FIELD OF INVENTION
This invention relates to the modulation of root hair development
in plants.
BACKGROUND OF THE INVENTION
In 1990, Schiefelbein and Somerville" published a paper
describing their work with Arabldopsls thaliana mutants in their
efforts to understand genetic control of root hair development.
They examined roots from 12,000 mutagenized Axabidopaia seedlings,
leading to identification of more than 40 mutants impaired in root
hair morphogenesis. Mutants were characterized as belonging to
four phenotypic classes which genetically were produced from
single nuclear recessive mutations in four different genes
designated RHDl, RHDP, RHD3, and RHD4. As a result of the
phenotypic analysis of the mutants and homozygous double mutants,
a model for root hair development was proposed, including the
stages at which the genes are normally required. The RHDl gene
product appears to be necessary for proper initiation of root
hairs, whereas the RHDS, RHD3, and RHD4 gene products are required
for normal hair elongation. These authors concluded that the
results they obtained demonstrate that root hair development in
Arabldopaia is amenable to genetic dissection and should prove to
be a useful model system to study the molecular mechanisms
governing cell differentiation in plants.
In 1994, Masucci and Schiefelbein7 extended those results by
identifying another mutant, the rhd6 mutant, concluding that root-
hair initiation in Arabidopsls thaliana provides a model for
studying cell polarity and its role in plant morphogenesis. They
observed that root hairs normally emerge at the apical end of root
epidermal cells, implying that these cells are polarized. The rhd6
mutant was characterized as displaying three defects: (a) a
reduction in the number of root hairs, (b) an overall basal shift
in the site of root hair emergence, and (c) a relatively high
frequency of epidermal cells with multiple root hairs. They
concluded that these defects implicate the RHD6 gene in root-hair
initiation and indicate that RHD6 is normally associated with the
establishment of, or response to, root epidermal cell polarity.
Similar alterations in the site of root-hair emergence, although
less extreme/ were also discovered in roots of the auxin-,
ethylene-, abscisic acid-resistant mutant axr2 and the ethylene-
resistant mutant etrl. All three rhd€ mutant phenotypes were
rescued when either auxin (indoleacetic acid) or an ethylene
precursor (1 -aminocyclopropane-1-carboxylic acid) was included in
the growth medium. The rhd6 root phenotypes could be phenocopied
by treating wild-type seedlings with an inhibitor of the ethylene
pathway (aminoethoxyvinylglycine). These results indicate that
RHD6 is normally involved in directing the selection or assembly
of the root-hair initiation site through a process involving auxin
and ethylene.
Root hairs play important roles in plant nutrition and water
uptake. In most soils they are important for phosphate and iron
uptake. In drought conditions they are important in the uptake of
other nutrients such as nitrate. Therefore the manipulation of
root hair traits will be important in developing crops that can
effectively extract nutrients from the soil. Until now this has
been difficult since no gene with a function limited to the root
hair has been identified.
EP0803572B1 discloses the identification, isolation, cloning, and
characterization of the CPC gene of Arabldopsls thallana, for
regulating initiation of root hair formation, as well as
transgenic plants over-expressing the CPC gene. The CPC gene is
not responsible for the rhd mutant phenotypes described above.
This is confirmed, for example, in US661749, as well as
EP0803572B1 itself.
SUMMARY OF INVENTION
The present invention relates to the finding that the over-
expression of ROOT HAIR DEFECTIVE 6 [RHD6) genes in plants alters

root hair development, for example leading to plants with an
increased number, length and/or longevity of root hairs.
Furthermore, over-expression of a different gene family [ROOT HAIR
DEFECTIVE SIX LIKE1 [RSD genes) produces a similar effect.
Modulation of the expression of these genes (collectively termed
1HKD6-related genes') in plants may be useful, for example, in
manipulating root hair traits in diverse groups of plant species
(including crops) to improve their ability to extract nutrients
from the soil.
An aspect of the invention provides an isolated ROOT HAIR
DEFECTIVE 6 (RHD6)-related gene.
RHD6-related genes include both ROOT HAIR DEFECTIVE 6 {RHD6) genes
and ROOT HAIR DEFECTIVE SIX LIKE1 (RSL1) genes, and functional
homologues thereof, as described herein. JUfDff-related genes
include genes capable of complementing the rhd6 mutation in
plants.
Another aspect of the invention provides an isolated gene encoding
an amino acid sequence encoded by the JUfDff-related gene or a gene
product that is sufficiently homologous thereto to permit, on
production thereof in an rhd6 mutant cell a functional
complementation of said mutation.
Another aspect of the invention provides an isolated product of
the expression of an isolated AJfD6-related gene.
Another aspect of the invention provides an isolated
polynucleotide which encodes a gene product comprising an amino
acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 to 26, 28,
30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,
62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,
94, 96, 98, 100, 102, 104, 106, 108, 110, 112 and 114.
Another aspect of the invention provides an isolated
polynucleotide which has at least 40% nucleic acid sequence
identity with one or more of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 27,
29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,
61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91,
93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, and 115.
Other aspects of the invention provide expression constructs,
plant cells, and plants or plant progeny, including seeds, which
comprise an isolated RHD6-related gene or polynucleotide described
herein.
Another aspect of the invention provides a method of modulating
root hair development in a plant comprising;
increasing the expression of an AifDff-related polypeptide
within cells of said plant relative to control plants.
An RHD6-related polypeptide may, for example, comprise an amino
acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 to 26, 28,
30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,
62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,
94, 96, 98, 100, 102, 104, 106, 108, 110, 112 or 114.
Another aspect of the invention provides a method of improving the
tolerance of a plant to nutrient-deficient conditions comprising;
increasing the expression of an AHD6-related polypeptide
within cells of said plant relative to control plants.
Another aspect of the invention provides a method of increasing
the production of a root-secreted phytochemical in a plant
comprising;
increasing the expression of an RHDff-related polypeptide
within cells of a plant which produces the root-secreted
phytochemical.
In some embodiments, expression of an JUTD6-related polypeptide may
be increased in a plant by expressing a heterologous nucleic acid
encoding said RHD6-related polypeptide within cells of said plant.
In some embodiments, expression of an JUZDff-related polypeptide may
be increased in a plant by;
crossing a first and a second plant to produce a
population of progeny plants;
determining the expression of the RHD6-zelated
polypeptide in the progeny plants in the population, and
identifying a progeny plant in the population in which
expression of the RHD6-related polypeptide is increased relative
to controls.
In some embodiments, expression of an RHD6-related polypeptide may
be increased in a plant by;
exposing a population of plants to a mutagen,
determining the expression of the RffDff-related
polypeptide in one or more plants in said population, and;
identifying a plant with increased expression of the
RHD6-related polypeptide.
Plants identified as having increased expression of the RHD6-
related polypeptide may be sexually or asexually propagated or
grown to produce off-spring or descendants showing increased
expression of the AHDff-related polypeptide.
Another aspect of the invention provides a method of producing a
plant with altered root-hair development comprising:
incorporating a heterologous nucleic acid which alters the
expression of a JUfD6-related polypeptide into a plant cell by
means of transformation, and;
regenerating the plant from one or more transformed cells.
Another aspect of the invention provides a plant produced by a
method described herein which displays altered root-hair
development relative to controls.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows that AtRHD6 is a positive regulator of root hairs
development in Arabidopsis. Figure la shows roots of Atrhd6-1,
Atrhd6-2 and Atrhd6-3 mutants with their respective wild type and
complementation of the Atrhd6-3 mutant with a genomic
AtRHD6p::GFP:AtRHD6 fusion. Figure lb shows a fluorescent image of
the genomic AtRHD6p: :GFP:AtRHD6 fusion in the Atrhd6-3 background
showing AtRHD6 protein in hair cells nuclei. Figure lc shows the
expression of the Atrhd6-2 enhancer trap GUS gene in root cross
section. Figure Id shows a whole mount longitudinal view of the
expression of the enhancer trap GUS gene in AtrhdS-2 and in
different backgrounds (cpc, war, ttgl and gl2). H, hair cell; N,
non hair cells; C, cortex. Scales bars, 500 urn (a), 50 urn (b), 25
urn (c) and 100 urn (d).
Figure 2 shows that AtRSLl positively regulates root hairs
development in Arabidopsis. Figure 2a shows roots of WT, Atrhd6-3
single mutant, Atrsll-1 single mutant, Atrhd6-3 Atrsll-1 double
mutant and Atrhd6-3 Atrsll-1 double mutant bearing the
AtRSLlp::GFP:AtRSLl transgene. Plants were grown on MS media with
sucrose overlaid with a cellophane disc to increase root hairs
production in the Atrhd6-3 mutant. Figure 2b shows a fluorescent
image of the genomic AtRSLlp: :GFP:AtRSLl fusion in the Atrhd€-3
Atrsll-1 background showing AtRSLl protein in hair cells nuclei.
H, hair cell; N, non hair cells. Scale bars, 500 urn (a) and 50 urn
(b).
Figure 3 shows the relationship between RHD6-LIKE proteins from
Arabidopsis and Physcomitrella. The tree is a strict consensus
tree of 12 most parsimonious tree generated using the alignment of
bHLH domains amino acids sequences shown in Tables 1 and 2. The
Arabidopsis genes used are the members of bHLH subfamily VIIIc,
except AtlND (INDEHISCENT)/At4gO0120 which was used as out-group
and belongs to the bHLH subfamily VIlib •• "• 2e. Physcomitrella
PpRSL 1 to 7 sequences were obtained by BLAST of the
Physcomitrella genomic sequence. PpINDl is a Physcomitrella
sequence similar to AtlND and a putative member of family VIlib in
Physcomitrella. Numbers are bootstrap values and indicates an 82
% level of confidence for the occurrence of the AtRHD6 clade. The
brackets indicates the AtRHD6 clade and the sister clade.
Figure 4 shows that PpRSLl and PpRSL2 positively control the
development of caulonemal cells and rhizoids in Physcomitrella and
PpRSLl and AtRHD6 have a conserved molecular function. Figure 4a
and b show eighteen day old protonema from WT, Pprsll and Ppral2
single mutants, and Pprsll Pprsl2 double mutant, grown from spores
on 0.8% agar. Figure 4a shows whole protonema growing from a
single spore. Figure 4b shows dissected filaments from protonema
shown in Figure 4a. Figure 4c shows isolated one month old
gametophores. Figure 4d shows roots of the Arabidopsis Atrhd6-3
mutant carrying the 35S:: PpRSLl transgene compared to WT and
Atrhd6-3 roots, ca, caulonemal cell; ch, chloronemal cell; rh,
rhizoid. Scale bars, 1 mm (a), 100 um (b), 1 mm (c), and 500 urn
(d).
Figure 5 shows the phenotype for the transformants:35S::RHD6
Figure 5A shows col-0 rhdS/rsll with 35S::RHD6; Figure 5B shows
col-0 rhd6/rsll with 35S::RHD6; Figure 5C. rhd6/rsll with
35S::RHD6
Figure 6 shows the phenotype for the transformants:35S::RSL2 and
35S::RSL3 Figure 6A shows col-0 rhd6/rsll with 35S::RSL2/3; Figure
6B shows root hypocotyls
Figure 7 shows the molecular basis of mutations in A. thaliana
AtRHD6 (A) and AtRSLl (B) genes. White boxes correspond to coding
regions (black boxes for the bHLH domain encoding region). Grey
triangles indicate the position of each insertion. Numbers in

