 df1-1) and
log2(pEXP7:DF1-GFP/gtl1-1 df1-1), respectively. Promoter binding
ofGTL1 and DF1 is based on ChIP-chip analysis using pGTL1:GTL1-GFP
and pDF1:DF1-GFP plants.++, direct targets with a cut-off Z-score
of 2.0; +, direct targets with a cut-off Z-score of 1.5; −, no
signal enrichment; NA, not applicable.RSL4-dependent
transcriptional regulation of these genes has been reported by Yi
et al. (2010)1, Vijayakumar et al. (2016)2 and Won et al.
(2009)3.These transcriptome data of RSL4 are taken using whole
roots, thus they are not necessarily specific to root hairs.
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iron uptake from soil (Vert et al., 2009), and UNUSUAL
SERINEPROTEASE INHIBITOR (UPI) is involved in pathogen
resistance(Laluk andMengiste, 2011). Since root hairs result from
outgrowth ofthe root epidermis, they directly interact with the
microenvironmentof the soil. It is thus likely that these
microenvironments influence theextent of root hair growth, and this
interaction also needs to beregulated by GTL1. Consistently, our GO
analysis of GTL1 targetssuggests that GTL1 affects ‘response to
stimulus’, ‘response to stress’and ‘response to chemical stimulus’
(Fig. S5B), suggesting thatGTL1 regulates diverse arrays of
physiological processes associatedwith root hair growth.
ConclusionsIn this study we demonstrate that the final size of
root hair cells isregulated by coordinated action of GTL1 and RSL4,
which serve asa repressor and activator, respectively, of root hair
growth. Ourmathematical analysis suggests that GTL1-RSL4 functions
as a coremodule in root hair growth and regulates cell expansion,
as well asseveral other physiological responses such as nutrient
uptake andpathogen response. Our data, in addition, suggest that
otherpreviously described root hair regulators, such as RHD6
andLRL1, may function within a larger GRN that controls root
hairgrowth downstream of the GTL1-RSL4 module.
MATERIALS AND METHODSPlant materials and growth conditionsThe
gtl1-1, ccs52a1-2, rsl4-1, pGTL1:GTL1-GFP,
pRSL4:RSL4-GFP,pEXP7:GTL1-GFP and pEXP7:NLS-GFP lines have been
previously
described (Breuer et al., 2009, 2012; Ikeuchi et al., 2015; Yi
et al., 2010).The df1-1 (SALK_106258) mutant was obtained from the
ArabidopsisBiological Resource Center. All mutants and transgenic
lines used in thisstudy were in the Columbia-0 background. Plants
were grown on half-strength Johnson media with 6 g/l gelzan (Sigma)
and final concentration ofphosphate adjusted to 1 mM (Johnson et
al., 1957; Ma et al., 2001). Forauxin and auxin inhibitor
treatment, a solution of 1 mM IAA and 30 mMauxinole (Hayashi et
al., 2012) in mixed DMSO and ethanol were added tothe Johnson media
to final concentrations of 10 nM and 10 μM,respectively. Phosphate
media were prepared based on recipes of Ma et al.(2001).
Plasmid construction and plant transformationFor the
construction of the pDF1:DF1-GFP vector, a 4900 bp genomicfragment
of the DF1 locus was amplified from the BAC clone F7O12 andcloned
into the pENTR/D-TOPO vector (Invitrogen). A SmaI restriction
sitewas introduced upstream of the translational stop codon by
site-directedmutagenesis. A SmaI-digested GFP fragment was then
inserted to create aC-terminal translational fusion construct and
the resulting construct wascloned into the pGWB1 binary vector
(Nakagawa et al., 2007). The pEXP7:DF1-GFP vector was generated by
combining pDONR-pEXP7 and pENTR-DF1-GFP together with the R4pGW501
destination vector (Nakagawaet al., 2008) as described by Ikeuchi
et al. (2015). A set of primers used forPCR amplification is
provided in Table S5. All plant transformation wascarried out using
the floral dip method (Clough and Bent, 1998).
RNA extraction and RT-qPCR analysisTotal RNAwas extracted from
7-day-old roots using an RNeasy Plant MiniKit (Qiagen). Extracted
RNA was reverse transcribed using a PrimeScriptRT-PCR kit with
DNase I (Perfect Real Time) (Takara) in accordance with
Fig. 5. Modeling predicts that GTL1 and RSL4 form a
negative-feedback loop and regulate a set of common target genes.
