A basic helix loop helix transcription factor controls ... · A basic helix loop helix...

A basic helix loop helix transcription factor controls cell growth

and size in root hairs

Keke Yi1,2, Benoît Menand

1,3, Elizabeth Bell

1, Liam Dolan

1,4

Supplementary note

Low soil phosphate availability (low phosphate stress) induces the elongation of root hairs 1

and enhances the capture of phosphate for plant growth 2. Plants grown in medium containing 10

µM phosphate are considered to be subject to low phosphate stress and root hairs are longer (698 ± 6 µm) than on plants grown with an adequate supply of phosphate (1000 µM phosphate) which develop shorter root hairs (338 ± 6 µm). To demonstrate a requirement for RSL4 in the

response to low phosphate, we compared root hair growth of rhd6-3 rsl1-1 double mutants and

rhd6-3 rsl1-1 rsl4-1 triple mutants in low phosphate. Low phosphate induces the development of a

few hairs in the rhd6-3 rsl1-1 mutant indicating that hairs could respond to the low phosphate

growth stimulus. In contrast no hairs developed on the rhd6-3 rsl1-1 rsl4-1 triple mutant

(Supplementary Fig. 8a). This indicates that RSL4 is required for the increase in root hair growth

that occurs when roots are grown in low phosphate. Further support for the role of RSL4 comes

from the observation that low phosphate stress did not induce growth in rsl2-1 rsl4-1 double

mutant (Supplementary Fig. 8a). Together, these data indicate that RSL4 activity is required for the

root hair growth response induced by low phosphate stress.

To confirm that low phosphate stress controls growth by modulating RSL4 activity, we

determined the RSL4 transcript and protein levels in roots grown under phosphate stress and

Nature Genetics: doi:10.1038/ng.529

control conditions. The levels of RSL4 transcript were 2 to 3 times higher in plants grown in

conditions where phosphate was in short supply compared to those grown in high phosphate,

while the transcript level of RSL2 did not change (Supplementary Fig. 8b). Furthermore, the

steady state levels of GFP-RSL4 protein increased under the low phosphate stress, while the levels

of GFP-RSL2 remain unchanged (Supplementary Fig. 8c). To verify that the induction of RSL4

was due to low phosphate and not another stress, we examined RSL4 induction under low

phosphate stress in plants with defective low phosphate stress root responses 3. LPR1 and LPR2

are multicopper oxidases involved in triggering the root response to low phosphate stress signal 3.

The root hair response to low phosphate is partially repressed in the lpr1 lpr2 double mutant (a

gift from Thierry Desnos, CEA Cadarache, France); wild type root hairs grown in low phosphate

stress are 100% longer than wild type hairs grown in control conditions. In contrast, the root hairs

of lpr1 lpr2 double mutants are only 40% longer in low phosphate than in high phosphate (lpr1

lpr2 HP: 350 ± 6 μm; lpr1 lpr2 LP: 489 ± 6 μm). As expected, the induction of RSL4

mRNA by low phosphate stress is defective in the lpr1 lpr2 double mutant background

(Supplementary Fig. 6b). These data indicate that low phosphate stress controls root hair length by

modulating steady state levels of RSL4 transcript and protein and requires the phosphate signalling

activity of LPR1 and LPR2.

1. Bates, T.R. & Lynch, J.P. Stimulation of root hair elongation in Arabidopsis thaliana by low

phosphorus availability. Plant, Cell & Environment 19, 529-538 (1996).

2. Bates, T.R. & Lynch, J.P. The efficiency of Arabidopsis thaliana (Brassicaceae) root hairs in

phosphorus acquisition. Am.J.Bot. 87, 964-970 (2000).

3. Svistoonoff, S. et al. Root tip contact with low-phosphate media reprograms plant root

architecture. Nat Genet 39, 792-796 (2007).


Supplementary Fig. 1


Supplementary Fig. 1 Expression pattern of RSL4. (a) The distribution of RSL4 protein partially

overlaps with RHD6. From left to right are GFP-RSL4, mCherry-RHD6, overlay and bright field;

(b) RSL4 mRNA was only present in the roots (R). No detectable mRNA transcript was present in

the rosette leaf (RL), cauline leaf (CL) stem (St) and flower (F); (c) GUS expression in an RSL4

gene trap line (CSHL_GT13756 from Cold Spring Harbor Laboratory) was detected only in the

root hair cells, not in any other tissues including shoots, leaves and flowers.