brackets indicate the distance between each T-DNA insertion and
the start codon. (C) RT-PCR showing that Atrhd6-3, Atrall-1 and
Atrhd6-3 Atrsll-1 are RNA null mutants. AtAPTl, Adenine
phosphoribosyltransferase 1.
Figure 8 shows that the Atrhd6-3 and Atrsll-1 single mutants and
the Atrhd6 Atrsll double mutant have no detectable pollen tube
growth defects. Ratios of resistance to antibiotic of F2 plants
from Atrhd6-3 Atrsll-1 double mutant backcrossed to WT are 76.7 %
(n=1404; x:(3/D - 2,19; P> 0,05) for Sulfadiazin (resistance
carried by the Atrhd6-3 allele) and 74.9 % (n=1289;
X:(3/l) - 0.0023; P > 0,05) for phosphinothricin (resistance
carried by the Atrsll-1 allele) showing normal segregation of the
single mutants and double mutant gametes. (Figure 8A shows pollen
of the genotype indicated below each picture was used to pollinate
WT stigma. Carpels were stained with aniline blue 4 hours after
pollination. The growth of each mutant pollen tubes in the WT
carpel is revealed by callose staining in blue (white arrows).
Similar pollen tube growth is observed in WT and mutant pollen
tubes. Figure 9B shows in vitro pollen tube growth experiment. WT
and mutants pollens were germinated on agar plates. Representative
plates are shown with the germination ratio (mean of 600 pollen
grains per line, with standard error). Similar germination ratios
are observed between WT and mutants pollen (Student1s-t-test p
values are 0.554 for Atrhd6-3 versus WT, 0.904 for Atrsll-1 versus
WT and 0.87 for Atrhd6-3 Atrsll-1 versus WT). Scale bars, 200 urn
(Figure 8A and B).
Figure 9 shows the molecular basis of P. patens Pprsll, Pprsl2 and
Pprsll Pprsl2 mutations (three independent mutants, named 1 to 3,
are shown in each case). Figure 9A and D show the structure of the
PpRSLl (A) and PpRSL2 (D) genes (up), and the expected result of
the homologous recombination (down). White boxes correspond to
coding regions (black boxes for the bHLH domain encoding region)
and the grey boxes correspond to the resistance gene cassette
{NptII and AphIV). The regions of homology used for gene
replacement are delimited by grey lines. The distance between the

restrictions sites used for Southern blots and the position of the
probes used are also shown. Figure 9B, C, E and F show southern
blots of WT and mutants DNA digested with Seal (B and C) or Ncol
(E and F) and hybridized with the probe indicated below the
picture. Blots C and F are hybridization of the same membrane used
for blot B and E respectively, after stripping of the gene
specific probe. The replacement of the WT band by a larger band of
expected size (see A and D) in mutants lines when hybridization is
performed with the gene specific probe (B and E), and the
hybridization of only the mutant band with the resistance gene
probe (C and F), demonstrate the presence of single insertions in
the PpRSLl and PpRSL2 loci. (G) RT-PCR showing that the mutants
are RNA null mutants. PpGAPDH, glyceraldehyde 3-phosphate
dehydrogenase. In each case, the three independent single
insertion mutants presented have the same phenotype and only the
mutant 1 is shown in Fig. 4.
Figure 10 shows the root hair system of an Arabidopsis plant over-
expressing RSL4 and displaying a root morphology resembling a
fungal symbiont, such as Mycorrhizae.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION
The present disclosure demonstrates the identification, isolation,
cloning and expression of the ROOT HAIR DEFECTIVE 6 (RHD6) and
ROOT HAIR DEFECTIVE SIX LIKE (RSL) genes (collectively termed
1JUfD6-related genes' herein) in plants. It shows complementation
of mutations by distantly related genes, providing the function of
root hair development in plants in which the distantly related
gene has been inactivated. Accordingly, those skilled in the art
will appreciate that, for the first time, the gene responsible for
previously identified mutant phenotypes has been isolated and
cloned according to this invention. It will also be appreciated
that from this disclosure functional benefits may be conferred on
plants by means of introduction into plants and expression of
these genes in such plants. Methods known in the art may be
utilized for this purpose. Thus, for example, those skilled in

the art will appreciate that the methods, for example, for
achieving the expression of the RHD6 and RSL genes of this
invention may be achieved according methods disclosed herein, and
by methods, for example, disclosed in, but not limited to,
EP0803572B1, which discloses the cloning and expression of the cpc
gene, which, like the RHD6 and RSL genes of this invention, is
also related to the control of root hair development in plants,
albeit at a different stage of plant and root hair development.
In various aspects, the invention provides ROOT HAIR DEFECTIVE 6
(RHD6)-related polypeptides encoded by ROOT HAIR DEFECTIVE 6
(RHD6)-related genes and nucleic acid sequences described herein.
ROOT HAIR DEFECTIVE 6 (RHD6)-related polypeptides include both
ROOT HAIR DEFECTIVE 6 (RHD6) polypeptides and ROOT HAIR DEFECTIVE
6-LIKE 1 (RSL1) polypeptides, and functional homologues thereof,
as described herein. RHD6-related polypeptides include may be
capable of complementing the rhd6 mutation upon expression in
plants.
A ROOT HAIR DEFECTIVE 6 (RHD6)-related polypeptide may fall within
the RHD6 clade comprising AtRHD6, AtRSLl, PpRSLl, PpRSL2, BdRSLb,
TaRSLa, OsRSLc, BdRSLc, OsRSLb, ZmRSLa, PtRSLa, PrRSLb, OsRSLa,
BdRSLa, SmRSLa, SmRSLb, SmRSLc and SmRSLd (the ROOT HAIR DEFECTIVE
6 (RHD6) clade) in a cladogram of protein sequences, for example
using the sequences of AtlND and PpINDa as an outgroup (see figure
3).
Alternatively, ROOT HAIR DEFECTIVE 6 (RHD6)-related polypeptide
may fall within the RSL clade comprising AtRSL3, CtRSLa, PtRSLe,
OsRSLi, AtRSL5, AtRSL4, PtRSLe, PtRSLd, AtRSL2, MtRSLa, OsRSLd,
OsRSLh, LsRSLa, MaRSLa, OsRSLe, GmRSLb, GmRSLa, ZmRSLb, ZmRSLd,
BdRSLd, ZmRSLc, OsRSLg, BdRSLe, 03RSLf, PpRSL3, PpRSL4, PpRSL5,
PpRSL6, PpRSL7, SmRSLg, SmRSLf, SmRSLh and SmRSLe (the ROOT HAIR
DEFECTIVE SIX LIKE (RSL) clade) in a cladogram of protein
sequences, for example using the sequences of AtlND and PpINDa as
an outgroup (see figure 3).
A dadogram may be produced using conventional techniques. For
example, a cladogram may be calculated using ClustalW to align the
protein sequences, Phylip format for tree output, with 1000
bootstrap replicates and TreeViewX (version 0.5.0) for
visualisation.
A suitable ROOT HAIR DEFECTIVE 6 (RHD6)-related polypeptide may
comprise the amino acid sequence shown of SEQ ID NO: 1, 3, 5, 7,
9, 11, 13 to 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,
52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,
84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112
or 114 or may be a fragment or variant of one of these sequences
which retains RHD6 activity.
In some preferred embodiments, the ROOT HAIR DEFECTIVE 6 (RHD6)-
RELATED polypeptide may be a ROOT HAIR DEFECTIVE 6 (RHD6)
polypeptide having the amino acid sequence of SEQ ID NO:1
(Atlg66470; NP_176820.1 Gi: 15219658) or may be a fragment or variant
of this sequence which retains RHD6 activity.
In other embodiments, the ROOT HAIR DEFECTIVE 6 (RHD6)-RELATED
polypeptide may be a ROOT HAIR DEFECTIVE SIX LIKE (RSL)
polypeptide having the amino acid sequence of any one of SEQ ID
NOS: 5, 7, 9, and 11 or may be a fragment or variant of any of
these sequences which retains RHD6 activity.
A ROOT HAIR DEFECTIVE 6 (RHD6)-RELATED polypeptide which is a
variant a reference sequence set out herein, such as SEQ ID NO: 1,
3, 5, 7, 9, 11, 13 to 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,
48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,
80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,
110, 112 or 114, may comprise an amino acid sequence which shares
greater than 20% sequence identity with the reference amino acid

sequence, preferably greater than 30%, greater than 40%, greater
than 50%, greater than 60%, greater than 65%, greater than 70%,
greater than 80%, greater than 90% or greater than 95%.
Particular amino acid sequence variants may differ from a RHD6-
related polypeptide sequence as described herein by insertion,
addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10,
10-20 20-30, 30-50, or more than 50 amino acids.
Sequence identity is commonly defined with reference to the
algorithm GAP (Wisconsin Package, Accelerys, San Diego USA). GAP
uses the Needleman and Hunsch algorithm to align two complete
sequences that maximizes the number of matches and minimizes the
number of gaps. Generally, default parameters are used, with a
gap creation penalty = 12 and gap extension penalty = 4.
Dse of GAP may be preferred but other algorithms may be used, e.g.
BLAST (which uses the method of Altschul et al. (1990) J. Mol.
Blol. 215: 405-410), FASTA (which uses the method of Pearson and
Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman
algorithm (Smith and Waterman (1981) J. Mol Blol. 147: 195-197),
or the TBLASTN program, of Altschul et al. (1990) supra, generally
employing default parameters. In particular, the psi-Blast
algorithm (Nucl. Acids Res. (1997) 25 3389-3402) may be used.
Sequence comparison may be made over the full-length of the
relevant sequence described herein.
Certain domains of a RHD6-related polypeptide may show an
increased level of identity with domains of a reference sequence,
such as SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 to 26, 28, 30, 32, 34,
36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,
68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,
100, 102, 104, 106, 108, 110, 112 or 114, relative to the RHD6-
related polypeptide sequence as a whole. For example, a RHD6-
related polypeptide may comprise one or more domains or motifs