(A) Gene regulatory network(GRN) of GTL1, RSL4 and their shared
downstream targets. Transcriptional activation and repression are
indicated by arrows and bars, respectively. A time-course
transcriptome of elongation and differentiation zones was used to
predict the transcriptional activation and repression of target
genes. Red edgesrepresent direct promoter binding validated using
ChIP-chip data. (B) Model simulation of GTL1 and RSL4 dynamics in
wild-type (left), pEXP7:GTL1-GFP(middle) and gtl1-1 df-1 mutant
(right) roots. Red and blue lines represent model solutions for the
RSL4 and GTL1 equations, respectively. The value of k2,
theproduction rate of GTL1, was varied from the wild-type case so
that the steady-state values of GTL1 in pEXP7:GTL1-GFP and gtl1-1
df-1 roots meet the valueestimated from our RT-qPCR data.
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the accompanying protocol. Transcript levels were determined by
RT-qPCRusing a THUNDERBIRD SYBR qPCR Mix kit (Toyobo) and an
Mx399PQPCR system (Agilent). The expression of the UBQ10 gene was
used as areference (Shibata et al., 2013). A set of primers used
for RT-qPCR isprovided in Table S5.
Fluorescence-activated cell sorting and microarray analysisTo
identify genes regulated by GTL1 and DF1 in root hairs,
GFP-positivecells were sorted from 5-day-old wild-type and gtl1-1
df1-1 roots carryingpEXP7:NLS-GFP, as well as from pEXP7:GTL1-GFP
and pEXP7:DF1-GFP roots. The root tips were dissected at ∼0.5 cm
from root tips and GFP-positive protoplasts were isolated by
fluorescence-activated cell sorting,following the protocol
described by Sozzani et al. (2010). Total RNA wasextracted from
three biological replicates and labeled probes were used
forhybridization on ATH1 chips (Affymetrix). Microarray data were
analyzedusing R software and the gcRMA implementation with
AffylmGUI(Wettenhall et al., 2006) of the Bioconductor package as
previouslydescribed (Morohashi and Grotewold, 2009; Sozzani et al.,
2010).Microarray data have been deposited in Gene Expression
Omnibus(https://www.ncbi.nlm.nih.gov/geo/) under accession number
GSE103917.
ChIP-chip and ChIP-qPCR analysisChIP-chip experiment was
performed using 5-day-old pGTL1:GTL1-GFPand pDF1:DF1-GFP roots as
previously described (Sozzani et al., 2010).GTL1-GFP and DF1-GFP
proteins were immunoprecipitated from threebiological replicates
using antibodies against GFP (ab290, Abcam). Bovineserum albumin
was used as a negative control. Labeled DNAwas hybridizedto a
custom-made Arabidopsis promoter microarray (Sozzani et al.,
2010).ChIP-chip datawere analyzed as previously described (Sozzani
et al., 2010).
Relatively low Z-scores were chosen to decrease the number of
falsenegatives and identify meaningful overlaps with the expression
data. ChIP-chip data have been deposited in Gene Expression Omnibus
(https://www.ncbi.nlm.nih.gov/geo/) under accession number
GSE104010. ChIP-qPCRanalysis was performed using 7-day-old
pGTL1:GTL1-GFP and pDF1:DF1-GFP roots as previously described
(Rymen et al., 2017). Data werenormalized against input DNA and
shown as relative enrichment of DNAimmunoprecipitated at the TA3
retrotransposon locus (Yamaguchi et al.,2014). A set of primers
used for ChIP-qPCR is provided in Table S5.
Promoter-luciferase assayTo construct the firefly luciferase
(LUC) reporter vector, a 1.8 kb promoterofGTL1was amplified by PCR
and introduced into the LUC reporter vector(Ohta et al., 2001), as
reported by Rymen et al. (2017). For the constructionof the
effector vector, the coding sequence of GTL1 was amplified by
PCRand cloned into the p35SSG vector (Mitsuda et al., 2005) using
the SmaI sitelocated between the CaMV p35S promoter-Omega and the
NOS terminatorsequence of the p35SSG vector. The set of primers
used for PCRamplification is provided in Table S5.
The p35S:GTL1 and the empty p35SSG vectors were used as an
effectorand control vector, respectively. The pRSL4:LUC and
pGTL1:LUC vectorswere used as reporters. As an internal control,
the pPTRL vector, whichdrives the expression of a Renilla LUC gene
under the control of the CaMVp35S promoter, was used. The
constructions were introduced intoArabidopsis MM2D culture cells
(Menges and Murray, 2002) using agold particle bombardment system.