Supplementary Fig. 2 qRT-PCR analysis of RSL4 relative expression levels in the RSL4 RNAi

lines. Values represent mean±s.d., n=3.



Supplementary Fig. 3: Constitutive expression of RSL4 results in constitutive growth until root

hair cells undergo programmed cell death. (a) Root hair phenotype of Col-0 (left side) and a plant

transformed with 35S:RSL4 (right side) grown in half strength Johnson media. (b) Gray scale

pictures are DIC images of root hairs of Col-0 (left side) and 35S:RSL4 (right side). Colour images

above each DIC image are pseudo colour of root hairs grown in half strength Johnson media with

10 nM Rhodamine 123 showing the mitochondrial membrane potential (∆ψm). The ruler above

indicates the fluorescent intensity of the pseudo colour images. The absence of fluorescence

intensity is an indicator of cell death.



Supplementary Fig. 4 Morphologies of 35S::RSL4 plants. Wild type (left) and 35S::RSL4 (right)

plants are morphologically identical except that the root hairs are much longer in the 35S::RSL4

plants.



Supplementary Fig. 5: Nuclear volume is the same in wild type and in plants transformed with

35S::RSL4. DAPI staining of Col-0 and plants transformed with 35S::RSL4 root hairs indicates

that the nuclear size is the same in these two genotypes.



Supplementary Fig. 6: RSL2 regulates root hair tip growth. (a) Root hair morphology of Col-0 (the

left panel) and rsl2-1 (the right panel). (b) Root hair length distributions of Col-0 and rsl2-1. (c)

GFP:RSL2 accumulates in initiating and growing root hair cells similarly to GFP:RSL4. (d) RSL2

is not a direct target of RHD6. qRT-PCR analysis of RNAs from rhd6-3 rsl1-1 harboring

GR:RHD6 treated with DEX for 2h (open bar), 24h (black bar) and DEX/CHX for 2h (hatched

bar).



Supplementary Fig. 7: RSL2 and RSL4 regulate root hair development. (a) Cryo-SEM images of

Col-0 (left panel) and rsl2-1 rsl4-1 (right panel); (b) Genomic DNA fragments of RSL2 and RSL4

complement the rsl2 and rsl4 mutations.



Supplementary Fig. 8: Low phosphate stress modulates RSL4 expression to control hair cell

growth. (a) Root hair morphologies of Col-0, rhd6-3 rsl1-1, rhd6-3 rsl1-1 rsl4-1 and rsl2-1 rsl4-1

under HP and LP treatments show that LP stress can induce root hair growth in Col-0 and rhd6-3

rsl1-1, but not in rhd6-3 rsl1-1 rsl4-1 and rsl2-1 rsl4-1. (b) qRT-PCR analysis of mRNAs isolated

from Col-0 and the lpr1 lpr2 double mutant under high phosphate and low phosphate treatments

show that the induction of RSL4 transcripts by low phosphate stress is compromised in the lpr1

lpr2 double mutant background. Values represent mean±s.d., n=3. (c) Pseudo colour images of GFP:RSL2 (upper panel) and GFP:RSL4 (lower panel) under high phosphate and low phosphate

treatments showing low phosphate stress induces the accumulation of RSL4 but not RSL2.



Supplementary Fig. 9 RSL4 positively regulates the expression of genes that promote growth in

root hair cells. (a) Hierarchical clustering of the 83 genes positively regulated by RSL4. (b)

qRT-PCR analysis of AtEXP7, MRH3 and MRH4 expression from mRNAs isolated from plants

with wild type levels of RSL4 expression (Col-0, rsl2-1), higher levels of RSL4 expression

(35S::RSL4) or plants that lack RSL4 expression (rsl4-1 and rsl2-1 rsl4-1). These data show that

RSL4 positively regulates AtEXP 7, MRH3 and MRH4 expression. Values represent mean±s.d., n=3.



Supplementary Fig. 10 A working model showing that RSL4 integrates the internal and external

signals to regulate root hair cell growth. The left diagram is a SEM picture of Arabidopsis primary

root. The white dot line marks the area where RHD6 and RSL1 are expressed in the root hair cells,

while the white line marks the area where RSL2 and RSL4 are expressed in the root hair cells. The

partial overlapping of these two white lines indicates the slight overlapping expression of RHD6

with RSL2 and RSL4. The black arrows indicate the genetic relationships.