consisting of an amino acid sequence which has at least 70%, at
least 75%, at least 80%, at least 90%f at least 95%, or at least
98% sequence identity or similarity, with an amino acid sequence
selected from the group consisting of SEQ ID NOS: 13 to 25 or
other RHD6-related polypeptide domain shown in tables 1 and 2.
In some preferred embodiments, a RHD6-related polypeptide may
comprise one or more domains or motifs consisting of an amino acid
sequence which is selected from the group consisting of SEQ ID
NOS: 13 to 25 or other RHD6-related polypeptide domain shown in
tables 1 and 2.
In various aspects, the invention provides ROOT HAIR DEFECTIVE 6
(RHD6)-related genes and nucleic acid sequences which encode ROOT
HAIR DEFECTIVE 6 (RHD6)-related polypeptides, as described herein.
A nucleic acid encoding a AHDff-related polypeptide may comprise or
consist of the nucleotide sequence of any one of SEQ ID NOS: 2, 4,
6, 8, 10, 12, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,
53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83,
85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113,
and 115 or may be a variant or fragment of any one of these
sequences which encodes a polypeptide which retains RHD6 activity.
In some preferred embodiments, a nucleic acid encoding a RHD6-
related polypeptide may comprise or consist of the nucleotide
sequence of SEQ ID NO: 2 or may be a variant or fragment of any
one of these sequences which encodes a polypeptide which retains
RHD6 activity.
A variant sequence may be a mutant, homologue, or allele of any
one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 27, 29, 31, 33, 35, 37, 39,
41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,
73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103,
105, 107, 109, 111, 113, and 115 and may differ from one of these
sequences by one or more of addition, insertion, deletion or

substitution of one or more nucleotides in the nucleic acid,
leading to the addition* insertion, deletion or substitution of
one or more amino acids in the encoded polypeptide. Of course,
changes to the nucleic acid that make no difference to the encoded
amino acid sequence are included. A nucleic acid encoding a RHD6-
related polypeptide, which has a nucleotide sequence which is a
variant of an KHD5-related nucleic acid sequence set out herein
may comprise a sequence having at least 30% sequence identity with
the nucleic acid sequence of any one of SEQ ID NOS: 2, 4, 6, 8,
10, 12, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,
55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,
87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, and
115, for example, preferably greater than 40%, greater than 50%,
greater than 60%, greater than 65%, greater than 70%, greater than
80%, greater than 90% or greater than 95%. Sequence identity is
described above.
A fragment or variant may comprise a sequence which encodes a
functional AHDff-related polypeptide i.e. a polypeptide which
retains one or more functional characteristics of the polypeptide
encoded by the wild-type RHD6 gene, for example, the ability to
stimulate or increase root hair number, growth or longevity in a
plant or to complement the rhd€ mutation.
In other embodiments, a nucleic acid encoding a RHD6 polypeptide,
which has a nucleotide sequence which is a variant of the sequence
of any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 27, 29, 31, 33, 35,
37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,
69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,
101, 103, 105, 107, 109, 111, 113, and 115 may selectively
hybridise under stringent conditions with this nucleic acid
sequence or the complement thereof.
Stringent conditions include, e.g. for hybridization of sequences
that are about 80-90% identical, hybridization overnight at 42°C
in 0.25M Na2HPO4, pH 7.2, 6.5% SDS, 10% dextran sulfate and a

final wash at 55 °C in 0.1X SSC, 0.1% SDS. For detection of
sequences that are greater than about 90% identical, suitable
conditions include hybridization overnight at 65°C in 0.25M
Na:HPO4, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at
60°C in 0.1X SSC, 0.1% SDS.
An alternative, which may be particularly appropriate with plant
nucleic acid preparations, is a solution of 5x SSPE (final 0.9 M
NaCl, 0.05M sodium phosphate, 0.005M EDTA pH 7.7), 5X Denhardt's
solution, 0.5% SDS, at 50°C or 65°C overnight. Hashes may be
performed in 0.2x SSC/0.1% SDS at 65'C or at 50-60°C in lx
SSC/0.1% SDS, as required.
Nucleic acids as described herein may be wholly or partially
synthetic. In particular, they may be recombinant in that nucleic
acid sequences which are not found together in nature (do not run
contiguously) have been ligated or otherwise combined
artificially. Alternatively, they may have been synthesised
directly e.g. using an automated synthesiser.
The nucleic acid may of course be double- or single-stranded, cDNA
or genomic DNA, or RNA. The nucleic acid may be wholly or
partially synthetic, depending on design. Naturally, the skilled
person will understand that where the nucleic acid includes RNA,
reference to the sequence shown should be construed as reference
to the RNA equivalent, with U substituted for T.
ROOT HAIR DEFECTIVE 6 (RHD6)-related polypeptides and nucleic
acids may be readily identified by routine techniques of sequence
analysis in a range of plants, including agricultural plants
selected from the group consisting of Llthospermum erythrorhizon,
Taxus spp, tobacco, cucurbits, carrot, vegetable brassica, melons,
capsicums, grape vines, lettuce, strawberry, oilseed brassica,
sugar beet, wheat, barley, maize, rice, soyabeans, peas, sorghum,
sunflower, tomato, potato, pepper, chrysanthemum, carnation,
linseed, hemp and rye.

A RHDff-related nucleic acid as described herein may be operably
linked to a heterologous regulatory sequence, such as a promoter,
for example a constitutive, inducible, root-specific or
developmental specific promoter.
"Heterologous" indicates that the gene/sequence of nucleotides in
question or a sequence regulating the gene/sequence in question,
has been linked to the RHD6 related nucleic acid using genetic
engineering or recombinant means, i.e. by human intervention.
Regulatory sequences which are heterologous to an RHD6 related
nucleic acid may be regulatory sequences which do not regulate the
RHD6 related nucleic acid in nature or are not naturally
associated with the RHD6 related nucleic acid. "Isolated" indicate
that the isolated molecule (e.g. polypeptide or nucleic acid)
exists in an environment which is distinct from the environment in
which it occurs in nature. For example, an isolated nucleic acid
may be substantially isolated with respect to the genomic
environment in which it naturally occurs.
Many suitable regulatory sequences are known in the art and may be
used in accordance with the invention. Examples of suitable
regulatory sequences may be derived from a plant virus, for
example the Cauliflower Mosaic Virus 35S (CaMV 35S) gene promoter
that is expressed at a high level in virtually all plant tissues
(Benfey et al, (1990) EMBO J 9: 1677-1684). Other suitable
constitutive regulatory elements include the cauliflower mosaic
virus 19S promoter; the Figwort mosaic virus promoter; and the
nopaline synthase (nos) gene promoter (Singer et al.. Plant Mol.
Biol. 14:433 (1990); An, Plant Physiol. 81:86 (1986)). For
example, RHD6-related genes such as AtRHD6, AtRSLl and AtRSL4 may
be expressed using constitutive promoters.
Constructs for expression of RHD6 and RSL genes under the control
of a strong constitutive promoter (the 35S promoter) are
exemplified below. Expression of AtRHD6, AtRSLl and AtRSL4 from

the 35S promoter is shown to modulate root hair development in
plants without causing additional phenotypic changes.
However, those skilled in the art will appreciate that a wide
variety of other promoters may be employed to advantage in
particular contexts. Thus, for example, one might select an
epidermal or root-specific promoter to ensure expression of these
constructs only in roots. Suitable root-specific promoters are
described for example in Qi et al PNAS (2006) 103(49) 18848-18853.
For example, RHD6-related genes such as AtRSL2 and ATRSL3, may be
expressed using root-specific promoters.
Alternatively, or in addition, one might select an inducible
promoter. In this way, for example, in a cell culture setting,
production of a particular gene product of interest may be
enhanced or suppressed by induction of the promoter driving
expression of the genes described herein. Inducible promoters
include the alcohol inducible ale gene-expression system (Roslan
et al.. Plant Journal; 2001 Oct; 28(2):225-35) may be employed.
RHD6-related nucleic acid may be contained on a nucleic acid
construct or vector. The construct or vector is preferably
suitable for transformation into and/or expression within a plant
cell.
A vector is, inter alia, any plasmid, cosmid, phage or
Agrobacteriwn binary vector in double or single stranded linear or
circular form, which may or may not be self transmissible or
mobilizable, and which can transform prokaryotic or eukaryotic
host, in particular a plant host, either by integration into the
cellular genome or exist extrachromasomally (e.g. autonomous
replicating plasmid with an origin of replication).
Specifically included are shuttle vectors by which is meant a DNA
vehicle capable, naturally or by design, of replication in two
different organisms, which may be selected from actinomyces and

related species, bacteria and eukaryotic (e.g. higher plant,
mammalia, yeast or fungal) cells.
A construct or vector comprising nucleic acid as described above
need not include a promoter or other regulatory sequence,
particularly if the vector is to be used to introduce the nucleic
acid into cells for recombination into the genome.
Constructs and vectors may further comprise selectable genetic
markers consisting of genes that confer selectable phenotypes such
as resistance to antibiotics such as kanamycin, hygromycin,
phosphinotricin, chlorsulfuron, methotrexate, gentamycin,
spectinomycin, imidazolinones, glyphosate and d-amino acids.
Those skilled in the art are well able to construct vectors and
design protocols for recombinant gene expression, in particular in
a plant cell. Suitable vectors can be chosen or constructed,
containing appropriate regulatory sequences, including promoter
sequences, terminator fragments, polyadenylation sequences,
enhancer sequences, marker genes and other sequences as
appropriate. For further details see, for example. Molecular
Cloning: a Laboratory Manual: 3rd edition, Sambrook & Russell,
2001, Cold Spring Harbor Laboratory Press.
Those skilled in the art can construct vectors and design protocols
for recombinant gene expression, for example in a microbial or
plant cell. Suitable vectors can be chosen or constructed,
containing appropriate regulatory sequences, including promoter
sequences, terminator fragments, polyadenylation sequences,
enhancer sequences, marker genes and other sequences as
appropriate. For further details see, for example. Molecular
Cloning: a Laboratory Manual: 3rd edition, Sambrook et al, 2001,
Cold Spring Harbor Laboratory Press and Protocols In Molecular
Biology, Second Edition, Ausubel et al. eds. John Wiley & Sons,
1992. Specific procedures and vectors previously used with wide
success upon plants are described by Bevan, Nucl. Acids Res. (1984)