Luciferase activities were quantifiedusing a Mithras LB940
microplate luminometer (Berthold Technologies)according to the
protocol described previously (Hiratsu et al., 2002).
MicroscopyRoot hair phenotypes were recorded using a Leica M165
FC dissectionmicroscope equipped with a digital Leica DFC 7000T
camera. The length of120 root hairs from at least six seedlings
were quantified for each genotypewith ImageJ version 1.50a. For
auxin and auxin inhibitor treatment, thelengths of 60 root hairs
from at least six seedlings were quantified. Toquantify the rate of
root hair growth, hair growth of 7-day-old wild-type andgtl1-1
df1-1 seedlings was recorded every 1 h and root hair length
wasquantified by ImageJ. To estimate the ploidy level of nuclei,
nuclei werestained with DAPI (Partec) and visualized using an
Olympus BX51fluorescence microscope equipped with a digital Olympus
DP70 cameraand Olympus DP Manager software version 1.2.1.107 (Zhang
andOppenheimer, 2004). Fluorescence signals of over 40 root cap
nuclei and120 root hair nuclei from at least five seedlings were
quantified per genotypewith ImageJ as previously described (Ikeuchi
et al., 2015). Expressionpatterns of GTL1-GFP, DF1-GFP and RSL4-GFP
proteins were examinedusing a SP5 confocal laser scanning
microscope (Leica). Recorded imageswere exported as a 16-bit TIFF
image. Fluorescence intensity was quantifiedusing ImageJ. To
produce a merged image from multiple images,‘photomerge’ was
applied on Photoshop CS3 Extended (Adobe). Contrastand brightness
were adjusted using Photoshop.
Gene regulatory network inferenceGene network inference with
ensemble of trees 3 (GENIE3) (Huynh-Thuet al., 2010) was used to
predict the downstream targets of GTL1 and RSL4.GENIE3 uses
regression tree inference to build the GRN that best fits
theexperimental data. Microarray data from gtl1-1, df1-1, gtl1-1
df1-1, pEXP7:GTL1-GFP and pEXP7:DF1-GFP were used to infer 10,000
directedregression trees using the Random Forest method (Breiman,
2001), and thetrees were then averaged to form the final GRN. Once
the final GRN wasobtained, a threshold was set on the number of
edges to increase theprecision. The threshold was chosen using
ChIP-chip data to validate thedownstream targets of GTL1 in the
network. The number of edges waschosen as floor (1.65×36)=59 edges
(1.65×number of genes), as this numberresulted in the highest
precision for the network (Table S3).
A time-course RNAseq dataset of the root meristem (de Luis
Balagueret al., 2017) was used to determine the sign of the
regulation from gene A togene B. According to the first-order
Markov assumption, if gene B increases(or decreases) at one time
point after gene A increases (decreases), the
Fig. 6. A schematic diagram of the proposed transcriptional
networkregulating root hair growth inArabidopsis.RHD6 inducesRSL4
expressionto promote root hair outgrowth. GTL1 binds the RSL4
promoter and directlyrepresses its expression to terminate root
hair growth. RSL4, in addition,activates GTL1, thus forming a
negative-feedback loop. RSL4 and GTL1regulate, both directly and
indirectly, a set of common downstream genesinvolved in cell wall
biosynthesis and remodeling, cellular signaling, membranetransport,
membrane trafficking, metabolism, pathogenesis and
unknownfunctions. Auxin promotes root hair growth by activating
RSL4 but not GTL1.
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regulation at that time point is assumed to be positive.
Similarly, if gene Bincreases (or decreases) after gene A decreases
(increases), the regulation atthat time point is assumed to be
negative (de Luis Balaguer et al., 2017). TheMarkov assumption was
used to calculate the sign for all the time points, andthen the
majority sign was used for the edge.
Mathematical modeling and sensitivity analysisThe mathematical
model consisted of two ordinary differential equations:one
measuring RSL4 concentration (R) and the other measuring
GTL1concentration (G). It was assumed that transcription and
translation happenquickly, such that transcription and protein
degradation could be modeled inthe same equation. Additionally, it
was assumed that GTL1 and RSL4proteins degrade linearly.