Supplementary Table 1 RSL4 positively regulated genes

Probe Set ID Fold change

(35S-RSL4 vs

Col-0)

P.Value Fold change

(Col-0 vs

rsl4-1)

P.Value AGI Gene Symbol Related to root

hair or not

261647_at 6.9774275 3.88E-08 32.11583 1.81E-08 AT1G27740 RSL4

261005_at 3.716045 6.76E-05 1.999287 0.004193 AT1G26420 FAD-binding domain-containing

protein

248374_at 3.6520593 0.002671 1.590485 1.31E-06 AT5G51870 AGL71

262564_at 3.1257668 0.000119 2.224049 0.000125 AT1G34330 putative peroxidase

246872_at 2.8688257 0.000416 2.035858 0.050767 AT5G26080 proline-rich family protein

253763_at 2.8459804 0.000111 1.561432 0.003106 AT4G28850 xyloglucan:xyloglucosyl transferase,

putative

261157_at 2.7505617 0.000111 2.409713 0.00033 AT1G34510 peroxidase, putative

259525_at 2.7038248 4.81E-05 1.614617 0.004123 AT1G12560 ATEXPA7 root hair

specific

expression

257418_at 2.6325424 0.000912 4.881911 1.4E-05 AT1G30850 RHS4 root hair

specific

expression

261185_at 2.6233034 7.64E-05 3.165163 0.001092 AT1G34540 CYP94D1

249934_at 2.5732727 0.001595 3.590064 0.000417 AT5G22410 RHS18 root hair

specific

expression

251176_at 2.5624392 0.004345 1.934799 0.00035 AT3G63380 calcium-transporting ATPase

263376_at 2.5410964 0.000209 2.443631 0.002455 AT2G20520 FLA6


256352_at 2.4212208 0.000341 3.854431 2.29E-06 AT1G54970 RHS7/ATPRP1 root hair

specific

expression

254534_at 2.4156563 0.000717 2.610299 0.000494 AT4G19680 IRT2

263482_at 2.4023705 0.000376 2.348453 0.000115 AT2G03980 GDSL-motif lipase/hydrolase family

protein

245966_at 2.327236 0.000399 1.643065 0.010408 AT5G19790 RAP2.11 root hair

specific

expression


specific

expression

254107_at 2.2860358 5.08E-05 1.567657 0.002999 AT4G25220 RHS15 root hair

specific

expression

247871_at 2.2854035 0.000414 7.870044 5.47E-08 AT5G57530 xyloglucan:xyloglucosyl transferase,

putative



specific

expression

254338_s_at 2.1527658 0.000399 2.509884 0.007497 AT4G22080

///

AT4G22090

RHS14 root hair

specific

expression

264567_s_at 2.1359873 0.001321 2.427884 0.000235 AT1G05240

///

peroxidase, putative


AT1G05250

245874_at 2.1276755 0.004988 3.314086 0.000955 AT1G26250 proline-rich extensin, putative


specific

expression

254025_at 2.0846293 0.000719 2.45537 1E-05 AT4G25790 allergen V5/Tpx-1-related family

protein

251410_at 2.056107 0.001227 3.141587 0.018312 AT3G60280 UCC3

247536_at 2.0454068 0.011561 1.905908 0.013521 AT5G61650 CYCP4;2

253613_at 1.987907 0.000794 3.441541 0.000265 AT4G30320 allergen V5/Tpx-1-related family

protein

249348_at 1.9292821 0.004165 2.502332 0.004223 AT5G40860 unknown protein

245967_at 1.9241055 0.00095 2.043032 0.000531 AT5G19800 hydroxyproline-rich glycoprotein

family protein

255307_at 1.907704 0.002567 3.185133 0.006978 AT4G04900 RIC10

259576_at 1.9030424 0.000208 1.852405 0.003631 AT1G35330 zinc finger (C3HC4-type RING finger)

family protein

249101_at 1.8985797 0.001383 2.088197 0.001704 AT5G43580 Predicted to encode a PR peptide

252833_at 1.894271 0.002298 2.06192 0.000693 AT4G40090 AGP3

263614_at 1.8699329 0.000777 2.407115 0.000189 AT2G25240 serine-type endopeptidase inhibitor

251970_at 1.8643738 0.04818 2.320804 0.059478 AT3G53150 UGT73D1

254044_at 1.8169075 0.002148 1.999282 0.000408 AT4G25820 XTR9


247581_at 1.7881445 0.006449 2.498752 6.96E-05 AT5G61350 protein kinase family protein