12, 8711-8721), and Guerineau and Mullineaux, (1993) Plant
transformation and expression vectors. In: Plant Molecular Biology
Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121-
148.
When introducing a chosen gene construct into a cell, certain
considerations must be taken into account, well known to those
skilled in the art. The nucleic acid to be inserted should be
assembled within a construct that contains effective regulatory
elements that will drive transcription. There must be available a
method of transporting the construct into the cell. Once the
construct is within the cell membrane, integration into the
endogenous chromosomal material either will or will not occur.
Finally, the target cell type is preferably such that cells can be
regenerated into whole plants.
Those skilled in the art will also appreciate that in producing
constructs for achieving expression of the genes according to this
invention, it is desirable to use a construct and transformation
method which enhances expression of the RHD6 gene, the RSL gene or
a functional homolog thereof. Integration of a single copy of the
gene into the genome of the plant cell may be beneficial to
minimize gene silencing effects. Likewise, control of the
complexity of integration may be beneficial in this regard. Of
particular interest in this regard is transformation of plant
cells utilizing a minimal gene expression construct according to,
for example, EP Patent No. EP 1 407 000 Bl, herein incorporated by
reference for this purpose.
Techniques well known to those skilled in the art may be used to
introduce nucleic acid constructs and vectors into plant cells to
produce transgenic plants with the properties described herein.
Agrobacterium transformation is one method widely used by those
skilled in the art to transform woody plant species, in particular
hardwood species such as poplar. Production of stable, fertile

transgenic plants is now routine in the art:(Toriyama, et al.
(1988) Bio/Technology 6, 1072-1074; Zhang, et al. (1988) Plant
Cell Rep. 7, 379-384; Zhang, et al. (1988) Theor Appl Genet 76,
835-840; Shimamoto, et al. (1989) Nature 338, 274-276; Datta, et
al. (1990) Bio/Technology 8, 736-740; Christou, et al. (1991)
Bio/Technology 9, 957-962; Peng, et al. (1991) International Rice
Research Institute, Manila, Philippines 563-574; Cao, et al.
(1992) Plant Cell Rep. 11, 585-591; Li, et al. (1993) Plant Cell
Rep. 12, 250-255; Rathore, et al. (1993) Plant Molecular Biology
21, 871-884; Fromm, et al. (1990) Bio/Technology 8, 833-839;
Gordon-Kamm, et al. (1990) Plant Cell 2, 603-618; D'Halluin, et
al. (1992) Plant Cell 4, 1495-1505; Walters, et al. (1992) Plant
Molecular Biology 18, 189-200; Koziel, et al. (1993) Biotechnology
11, 194-200; Vasil, I. K. (1994) Plant Molecular Biology 25, 925-
937; Weeks, et al. (1993) Plant Physiology 102, 1077-1084; Somera,
et al. (1992) Bio/Technology 10, 1589-1594; WO92/14828; Nilsson,
0. et al (1992) Transgenic Research 1, 209-220).
Other methods, such as microprojectile or particle bombardment (US
5100792, EP-A-444882, EP-A-434616), electroporation (EP 290395, WO
8706614), microinjection (WO 92/09696, WO 94/00583, EP 331083, EP
175966, Green et al. (1987) Plant Tissue and Cell Culture,
Academic Press), direct DNA uptake (DE 4005152, WO 9012096, DS
4684611), liposome mediated DNA uptake (e.g. Freeman et al. Plant
Cell Physio1. 29: 1353 (1984)), or the vortexing method (e.g.
Kindle, PNAS U.S.A. 87: 1228 (1990d)) may be preferred where
Agrobacterium transformation is inefficient or ineffective, for
example in some gymnosperm species.
Physical methods for the transformation of plant cells are
reviewed in Oard, 1991, Biotech. Adv. 9: 1-11.
Alternatively, a combination of different techniques may be
employed to enhance the efficiency of the transformation process,
e.g. bombardment with Agrobacterium coated microparticles (EP-A-
486234) or microprojectile bombardment to induce wounding followed
by co-cultivation with Agrobacterium (EP-A-486233).
Following transformation, a plant may be regenerated, e.g. from
single cells, callus tissue or leaf discs, as is standard in the
art. Almost any plant can be entirely regenerated from cells,
tissues and organs of the plant. Available techniques are
reviewed in Vasil et al.. Cell Culture and Somatic Cell Genetics
of Plants, Vol I, II and III, Laboratory Procedures and Their
Applications, Academic Press, 1984, and Heissbach and Weissbach,
Methods for Plant Molecular Biology, Academic Press, 1989.
The particular choice of a transformation technology will be
determined by its efficiency to transform certain plant species as
well as the experience and preference of the person practising the
invention with a particular methodology of choice. It will be
apparent to the skilled person that the particular choice of a
transformation system to introduce nucleic acid into plant cells
is not essential to or a limitation of the invention, nor is the
choice of technique for plant regeneration.
Other aspects of the invention relate to the modulation of plant
root hair development using RHD6 related polypeptides and nucleic
acids as described herein.
A method of modulating root hair development or altering the root
hair phenotype in a plant may comprise;
increasing the expression of a RHD6-related polypeptide
within cells of said plant relative to control plants.
Modulation of root hair development in a plant may include
increasing one or more of: root-hair growth, number of root-hairs,
length of root-hairs, rate of growth of root-hairs, and longevity
of individual root-hairs on the plant.
RHD6-related polypeptides are described in more detail above.

Expression of an RHD6-related polypeptide may be increased by any
suitable method. In some embodiments, the expression of a RHD6-
related polypeptide may be increased by expressing a heterologous
nucleic acid encoding the RHD6-related polypeptide within cells of
said plant.
Suitable controls will be readily apparent to the skilled person
and may include plants in which the expression of the RHD6-related
polypeptide is not increased.
A method of producing a plant with altered root hair phenotype may
comprise:
incorporating a heterologous nucleic acid which alters the
expression of a RHD6-related polypeptide into a plant cell by
means of transformation, and;
regenerating the plant from one or more transformed cells.
Suitable RHD6-related polypeptides are described in more detail
above.
In some embodiments, the plant may be a plant whose roots are not
naturally colonised by symbiotic fungi, such as Mycorrhizae.
Plants whose roots are not naturally colonised by fungi include
non-mycorrhizal plants such as Brassleas.
Plants for use in the methods described herein preferably lack
mutations in RHD6-related genes. For example the plant may be a
wild-type plant.
A plant with altered root hair phenotype produced as described
above may show improved tolerance to nutrient-deficient growth
conditions, increased production of phytochemicals and/or
increased phytoremediation properties, such as absorption of heavy
metals.
Nucleic acid encoding RHD6-related polypeptides and their
expression in plants is described in more detail above.
In other embodiments, the expression of an RHD6-related
polypeptide may be increased by increasing the expression of an
endogenous nucleic acid encoding the RHD6-related polypeptide
within cells of said plant.
The expression of an endogenous nucleic acid encoding the RHD6-
related polypeptide within cells of said plant may be increased by
recombinant means, such as the targeted insertion of regulatory
factors,
The expression of an endogenous nucleic acid encoding the RBD6-
related polypeptide within cells of said plant may be increased by
non-recombinant means. For example, expression of the RHD6-related
polypeptide may be increased in a plant by selective plant
breeding methods which employ the RHD6-related amino acid or
nucleic acid sequence as a molecular marker in order to produce a
plant having an altered root hair phenotype, for example increased
size, number or longevity of root hairs relative to controls.
A method of producing a plant having altered root hair phenotype
may comprise:
providing a population of plants,
determining the amount of expression of a RKDff-related
polypeptide as described herein in one or more plants in the
population, and
identifying one or more plants in the population with
increased expression of the RHD6-related polypeptide relative to
other members of said population.
The identified plants may be further propagated or crossed, for
example, with other plants having increased RHD6-related
polypeptide expression or self-crossed to produce inbred lines.
The expression of an RHD6-related polypeptide in populations of

progeny plants may be determined and one or more progeny plants
with reduced expression of the RHD6-zelated polypeptide
identified.
The expression of an RHD6-related polypeptide in a plant may be
determined by any convenient method. In some embodiments, the
amount of expression of the RHD6-related polypeptide may be
determined at the protein level. A method of producing a plant
with altered root hair development may comprise:
providing a population of plants,
determining the amount of RHD6-related polypeptide in
one or more plants of said population, and
identifying one or more plants in the population with
increased amounts of an RHD6-related polypeptide relative to other
members of said population.
The amount of RHD6-related polypeptide may be determined in one or
more cells of the plant, preferably cells from a below-ground
portion or tissue of the plant, such as the root.
The amount of RHD6-related polypeptide may be determined using any
suitable technique. Conveniently, immunological techniques, such
as Western blotting may be employed, using antibodies which bind
to the RHD6-related polypeptide and show little or no binding to
other antigens in the plant. For example, the amount of an RHD6-
related polypeptide in a plant cell may be determined by
contacting a sample comprising the plant cell with an antibody or
other specific binding member directed against the RHD6-related
polypeptide, and determining binding of the RHD6-related
polypeptide to the sample. The amount of binding of the specific
binding member is indicative of the amount of RHD6-related
polypeptide which is expressed in the cell.
In other embodiments, the expression of the RHD6-related
polypeptide may be determined at the nucleic acid level. For
example, the amount of nucleic acid encoding an RHD6-related

polypeptide may be determined. A method of producing a plant
having altered root hair development may comprise:
providing a population of plants,
determining the level or amount of nucleic acid, for example
mRNA, encoding the RHD6-related polypeptide in a cell of one or
more plants of said population, and,
identifying one or more plants in the population with
increased amount of nucleic acid encoding an RHD6-related
polypeptide relative to other members of said population.
The level or amount of encoding nucleic acid in a plant cell may
be determined for example by detecting the amount of transcribed
encoding nucleic acid in the cell. Numerous suitable methods for
determining the amount of a nucleic acid encoding an RHD6-related
polypeptide in a plant cell are available in the art, including,
for example. Northern blotting or RT-PCR (see for example
Molecular Cloning: a Laboratory Manual: 3rd edition, Sambrook &
Russell (2001) Cold Spring Harbor Laboratory Press NY; Current
Protocols in Molecular Biology, Ausubel et al. eds. John Wiley £
Sons (1992); DNA Cloning, The Practical Approach Series (1995),
series eds. D. Rickwood and B.D. Hames, IRL Press, Oxford, UK and
PCR Protocols: A Guide to Methods and Applications (Innis, et al.
1990. Academic Press, San Diego, Calif.).
A suitable cell may be from a below-ground portion or tissue of
the plant, such as the root.
A progeny plant identified as having increased RHD6-related
polypeptide expression may be tested for altered root hair
development relative to controls, for example increased growth,
number or longevity of root hairs, or may be tested for other
properties, such as increased resistance to nutrient deficient
conditions, increased phytochemical production, increased
phytoremediation properties or a consitutive low phosphate
response.
A method of producing a plant having an altered root hair
phenotype may comprise:
crossing a first and a second plant to produce a
population of progeny plants;
determining the expression of a RHD6-related polypeptide
in the progeny plants in the population, and
identifying a progeny plant in the population in which
expression of the RHD6-related polypeptide is increased relative
to controls.
A progeny plant having an altered root hair phenotype may show
increased growth, number or longevity of root hairs relative to
controls (e.g. other members of the population with a wild-type
phenotype).
The identified progeny plant may be further propagated or crossed,
for example with the first or second plant (i.e. backcrossing) or
self-crossed to produce inbred lines.
The identified progeny plant may be tested for increased tolerance
to nutrient-deficient conditions relative to controls.
Other aspects of the invention provide the use of an RHD6-related
polypeptide or encoding nucleic acid as described herein as a
marker for the selective breeding of a plant which has an altered
root hair phenotype relative to control plants, and a method of
selective breeding of a plant which has an altered root hair
phenotype relative to control plants, which employs the RHD6-
related amino acid or encoding nucleic acid sequence.
In some embodiments, plants having reduced expression of the RHD6-
related polypeptide may be produced by random mutagenesis,
followed by screening of mutants for reduced RHD6-related
polypeptide expression. Suitable techniques are well known in the
art and include Targeting Induced Local Lesions IN Genomes
(TILLING). TILLING is a high-throughput screening technique that