The gene regulatory network and experimental data predicted that
GTL1represses RSL4. Thus, a Hill equation was used to model
RSL4transcription, where increased levels of GTL1 result in
decreased levels ofRSL4. As the oligomeric state of GTL1 is
unknown, there was a possibilityof GTL1 forming higher oligomers,
where n1 is the number of GTL1proteins bound to each other.
dR
dt¼ k1 K
n11D
Kn11D þ Gn1� �
� d1R
The gene regulatory network predicted that RSL4 activates GTL1.
Inaddition, the experimental data suggested that GTL1 represses
itself. Thus,the Hill equation for GTL1 takes into account what
happens when bothRSL4 and GTL1 bind the TL1 promoter at the same
time. One possibility isthat GTL1 inhibition overcomes the RSL4
activation. This means that, inorder for GTL1 to be transcribed,
GTL1 must be unbound (Eqn 1). In theother case, RSL4 activation
could overcome GTL1 inhibition. This meansthat, as long as RSL4 is
bound, GTL1 will be transcribed, even if GTL1 isbound to its own
promoter (Eqn 2). As in the RSL4 equation, there is apossibility of
higher oligomeric states.
dG
dt¼ k2 K
n22DK
n33D þ Kn33DRn2
Kn22DKn33D þ Kn33DRn2 þ Kn22DGn3 þ Rn2Gn3
� �� d2G ð1Þ
dG
dt¼ k2 K
n22DK
n33D þ Kn33DRn2 þ Rn2Gn3
Kn22DKn33D þ Kn33DRn2 þ Kn22DGn3 þ Rn2Gn3
� �� d2G ð2Þ
After constructing the two models, nullcline analysis was
performed tocheck for steady-state solutions. Mathematica was used
to analytically solvefor the nullclines as well as for steady
states. As concentrations must bepositive values, only the region
where R≥0 andG≥0 was considered. In thisregion, the model with Eqn
1 has no steady states, whereas the model withEqn 2 has one steady
state. In a wild-type case, there should be a steady statevalue of
R and G that produces root hairs with normal length. Thus, themodel
with Eqn 2 was used for the simulations.
A sensitivity analysis was used to identify the parameters that
most greatlyaffect the model outcome, as these parameters could
give insight into theeffects of RSL4 and GTL1 mutants and
overexpression lines. Soboldecomposition, which quantifies
sensitivity by calculating the variance inthe model outcome as the
parameters are changed (Sobol, 2001), was used tocalculate the
sensitivity indices. The index was calculated for eachparameter
using 1000 Monte Carlo evaluations and repeated 10 times
fortechnical replicates (Clark et al., 2016).
AcknowledgementsWe are grateful to the members of K.S.’s lab for
discussions and to Momoko Ikeuchiand David Favero for comments on
the manuscript. We thank Mariko Mouri, ChikaIkeda and Noriko Doi
for their technical assistance.
Competing interestsThe authors declare no competing or financial
interests.
Author contributionsConceptualization: M.S., C.B., K.S.;
Validation: M.S., C.B., N.M.C., B.R., L.B., K.M.;Formal analysis:
M.S., C.B., N.M.C., K.M., W.B., R.S.; Investigation: M.S.,
C.B.,A.K., N.M.C., B.R., L.B., K.M.; Resources: A.K., B.R.; Data
curation: M.S., C.B.,N.M.C., B.R., W.B., P.N.B., R.S.; Writing -
original draft: M.S., N.M.C., R.S., K.S.;
Writing - review & editing: M.S., C.B., N.M.C., B.R., L.B.,
K.M., W.B., P.N.B., R.S.,K.S.; Visualization: M.S., C.B., K.S.;
Supervision: R.S., K.S.; Project administration:P.N.B., K.S.;
Funding acquisition: M.S., N.M.C., K.S.
FundingThis work was supported by a National Science Foundation
CAREER grant (MCB-1453130) to R.S., by a National Science
Foundation EAPSI grant (1514779) toN.M.C. and by grants from
Ministry of Education, Culture, Sports and Technology ofJapan to
M.S. (16J07464) and to K.S. (26291064 and 15H05961). N.M.C.
issupported by a National Science Foundation GRF (DGE-1252376) and
M.S. issupported by a Japan Society for the Promotion of Science
postdoctoral fellowship.Deposited in PMC for immediate release.
Data availabilityMicroarray and ChIP-chip data have been
deposited in Gene Expression
Omnibus(https://www.ncbi.nlm.nih.gov/geo/) under accession numbers
GSE103917 andGSE104010, respectively.
Supplementary informationSupplementary information available
online
athttp://dev.biologists.org/lookup/doi/10.1242/dev.159707.supplemental
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