250683_x_at 1.7841561 0.002009 2.10878 0.000611 AT5G06640 proline-rich extensin-like family

protein

249950_at 1.7841265 0.01353 2.024164 0.010165 AT5G18910 protein kinase family protein

262756_at 1.7839128 0.004474 1.681966 0.016099 AT1G16370 ATOCT6

247914_at 1.7706201 0.002671 5.805789 1.31E-06 AT5G57540 xyloglucan:xyloglucosyl transferase,

putative

266299_at 1.766686 0.003644 1.550043 0.013554 AT2G29450 ATGSTU5

264029_at 1.7581513 0.0051 2.433681 0.000408 AT2G03720 MRH6 defective in root

hair growth


251226_at 1.73925 0.00132 2.14811 0.000154 AT3G62680 PRP3 root hair

specific

expression

255432_at 1.7323341 0.009001 1.533634 0.007428 AT4G03330 SYP123 root hair

specific

expression

261099_at 1.7313373 6.76E-05 1.516547 0.004193 AT1G62980 ATEXPA18 root hair

specific

expression


262898_at 1.7206964 0.007433 2.236743 0.002399 AT1G59850 binding, biological process unknown

249868_at 1.7085866 0.006617 2.77432 0.004052 AT5G23030 TET12

245636_at 1.7032975 0.004988 2.074212 0.000955 AT1G25240 epsin N-terminal homology (ENTH)

domain-containing protein


262412_at 1.6918312 0.000829 1.500515 0.030982 AT1G34760 RHS5/GRF11 root hair


specific

expression


253244_at 1.6673543 0.007162 2.140779 0.001864 AT4G34580 COW1 defective in root

hair growth

254772_at 1.6536554 0.029042 4.478019 7.46E-07 AT4G13390 proline-rich extensin-like family

protein

255541_s_at 1.6480447 0.001516 1.59744 0.000404 AT4G01820

///

AT4G01830

PGP5


266765_at 1.606342 0.006123 2.501496 7.49E-05 AT2G46860 ATPPA3

262828_at 1.5926627 0.004474 1.504132 0.016099 AT1G14950 major latex protein-related /

MLP-related




255140_x_at 1.5790453 0.00322 2.398313 0.015543 AT4G08410 proline-rich extensin-like family

protein


261453_at 1.5729337 0.002537 3.329812 0.009046 AT1G21130 O-methyltransferase, putative

265102_at 1.5696362 0.005322 3.471569 0.001748 AT1G30870 cationic peroxidase

262517_at 1.5655074 0.007903 3.43951 1.79E-06 AT1G17180 ATGSTU25

261541_at 1.5557078 0.018363 1.861052 0.048122 AT1G63600 protein kinase-related

264775_at 1.554045 0.003211 1.510692 0.000189 AT1G22880 ATCEL5



specific

expression

255632_at 1.5423801 0.033234 1.786571 0.002352 AT4G00680 ADF8 root hair

specific

expression

248564_at 1.5420673 0.055904 2.058809 0.02273 AT5G49700 DNA-binding protein-related