results in the systematic identification of non-GMO-derived
mutations in specific target genes (Comai and Henikoff, The Plant
Journal (2006) 45, 684-694 Till et aI_BMC Plant Biol. 2007 Apr 7, 19.
Those skilled in the art will also appreciate that, based on the
genetic information disclosed herein. Targeted Induced Local
Lesions IN Genomes ("TILLING", e.g. utilizing PCR-based screening
of plants generated through chemical mutagenesis (generally via
ethyl methane sulfonate (EMS) treatment), often resulting in the
isolation of missense and nonsense mutant alleles of the targeted
gene(s); TILLING permits the high-throughput identification of
mutations in target genes without production of genetically
modified organisms and it can be an efficient way to identify
mutants in a specific gene that might not confer a strong
phenotype by itself), may be carried out to produce plants and
offspring thereof with a change in the RHD6 or RSL gene, thereby
permitting identification of plants with specific phenotypes
relevant to plant root hair production.
A method of producing a plant having an altered root hair
phenotype may comprise:
exposing a population of plants to a mutagen,
determining the expression of a RHD6-related polypeptide
or nucleic acid in one or more plants in said population, and
identifying a plant with increased expression of the
RHD6-related polypeptide relative to other members of said
population.
Suitable mutagens include ethane methyl sulfonate (EMS).
Methods for determining the expression of RHD6-related polypeptide
or nucleic acid in plants is described in more detail above.
The identified plant may be further tested for increased tolerance
or resistance to low-nutrient conditions relative to controls,
increased production of phytochemicals or increased
phytoremediation.
A plant identified as having increased expression of the RHD6-
related polypeptide relative to controls (e.g. other members of
the population) may display increased growth, number or longevity
of root hairs relative to the controls.
A plant produced or identified as described above may be sexually
or asexually propagated or grown to produce off-spring or
descendants. Off-spring or descendants of the plant regenerated
from the one or more cells may be sexually or asexually propagated
or grown. The plant or its off-spring or descendents may be
crossed with other plants or with itself.
Expression of RHD6-related genes such as RSL4 is shown herein to
produce a phenotype in which the root hairs display a fungus-like
morphology. This morphology is characterised by extensive
indeterminate masses of growing cells which resemble fungal like
colonies (figure 10) and results in a greatly increased surface
area of root hairs. This phenotype may confer significantly
enhanced root uptake of phosphate and iron, which is largely
limited by the length and surface area of the root hair.
By means of expression of the genes described herein in a plant, a
plant may be made to exhibit enhanced absorption of otherwise less
efficiently absorbed nutrients. Thus, for example, it is known in
the art that phosphate and iron absorption from the soil is
achieved primarily by plant root hairs. By enhancing the number,
length, time of production or duration of survival of plant root
hairs, by expression, overexpression, or targeted misexpression
(expression in cells that otherwise may not produce root hairs) of
RHO6-related genes, absorption of iron or phosphate or both, as
well as other nutrients, may be enhanced. In particular,
absorption may be increased by the fungus-like root hair
morphology produced by overexpression of RHD6-related genes such

as AtRSL4 (see figure 10). Thus, it has been shown in the
literature that rhd6 mutants are compromised in their ability to
absorb phosphate, see Plant Growth and Phosphorus Accumulation of
Wild Type and Two Root Hair Mutants of Arabidopals thallana
(Brassicaceae)", Terence R. Bates and Jonathan P. Lynch, American
Journal of Botany 87(7): 958-963. 2000. This effect may be
reversed by supplementation with the functional gene described
herein. Aspects of the invention would be of particular benefit,
for example, in low-iron or low-phosphate containing soils, such
as those found in China, sub-Saharan Africa and Australia
A method of improving the tolerance or resistance of a plant to
nutrient deficient conditions may comprise;
increasing the expression of an RHD6-related polypeptide
within cells of said plant relative to control plants.
Nutrient deficient conditions include conditions which contain
levels of one or more nutrients such as nitrate, phosphate and/or
iron, which are insufficient to fulfil the nutritional
requirements of the wild-type plant. A wild-type plant subjected
to nutrient deficient conditions may adopt a nutrient deficient
phenotype, such as reduced growth resulting in greatly reduced
yield and crop quality.
For example, the plant may show improved growth in soil which
contains low levels of one or more nutrients such as nitrate,
phosphate and/or iron, relative to control plants (i.e. plants in
which RHD6-related polypeptide expression is unaltered).
Furthermore, phosphate deficiency increases the expression of
RH06-related polypeptides such as AtRHD6 and AtRSLl in the root
epidermis of a plant. Expression of a RHD6-related polypeptide in
the root epidermis may therefore be useful in producing a
constitutive "low phosphate" response in a plant.
The genes described herein may be utilized to achieve enhanced
production of compounds of interest, including medicinally
relevant compounds. Thus, for example, it is known that plant
root hairs are responsible for production of antibiotic compounds.
In nature, these compounds are secreted by plant roots and
especially plant root hairs, to thereby modify or otherwise
control the microflora and microfauna surrounding the plant roots.
Production of these phytochemicals is enhanced in plants in which
the number, length, duration of production, time of production and
other characteristics of root hair development and growth may be
modified at will according to the methods of this invention.
A method of increasing the production or secretion of a root-
secreted phytochemical in a plant may comprise;
increasing the expression of a RHD6-related polypeptide
within cells of a plant which secretes the phytochemical through
its roots.
Root-secreted phytochemicals include shikonin (Brigham LA, et al
Plant Physiol. 1999 Feb;119(2):417-28) which may be produced by
Llthospermvm erythrorhizon. and paclitaxel, which may be produced
by Taxus spp.
Heavy metals are an important environmental pollutant and may be
removed by growing plants on contaminated soils. In
phytoremediation or phytoextraction, plants absorb contaminating
substances such as heavy metals from the soil and the plants are
harvested at maturity, thereby removing these contaminants from
the area. The long root hair phenotypes conferred by increased
expression of RHD6-related polypeptides may enhance the
phytoremediation properties of plant species.
A method of reducing the amount of a contaminating substance in
soil comprising;
increasing the expression of a RHD6-related polypeptide
within cells of a plant which absorbs the contaminating substance
through its roots,
growing the plant or a descendant thereof in soil which
comprises the contaminating substance such that the plant or
descendent absorbs the contaminating substance from the soil, and
harvesting said plant or descendent thereof.
Contaminating substances include uranium, polychlorinated
biphenyls, salt, arsenic and heavy metals such as cadmium, zinc
and lead.
A plant suitable for use in the present methods is preferably a
higher plant, for example an agricultural plant selected from the
group consisting of Lithospermum erythrorhizon, Taxus spp,
tobacco, cucurbits, carrot, vegetable brassica, melons, capsicums,
grape vines, lettuce, strawberry, oilseed brassica, sugar beet,
wheat, barley, maize, rice, soyabeans, peas, sorghum, sunflower,
tomato, potato, pepper, chrysanthemum, carnation, linseed, hemp
and rye.
In embodiments relating to phytochemical production, Lithospermum
erythrorhizon and Taxus spp may be preferred.
In embodiments relating to phytoremediation, sunflower [Hellanthus
annuus), Chinese Brake fern, alpine pennycress (Thlaspl
caerulescens), Indian mustard (Brassica juncea), Ragweed (Ambrosia
artemlsllfolla) Hemp Dogbane (Apocymun cannabinum) and Poplar may
be preferred.
Another aspect of the invention provides a plant which is produced
by a method described herein, wherein said plant shows altered
root hair phenotype relative to controls.
For example, a plant may display increased growth, number or
longevity of root hairs relative to controls (e.g. other members
of the population with a wild-type phenotype).
A plant may display increased tolerance to nutrient deficient
conditions and/or increased production of root-secreted
phytochemicals.
Also provided is any part or propagule of such a plant, for
example seeds, selfed or hybrid progeny and descendants.
A plant according to the present invention may be one which does
not breed true in one or more properties. Plant varieties may be
excluded, particularly registrable plant varieties according to
Plant Breeders Rights.
In addition to a plant produced by a method described herein, the
invention encompasses any clone of such a plant, seed, selfed or
hybrid progeny and descendants, and any part or propagule of any
of these, such as cuttings and seed, which may be used in
reproduction or propagation, sexual or asexual. Also encompassed
by the invention is a plant which is a sexually or asexually
propagated off-spring, clone or descendant of such a plant, or any
part or propagule of said plant, off-spring, clone or descendant.
While the foregoing disclosure provides a general description of
the subject matter encompassed within the scope of the present
invention, including methods, as well as the best mode thereof, of
making and using this invention, the following examples are
provided to further enable those skilled in the art to practice
this invention and to provide a complete written description
thereof. However, those skilled in the art will appreciate that
the specifics of these examples should not be read as limiting on
the invention, the scope of which should be apprehended from the
claims and equivalents thereof appended to this disclosure.
Various further aspects and embodiments of the present invention

will be apparent to those skilled in the art in view of the
present disclosure.
All documents mentioned in this specification are incorporated
herein by reference in their entirety.
"and/or" where used herein is to be taken as specific disclosure
of each of the two specified features or components with or
without the other. For example "A and/or B" is to be taken as
specific disclosure of each of (i) A, (ii) B and (iii) A and B,
just as if each is set out individually herein.
Unless context dictates otherwise, the descriptions and
definitions of the features set out above are not limited to any
particular aspect or embodiment of the invention and apply equally
to all aspects and embodiments which are described.
Certain aspects and embodiments of the invention will now be
illustrated by way of example and with reference to the figures
described above and table described below.
Tables 1 and 2 show a sequence alignment of bHLH amino acid
sequences (Helm et al. Nol. Biol. Evol. 2003) generated by
ClustalW (http://www.ebi.ac.uk).
Table 3 shows % identities of RHD6-related proteins as determined
by DNA Strider (Christain Mark, Center. dfEtudes de Saclay).
Table 4 shows relative identities of the bHLH domains of RHD6-
related proteins to RHD6.
Table 5 shoes the correspondence between the names on the tree of
figure 3 and the alignments of tables 1 and 2 with respective
species and locus or GI accession number.
EXAMPLES
Root hairs are highly polarised cells that increase the surface
area of the plant that is in contact with the soil. They play
important roles in nutrient acquisition and anchorage in those
land plants that have roots" -. Other tip growing cells such as
rhizoids and caulonemal cells have a similar function in more
basal groups of land plants that lack roots3' *. Here we identify
and characterise two basic helix loop helix transcription factors
that control the development of root hair cells in Arabidopsis
sporophyte and show that their closest homologs in Physcomltrella
patens are required for the development of both rhizoids and
caulonemal cells in the gametophyte of this moss. This indicates
that an ancient mechanism controls the development of functionally
and morphologically similar but non-homologous cell types in these
divergent groups of land plants. This suggests that the evolution
of the land plant body over the past 475 million years5' 6 has
resulted at least in part from the independent recruitment of
genes from the gametophyte to the sporophyte.
Unless, stated otherwise, standard techniques were as follows:
RT-PCR
Total RNA (5 ug for A. thaliana and 1 ug for P. patens) was
reverse transcribed with the Superscript First Strand synthesis
system (Invitrogen, Carlsbad, USA) in a 20 ul reaction containing
oligo d (T)12-18 primer. One ul of this product was used for PCR
in 20 ul reactions containing primers described herein.
Pollen growth experiments
In vivo and in vitro pollen tube growth experiments were done as
described previously (44, 45).
Southern analysis of Inserts
Southern blots were performed with the DIG System for PCR
labelling of DNA probes (Roche Diagnostics, Penzberg, Germany)
according to manufacturer protocol Hybridization was done at 42°C
in DIG Easy Hyb hybridization buffer.
Arabidopsis growth conditions.
Arabldopsls thaliana (L.) Heyn. lines were grown vertically for 4
days on MS medium + 2% sucrose solidified with 0.5% Phytagel at 24
°C under continuous illumination. For the cellophane disc
experiment, the agar was overlaid with a cellophane disk (AA
packaging, Preston, UK) before application of the seeds.
Enhancer trapping and cloning of the AtRHD6 gene
Atrhd6-2 is an enhancer trap line (1261) of Arabidopsis (ecotype
Lansberg erecta) generated with the DsE element ld that was
screened for root hairless phenotype and reporter gene expression
in hair cells. Failure to complement Atrhd6-1 1 indicated that
line 1261 carries a mutation that is allelic to Atrhd€-1. The DNA
sequence flanking the DsE element insertion was identified by
inverse-PCR 19. Genomic DNA of the 1261 line was digested by 5au3A
I and subsequently ligated using T4 DNA ligase. The ligated DNA
was used for PCR with Ds element-specific primers. This showed
that the DsE is inserted 111 bp upstream the ATG site of Atlg66470
gene (Fig. 7).
GUS staining of Arabidopsis thaliana roots and embedding
Four-days-old seedlings were stained for 12 hr at 37°C in 1 mN 5-
bromo-4-chloro-3-indolyl-glucuronide, 0.5 mM potassium
ferricyanide, 0.5 mM potassium ferrocyanide, and 10 mM sodium
phosphate buffer (pH 7). Seedlings were embedding in Technovit
71009 resin (Kulzer GmbH, Germany) according to the manufacturer
instructions and 10 urn transverse sections were taken from roots.
Identification of A. thaliana mutants and generation of transgenic
plants
Verification of the T-DNA insertion sites in mutants used in this
work (Fig. 7) was carried out by sequencing PCR fragments
amplified with primers described herein. Atrhd6-3 (ecotype

Columbia 0) correspond to the GABI-Kat line 475E09 (10). Atrsll-1
(ecotype Columbia 0) corresponding to line WiscDsLox356A02 comes
from the Biotechnology centre of the University of Wisconsin, cpc,
wer, ttgl and gl2 mutants have been described previously (32-35).
The genomic constructs AtRHO6p::GFP:AtRHD6 and AtRSLlp::GFP:AtRSLl
contain the promoter and 5'UTR of AtRHD6 or AtRSLl upstream of the
GFP coding sequence fused in N-terminal to the AtRHD6 or AtRSLl
coding region including introns and the AtRHD6 or AtRSLl 3'UTR
with terminator. These constructs were generated using the Gateway
system (Invitrogen, Carlsbad, USA). The AtRHD6 or AtRSLl promoter
+ 5'UTR and the AtRHD6 or AtRSLl coding region + 3'UTR +
terminator were amplified with PCR primers containing
recombination sequences and cloned into pDONR P4-P1R and pDONR
P2R-P3. A GATEWAY multisite reaction was then performed with the
two resulting pDONR plasmids, the plasmid p207-GFP2.5 and the
binary vector pGWBmultisite (destination vector). The binary
vector pGWBmultisite was generated by replacing the Rl-CmR-ccdB-R2
cassette of pGWBl into R4-CmR-ccdB-R3 (pGWBl is from Tsuyoshi
Nakagawa, Shimane University, Japan). AtRHD6p::GFP:AtRHD6 and
AtRSLlp::GFP:AtRSLl were transformed respectively in Atrhd6-3 and
in Atrhd6-3 Atrsll-1 double mutant by floral dip (36) and
transformants were selected on kanamycin (50 ug/ml) and hygromycin
(50 iil/ml). Nine independent transgenic lines containing the
AtRHD6p::GFP:AtRHD6 construct in the Atrhd6-3 background were
obtained. They show different levels of complementation of the
AtRhd6- hairless phenotype but all lines express the GFP in hair
cells before the emergence of root hair. Five independent lines
having the same GFP expression pattern and restore the Atrhd6-3
phenotype were obtained for AtRSLlp::GFP:AtRSLl transformation in
Atrhd6 Atrsll.
For the p35S::PpRSLl construct, the PpRSLl coding sequence was
amplified from protonema cDNA. This fragment was cloned between
the BamHI and Sail sites of a modified pCAMBIA1300 plasmid
containing the CaMV 35S promoter and the terminator of pea Rubisco

small subunit E9 from 35S-pCAMBIA1301 cloned into its EcoRI and
PstI sites (37). The p35S::PpRSLl construct was transformed by
floral dip in Atrhd6-3 and transformants were selected on
hygromycin (50 ug/ml). Ten independent cransgenic lines that
complement the Atrhd6-3 hairless phenotype were obtained.
Physcomitrella genes isolation and phylogenetic analyses
Physcomitrella RSLs and the PpINDl genome sequences where obtained
from BLAST of the available genome sequence assembled into contigs
(http://moss.nibb.ac.jp/). The splice sites were predicted with
NetPlantGene 20 and the bHLH coding sequences were confirmed by
RTPCR and sequencing. The full length coding sequence of BpRSLl
was obtained by sequencing EST clone pdp31414, provided by the
RIKEN BioResource Center -1 and the full length coding sequence of
PpRSL2 was obtained by RT-PCR. Sequences have been deposited to
GenBank as follows: PpRSLl (EF156393), PpRSL2 (EF156394), PpRSL3
(EF156395), PpRSL4 (EF156396), PpRSL5 (EF156397) PpRSL6
(EF156398), PpRSL7 (EF156399) and PpINDl (EF156400). The
phylogenetic analysis was performed with PAUP* software as
described previously ¦¦.
Physcomitrella growth conditions
The Gransden wild type strain of Physcomitrella patens (Hedw.)
Bruch and Schimp 23 was used in this study. Cultures were grown at
25°C and illuminated with a light regime of 16 h light /8h
darkness and a quantum irradiance of 40 uE m-2s-l. For the
analysis of protonema phenotype, spores kept at 4°C for at least 1
month were germinated in a 5 ml top agar (0.8%) plated on 9 cm
Petri dish containing 25 ml of 0.8% agar overlaid with a
cellophane disk (AA packaging, Preston, UK). Leafy gametophores
were grown on 100 times diluted minimal media 24 supplemented with
5 mg/L NH4 tartrate and 50 mg/L Glucose.
Constructing mutants in Physcomitrella genes
The constructs for Physcomitrella transformation were made in
plasmids pBNRF and pBHSNR. pBNRF carries a NptII gene driven by a

35S promoter cloned in the EcoRI site of pBilox, a derivative of
pMCS5 (MoBiTec, Goettingen Germany) carrying two direct repeats of
the loxP sites cloned in the Xhol-Rpnl and Bglll-Spel sites.
pBHSNR contains a AphlV gene driven by a 35S promoter clone
between the 2 loxP sites of pBilox using Sad and Notl. pPpRSLl-KO
was made by cloning PpRSLl genomic fragment 1 in pBNRF digested
with Xbal and Xhol and then cloning PpRSLl genomic fragment 2 in
the resulting plasmid digested with Hpal and Ascl. pPpRSL2-KO was
made by cloning PpRSL2 genomic fragment 1 in pBHSNR digested with
Mlul and Spel and then cloning PpRSL2 genomic fragment 2 in the
resulting plasmid digested with BamHI and HlndLII.
PEG transformation of protoplasts
PEG transformation of protoplasts was done as described previously
25. pPpRSLl-KO was linearised with Seal and Sspl before protoplast
transformation and transformants were selected on G418 (50 ul/ml).
pPpRSL2-KO was linearised with Bell and Sspl before protoplast
transformation and transformants were selected on Hygromycin B (25
lil/ml). The Pprsll Pprsl2 double mutants were obtained by
transformation of Pprsll line 1 with the pPpRSL2-KO construct.
Stable transformants were first selected by PCR using primers
flanking the recombination sites and then analysed by Southern
blot and RT-PCR. For each transformation, three independent lines
having the expected single insertion pattern and being RNA null
mutants were selected (Fig. 9). In each case, the 3 transformants
selected had the same phenotype.
Plants overexpressing RHD6 or RSL Genes
For the 35S::RHD6 and RSL2, 3, and 4 constructs, the coding
sequence of each gene was amplified from root cDNA with primers as
listed below. This fragment was subcloned into a modified
pCAMBIA1300 plasmid containing the CaMV 35S promoter and the
terminator of pea Rubisco small subunit E9. All these
overexpression constructs were transformed by floral dip in
rhd6/rsll and transformants were selected on hygromycin (50 pi/ml)
Results
The Arabidopsis root epidermis is organised in alternate rows of
hair forming cells (H cells) that produce a tip growing
protuberance (root hairs) and rows of non-hair cells (N cells)
that remain hairless. AtRHD6 [ROOT HAIR DEFECTIVE 6) positively
regulates the development of H cells - Atrhd6 mutants develop few
root hairs (Fig. la)".
We cloned AtRHD6 using an enhancer trap line (Atrhd6-2) in which
the GUS reporter gene is expressed in H cells but not in N cells
(Fig.l c, d. Fig. 7). AtRHD6 encodes the basic-Helix-loop-helix
(bHLH) transcription factor Atlg66470*. The identification of
another independent allele (Atrhd6-3) with a similar phenotype and
the complementation of the Atrhd6-3 mutation with a whole gene
AtRHD6p::GFP:AtRHD6 fusion confirmed that the defect in root hair
development observed in this mutant is due to mutation of
Atlg66470 (Fig. la). This complementing AtRHD6p::GFP:AtRHD6 fusion
indicates that AtRHD6 protein accumulates in H cell nuclei in the
meristem and elongation zones (Fig. lb) but disappears before the
emergence of the root hair. The spatial pattern of N cells and H