267051_at 1.5304104 0.006123 2.006302 7.49E-05 AT2G38500 unknown protein

266743_at 1.5287263 0.005096 1.719269 0.013834 AT2G02990

///

AT2G16300

RNS1


250632_at 1.5085515 0.002206 2.793871 3.1E-05 AT5G07450 CYCP4;3

263876_at 1.5048211 0.000777 1.551579 0.000189 AT2G21880 AtRABG2


Supplementary Table 2 Primers used in this study

rsl2-1 LB F: CTCGTCCCCAATGGAACAAAGGT

rsl2-1 LB R: TAGCATCTGAATTTCATAACCAACTCGATACAC

rsl2-1 homozygote F: CTCGTCCCCAATGGAACAAAGGT

rsl2-1 homozygote R: CGAACGTGACTCTCCATTTCTCTCA

rsl4-1 LB F: GGGAACCGGTATTTTTGTTCGG

rsl4-1 LB R: TTGTAAGCCAATGGTGCGTACAT

rsl4-1 homozygote F: GAAAGCTTCGGTCACAAGTGTTAAA

rsl4-1 homozygote R: TTGTAAGCCAATGGTGCGTACAT

35SRSL4F(KpnI): cgg ggtacc ATGGACGTTTTTGTTGATGGT

35SRSL4R(BamHI): cgc ggatcc TCACATAAGCCGAGACAAAAG

PRSL2::GFP:RSL2

GFPattB1F GGGG ACA AGT TTG TAC AAA AAA GCA GGC TCA ATG AGT AAA GGA GAA GAA CTT TTC


GFPattB2R GGGG AC CAC TTT GTA CAA GAA AGC TGG GTA TTT GTA TAG TTC ATC CAT GCC

RSL2P_ATTB4F: GGGG ACA ACT TTG TAT AGA AAA GTT G tgc atg tca cct ttc ttt gc

RSL2P _ATTB1R: GGGG AC TGC TTT TTT GTA CAA ACT TG a ttc tcc cat ggc ttc cat t

RSL2:3’UTRATTB2F: GGGG ACA GCT TTC TTG TAC AAA GTG G gg agc aac aac ctc gga gga at

RSL2:3’UTRATTB3R: GGGG AC AAC TTT GTA TAA TAA AGT TG g cca ctt tta aat gct ttg gac

PRSL4::GFP:RSL4

RSL4P_ATTB4F: GGGG ACA ACT TTG TAT AGA AAA GTT G at acg cgt tgg gct taa atg

RSL4P _ATTB1R: GGGG AC TGC TTT TTT GTA CAA ACT TG c aaa aac gtc cat cgc tct

RSL4:3’UTRATTB2F: GGGG ACA GCT TTC TTG TAC AAA GTG G tg gac gtt ttt gtt gat ggt

RSL4:3’UTRATTB3R: GGGG AC AAC TTT GTA TAA TAA AGT TG acgatacggtttggtttgactaat

PRHD6::mCherry:RHD6

mCherryattB1F GGGG ACA AGT TTG TAC AAA AAA GCA GGC TTA atg gtg agc aag ggc gag ga

mCherryattB2R GGGG AC CAC TTT GTA CAA GAA AGC TGG GT A CTT GTA CAG CTC GTC CAT GCC G


RHD6P_ATTB4F GGGG ACA ACT TTG TAT AGA AAA GTT Gtt ctc aaa gag gga caa gac caa agc cca tga c

RHD6P_ATTB1R GGGG AC TGC TTT TTT GTA CAA ACT TGc tag aca cta ata agt ttg ata agt gat ttt ttg t

RHD6:3’UTRATTB2F GGGG ACA GCT TTC TTG TAC AAA GTG Gcc atg gca ctc gtt aat gac cat ccc aac gag a

RHD6:3’UTRATTB3R GGGG AC AAC TTT GTA TAA TAA AGT TGc tga taa atc gag atc tta ggt atg tcg tcc

GR ATTB1F GGGG ACA AGT TTG TAC AAA AAA GCA GGC T CT ATG GAT CCT GAA GCT CGA AAA ACA

AA

GR ATTB2R GGGG AC CAC TTT GTA CAA GAA AGC TGG GTT TTT TTG ATG AAA CAG AAG CTT TTT G

RSL2 genomic DNA

RSL2genomicUATTB1: GGGG ACA AGT TTG TAC AAA AAA GCA GGC T tgcatgtcacctttctttgc

RSL2genomicLATTB2: GGGG AC CAC TTT GTA CAA GAA AGC TGG GT gccacttttaaatgctttggac

RSL4 genomic DNA

RSL4genomicUATTB1: GGGG ACA AGT TTG TAC AAA AAA GCA GGC T at acg cgt tgg gct taa atg

RSL4genomicLATTB2: GGGG AC CAC TTT GTA CAA GAA AGC TGG GT acgatacggtttggtttgactaat


qRT-PCR primers

RSL2-F TCCCCAATGGAACAAAGGTC

RSL2-R TCTCGGTGAGCTGAGACCAA

RSL4-F GTGCCAAACGGGACAAAAGT

RSL4-R TTGTGATGGAACCCCATGTC

AtEXP7-F ATCCCAGTTGCATACCGAAG

AtEXP7-R TATCCAATTCGTCCGGCTAC

MRH3-pair1-F GATGACCTAGACCACCACTAT

MRH3-pair1-R GCCTTCAATTCCAGGACTTGAC

MRH4-pair1-F CGAGGGTTGGCTCTGTCC

MRH4-pair1-R GGTGGTGATTGTTGTGTTGAC

EF1α-F GGTGGTGGCATCCATCTTGTTACA

EF1α-R TGAGCACGCTCTTCTTGCTTTCA


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