cells in the Arabidopsis root epidermis is controlled by a
transcriptional network including the positive regulator of H cell
identity CPC and the negative regulators of H cell identity WER,
TTG and GL2*.
To determine if AtRHD6 is regulated by these genes we analysed the
promoter activity of the Atrhd6-2 enhancer trap in different
mutant backgrounds. While the Atrhd6-2 enhancer trap expresses GDS
in cells in the H position this expression spreads to the cells in
the N position in the wer, ttg and gl2 mutant backgrounds
indicating that WER, TTG and GL2 negatively regulate transcription
of AtRHD6 in the N position (Fig. Id). No expression was observed
in the cpc mutant indicating that CPC positively regulates AtRHD6
expression (Fig. Id). Thus, AtRHD6 controls the development of
root hair cells and acts downstream of the genes involved in
epidermal pattern formation.
AtRHD6 is a member of sub family VIIIc of bHLH transcription
factors that comprises five other members *' 10. One of these genes,
At5g37800, hereafter named RHD SIX-LIKE1 (AtRSLl), is very similar
to AtRHD6 and these two genes may derive from a relatively recent
duplication event *. This provides indication that AtRSD6 and
AtRSLl might have redundant functions. To determine if AtRSLl is
also required for root hair development we identified a line
(Atrsll-1) carrying a complete loss of function mutation in the
AtRSLl gene and created the Atrhd6-3 Atrsll-1 double mutant (Fig.
7).
Because no new phenotypes were observed when these mutants were
grown in our standard growth conditions, we grew them on the
surface of cellophane discs, where small numbers of root hairs
develop in the Atrhd6-3 single mutant (Fig. 2a). Plants homozygous
for the Atrsll-1 mutation had wild type root hair morphology when
grown on cellophane discs (Fig. 2a). However, the Atrhd6-3 Atrsll-
1 double mutant did not develop root hairs, indicating that AtRHD6
and AtRSLl have partially redundant functions in root hair

development (Fig. 2a). Atrhd6-3 Atrsll-1 double mutant plants
carrying the genomic construct AtRSLlp::GFP:AtRSLl displayed the
AtRhd6-3- mutant phenotype, confirming that the extreme hairless
phenotype of the Atrhd6-3 Atrsll-1 double mutant is the result of
a loss of function of both AtRHD6 and AtRSLl genes (Fig. 2a). The
complementing GFP:AtRSLl fusion protein accumulates in hair cells
nuclei in the meristem and elongation zones, indicating that
AtRHD6 and AtRSLl have similar expression patterns (Fig. 2b).
These data indicate that AtRSLl and AtRHDS act together to
positively regulate root hair development.
To determine if AtRHD6 and AtRSLl are required for the development
of the only other tip growing cell in flowering plants, the pollen
tube, we characterised the phenotypes of pollen tubes in Atrhd6-3,
Atrsll-1 and Atrhd6-3 Atrsll-1 mutants both in vitro and In vivo.
He detected neither a defect in pollen tube growth nor in the
segregation of mutant alleles in the F2 progeny of backcrosses to
wild type (Fig. 8). No other defective phenotype was detected in
any other part of Atrhd6-3, Atrsll-1 or Atrhd6-3 Atrsll-1 mutants.
Together these data indicate that AtRHD6 and AtRSLl are bHLH
transcription factors that are specifically required for the
development of root hairs and act downstream of the genes that
regulate epidermal pattern formation in the flowering plant
Arabidopsis.
The most ancestral grade of land plants are the bryophytes - the
earliest micro fossils of land plants from the middle Ordovician
circa 475 Ma have bryophyte characteristics *. Bryophytes do not
have roots but possess tip-growing cells that are morphologically
similar to root hairs and fulfil rooting functions. In mosses,
caulonemal cells increase the surface area of the filamentous
protonema tissue in contact with the substrate and rhizoids anchor
the leafy gametophore to their growth substrate 3> * and both cell
types are hypothesised to be involved in nutrient acquisition 3.
However, rhizoids and caulonema develop from the gametophyte of

mosses whereas root hairs develop from the sporophyte of modern
vascular plants. Thus, according to the current view that land
plants evolved by the intercalation of a sporophytic generation
from a haplontic algal ancestor followed by the progressive
increase of size and complexity of the sporophyte in parallel to a
reduction of the gametophyte 11,12, neither rhizoids nor caulonema
are homologous to root hairs.
To determine if the developmental mechanism that controls the
development of root hairs in angiosperms also controls the
development of non-homologous tip growing cells with a rooting
function in bryophytes, we identified RHD6-LIKE genes from the
moss Physcomitrella patens. We identified seven members of the
AtRHD6 subfamily of bHLH genes from the publicly available
Physcomitrella genomic sequence (http://moss.nibb.ac.jp/)
providing indication that these genes have been conserved through
the land plant evolution. These were designated Physcomitrella
patens RHD6-LIKE 1 to 7 (PpRSLl to PpRSL7). To analyse the
relationship between Physcomitrella and Arabidopsis RSL genes we
constructed phylogenetic trees by maximum parsimony. A strict
consensus tree is presented in Fig. 3. This shows that AtRHD6,
AtRSLl and the two Physcomitrella PpRSLl and PpRSL2 genes are
closely related and together form a monophyletic clade (AtRHD6
clade) that is sister to the clade comprising all the other
members of the subfamily (sister clade} (Fig. 3). This indicates
that the AtRHD6 clade evolved before the separation of the
bryophytes and the vascular plants from a common ancestor.
To characterize the function of the RHD6-LIKE genes in moss we
constructed deletion mutants that lacked the function of PpRSLl
and PpRSL2 genes and determined if they develop morphological
defects. Three independent RNA null mutants with single insertions
in PpRSLl and in PpRSL2 were made. Double mutants with single
insertions into both genes were also generated (Fig. 9). The
phenotypes of each of these mutants were then analysed. A haploid
protonema develops upon germination of a wild type Physcomitrella

spore 3. This filamentous tissue comprises two cell types, the
chloronema and the caulonema (Fig. 4a, b). Chloronemal cells
contain large chloroplasts and grow by a slow tip growth
mechanism. Caulonemal cells are more elongated, contain few
smaller chloroplasts, grow by rapid tip growth and are involved in
the colonization of the substrate. Leafy gametophores usually
develop from caulonema and are anchored to their substrate by tip
growing multicellular rhizoids that are morphologically similar to
caulonema (Fig. 4c). The Pprall and Pprsl2 single mutants have
slightly smaller and greener protonema cultures than WT and this
phenotype is much stronger in the Pprall Pprsl2 double mutant
which produces small dark green protonema (Fig. 4a). Pprall and
Pprsl2 single mutants produce fewer caulonemal cells than the WT
indicating that the greener protonema phenotype is the result of a
defect in the development of caulonemal cells (Fig. 4b). No
caulonemal cells develop in the Pprall Pprsl2 double mutant and
the protonema of this mutant comprises chloronemal cells only
(Fig. 4b). In wild type plants gametophores develop from caulonema
but in the Pprall Ppral2 double mutants the gametophores develop
from chloronema, as previously observed in another caulonema
defective mutant ". The gametophores of the Pprsll Ppral2 double
mutant develop few very short rhizoids (Fig. 4c). No other
defective phenotypes were detected in the chloronema, in the leafy
part of the gametophore or in the sporophyte in the single or
double mutants. This indicates that PpRSLl and PpRSL2 together
regulate the development of caulonemal cells and rhizoids in the
moss gametophyte. To determine if protein function is conserved
across the land plants we performed a cross-species
complementation experiment. Expression of PpRSLl under the CaMV35S
promoter in the Atrhd6-3 mutant resulted in the formation of wild
type root hairs (Fig. 4d). Thus, the moss PpRSLl gene can
substitute for loss of AtRHD6 function in Arabidopsis. This
indicates that the molecular function of PpRSLl and AtRHD6 has
been conserved since the divergence of seed plants and mosses from
a common ancestor and suggests that the same molecular mechanism
controls the development of Arabidopsis root hairs and
Physcomitrella caulonema and rhizoids.
Plants were engineered to over-express RHD6 or RSL Genes using a
constitutive promoter, as described above. The phenotype of these
transformants is described below:
35S::RHD6
The deficient of root hair phenotype of rhd6/rsll can be rescued
by the over-expression of RHD6. The transformants get longer root
hair and higher percent of ectopic root hair (root hairs developed
on the non hair cells) than col-0. A few root hairs can be
observed on the hypocotyls.
Phosphate deficiency alters AtRHDS and AtRSLl gene expression.
When grown in the presence of sufficient phosphate these genes are
expressed in the meristem and elongation zone and transcription is
down regulated in the regions where hairs form. When growing in
conditions where phosphate is limiting, AtRHD6 and AtRSLl are
expressed in the root hair forming zone where they positively
regulate the development of root hairs. This shows that phosphate
deficiency promotes expression. Therefore, expressing high levels
of AtRSLl and AtRHD6 in the root epidermis results in a
constitutive "low phosphate" response.
3SS::RSL2 and 35S::RSL3
The rhd6/rsll plants harbouring 35S::RSL2 or 35S::RSL3 constructs
also develop some root hair. The root hairs are longer than the
col-0. Some transgenic lines showed swollen epidermal cells on the
roots and hypocotyls. There are also some root hairs on the
hypocotyls.
Over-expression of AtRSL2 and AtRSL3 using CaMV35S promoter in
wild type plants results in the development of long root hairs.
These hairs are not as long as those that form fungal like
colonies upon over expression of AtRSL4. Plants over-expressing

AtRSL2 and AtRSL3 develop stunted phenotypes. This may be due to
expression in non-root hair cells.
35S::RSL4
The deficient of root hair phenotype of rhd6/rsll can also be
partially complemented by introducing the over expression of RSL4.
The transformants show longer root hair than col-0. A few root
hairs were also detected on the hypocotyls, which is quite similar
with that of RHD6 overexpression transformants.
Over-expression of AtRSL4 using CaMV35S promoter in wild type
plants, causes the formation of long root hairs which can form
extensive indeterminate growing masses of cells resembling fungal
like colonies (figure 10). The root hair system of a plant
overexpressing RSL4 is shown in figure 10. The root is surrounded
by a mass of fungus-like cells, which resemble mycorrhizae, the
nutrient scavanging fungi that form associations with roots.
Furthermore, when the RSL4 is expressed by the 35S promoter, this
phenotypic effect (long root hairs) was found to be restricted to
the root hair cells. No defective phenotypes resulting from the
RSL4 expression were observed elsewhere in the plant.
Plants overexpressing RHO6-related genes may therefore have
increased nutrient uptake ability because of their increased
surface area resulting from enhanced root hair growth. This effect
may be marked in plants, such as Brassicas, which are devoid of
mycorrhizae throughout their entire life cycle.
Here we show how closely related transcription factors control the
development of tip growing cells that have a rooting function in
the seed plant sporophyte and the bryophyte gametophyte. These
data indicate that we have identified an ancient developmental
mechanism that was present in the common ancestor of the mosses
and vascular plants (tracheophytes). These genes will have been
important for the invasion of land by plants when nutrient
acquisition and anchorage to the solid substrate of the

continental surface was necessary. The observation that rhizoids
have been found on some of the oldest macro fossils of land plants
is consistent with this view """.
Our results provide indication that RHD6-LIKE genes functioned in
the haploid generation (gametophyte) of trie early land plant life
cycle which may have been bryophyte-like ~4, where they controlled
the formation of cells with a rooting function. We propose that
during the subsequent radiation of the land plants these genes
were deployed in the development of the diploid generation
(sporophyte) of vascular plants where they control the development
of root hairs in angiosperms and we predict they control the
development of root hairs and rhizoids in lycophytes (clubmosses
and allies) and monilophytes (ferns and horse tails). It is likely
that such independent recruitment of genes from haploid to diploid
phases of the life cycle was in part responsible for the explosion
in morphological diversity of the diploid stage of the life cycle
(sporophyte) that occurred in the middle Palaeozoic when green
plants colonised the continental surfaces of the planet 1-l.
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WE CLAIM :
1. A method of modulating root haiz development in plants
comprising:
incorporating a heterologous nucleic acid which encodes a
RHD6-related polypeptide comprising and amino acid sequence
selected from the group consisting of SEQ ID NOS: 13 to 25
into a plant ceil by means of transformation, and,
regeneraling the plant from one or more transformed cells.
2. A method according to claim 1 wherein the plant has increased
tolerance to nutrient-deficient conditions relative to control
plants.
3. A method according to claim 2 wherein the nutrient-deficient
conditions are selected from the group consisting of nitrate-
deficient, phosphate-deficient or iron-deficient conditions.
4. A method according to claim 1 wherein the plant has increased
production or secretion of root-secreted phytochemical.
5. A method according to claims 1 to 4 wherein the RHD6-related
polypeptide comprises an amino acid sequence having at least
55% sequence identity to SEQ ID NO:1 or SEQ ID NO: 3.
6. A method according to claims 1 to 5 wherein the RHD6-related
polypeptide comprising an amino acid sequence having at least
55. sequence identity to any one of SEQ ID NOS: 5, 7, 9, or
11, 26, 28, 30, 32. 34, 35, 36, 40, 42, 44, 46, 48, 50, 52,
54, 56, 58, 60, 62, 64, 68, 68,70, 72, 74, 76, 78, 80, 82, 84,
86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112
and 114.
7. A method according to any one of claims 1 to 6 wherein the
length, number and/or longevity of root hairs is increased in
the plant.
8. A method according to any one of claims 1 to 7 wherein
expression is increased by expressing a heterologots nucleic
acid encoding said RHDE-related polypeptide within cells of
said plant..
9. A method according to claim 8 wherein the heteroiogous nucleic
acid comprises a nucleotide sequence which has a*.: least 4Ci
sequence identity with any one of SF.Q ID NO: 2 or SEQ ID NO:
4.
10. A method according to claim 8 wherein the heteroiogous
nucleic acid comprises a nucleotide sequence which has at
least 40% sequence identity with any one of SEQ ID NOS: 6, 8,
10, 12, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,
53, 55, 57, 59, 61, 62, 6b, 67, 69, 71, 73, 75, 77, 79, 81,
83, 95, 87, 89, 91, 92, 95, 97, 99, 101, 103, 105, 107, 109,
111, 113 and 115.
11. A method according t.o any one of claims a to 10 wherein the
hetorologous nucleic acid is operably linked l:o a promoter.
12. A method according to claim 11 wherein the promoter is a
constitutive promoter.
13. A method according to claim 11 wherein the promoter is a
root-specific promoter.
14. A method according t.o claim 11 wherein the promoter is a
inducible promoter.
15. A method according to any one of claims 8 to 14 wherein the
heteroiogous nucleic acid is comprised in a vector.

16. A method according to any one of claims 1 to 7 wherein
expression is increased by a method comprising;
exposing a population of plants to a mutagen, determining
the expression of the RHD6-related polypeptide in one or
more plants in said population, and
identifying a plant with increased expression of the RHD6-
related polypeptide.
17. a method according to any one of claims 1 to 16 wherein the
plant is a higher plant.
18. A method according to claim 17 wherein the plant is an
agriculture plant selected from the group consisting of
Lithzspermum erythrorhizon, Taxus spp, tobacco, cucurbits,
carrot, vegetable brassica, melons, capsicums, grape vines.
Lettuce, strawberry, oilseed brassica, sugar beet, wheat,
barley, maize, rice, soyabeans, peas, sorghum, sunflower,
tomato, potato, pepper, chrysanthemum, carnation, linseed,
hemp and rye.
19. A method of modulating root hair development in a plant
comprising;
increasing the expression of a RHD6-related polypeptide
comprising an amino acid sequence having at least 50%
sequence identity to an amino acid sequence selected from
Lhe group consisting of SEQ ID IIOS: 13 to 25 within cells of
said plant relative to control plants.
20. A method of increasing the tolerance of a plant to nutrient-
deficient conditions comprising; increasing the expression of a
RHD6-related polypeptide comprising an amino acid sequence
having at least 50% sequence identity to an amino acid sequence
selected from the group consisting of SEQ ID NOS: 13 to 25
within cells of said plant relative to control plants.
21. A method according to claim 20 wherein the nutrient-
deficient conditions are selected from the group consisting of
nitrate-deficient, phosphate-deficient or iron-deficient
conditions.
22. A method of increasing the production or secretion of a
root-secreted phytochemical in a plant comprising;
increasing the expression of a RHD6-related polypeptide within
cells of a plant which secretes the phytochemical through its
roots.
23. A method according to any one of claims 26 to 29 wherein the
RHDe-reiated polypeptide comprising an amino acid sequence
havir.-? at least 55% sequence identity to SEQ ID NO:1 or SEQ ID
NO: 3.
24. A method according to any one of claims 19 to 21 wherein the
RHD6-related polypeptide comprising an amino acid sequence
havinq at least 55% sequence identity to any one of SEQ ID NOS:
5, 7, 9, or 11, 26, 28, 30, 32, 3-1, 36, 38, 40, 42, 44, 46, 45,
50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 30,
82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 103,
110, 112 and 114.
25. A method according to any one of claims 19 to 24 wherein one
or more of the length, number and/or longevity of root hairs is
increased in the plant.
26. A method according to any one of claims 19 to 25 wherein
expression is increased by expressing a heterologous nucleic
acid encoding said RHD6-related polypeptide within cells of
said plant.
2". A method according to claim 26 wherein the heterologous
nucleic acid comprises a nucleotide sequence which has at least
40* sequence identity with any one of SEQ ID NO: 2 or SEQ ID
WO: 4.
18. A method according to claim 25 wherein the heterologous
nucleic acid comprises a nucleotide sequence which has at least
4O, sequence identity with any one of SEO ID NOS: 6, 8, 10, 12,
27, 15, 31, 33, 35, 37, 33, 41, 43, 45, 47, 49, 51, 53, 55,
57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 37,
89, 91, 93, 95, 57, 99, 101, 103, 105, 107, 109, 111, 113 and
115.
29. A method according to any one of claims 26 to 28 wherein
the haterclogcus nucleic acid is operably linked to a promoter.
5C-. A method according to claim 29 wherein the promoter is a
constitutive promoter.
31. A method according to claim 29 wherein the promoter is a
root-specific promoter.
32. A method according to claim 29 wherein the promoter is a
inducible promoter.
53. A method according to any one of claims 29 to 32 wherein the
heterologous nucleic acid is comprised in a vector.
34. A method according to any one of claims 19 to 25 wherein
expression is increased by a method comprising; exposing a
population of plants to a mutagen, determining the expression
of the PHD6-related polypeptide in one or more plants in said
population, and identifying a plant with increased expression
of the RHD6-related polypeptide.
35. A method of reducing the amount of contaminant in
contaminated soil comprising;
increasing the expression of a RHD6-related polypeptide
within cells cf a plant which absorbs the contaminant
through its roots,
growing the plant or a descendent thereof in soil which
comprises the contaminant such that the plant or descendent
absorbs the contaminant from the soil, and
harvesting said plant or descendent thereof.
36. Ar. isolated RCOT HAIR DEFECTIVE 6 (RHD6)-related gene
selected from RSL2, RSL 3, RSL 4 and RSL5.
37. An isolated product of the expression of the gene according
to claim 36.
38. An expression constructs comprising the gene according to
any one of claims 36 to 37.
39. An expression construct according to claim 38 wherein the
gene is operably linked to a heterologous regulatory sequence.
40. An isolated nucleic acid according to claim 36 which encodes
a polypeptide comprising an amino acid sequence selected from
the group consisting of SEQ ID HOS: 5, 7, 9 and 11.

This invention relates to the modulation of root hair development
in plants by altering the expression of RHD6-related genes, for
example to increase the number, length and/or longevity of root
hairs in the plant. This may be useful, for example, in improving
the ability of plants to extract nutrients from the soil.