plntgenes3

embryo, the stem and the root established

and maintained?

Acknowledgements

My sincere thanks to Philip Benfey,

Hidehiro Fukaki and Mituhiro Aida for

critical reading and comments on my

manuscript.

References

1 Wysocka-Diller, J.W. et al. (2000) Molecular

analysis of SCARECROW function reveals a

radial patterning mechanism common to root and

shoot. Development 127, 595–603

2 Di Laurenzio, I. (1996) The SCARECROW gene

regulates an asymmetric cell division that is

essential for generating the radial organization of

Arabidopsis roots. Cell 86, 423–433

3 Helariutta, Y. (2000) The SHORT-ROOT

gene controls radial patterning of Arabidopsis

root through radial signaling. Cell 101,

555–567

4 Long, J.A. and Barton, M.K. (1998) The

development of apical embryonic pattern in

Arabidopsis. Development 125, 3027–3035

5 Aida, M. et al. (1999) Shoot apical meristem and

cotyledon formation during Arabidopsis

embryogenesis: interaction among the

CUP-SHAPED-COTYLEDON and SHOOT

MERISTEMLESS genes. Development

126, 1563–1570

6 Lynn, K. et al. (1999) The PINHEAD/ZWILLE

gene acts pleiotropically in Arabidopsis

development and has overlapping functions with

ARGONAUTE1 gene. Development 126, 469–481

7 Prigge, M.J. and Wagner, D.R. (2001)

The Arabidopsis SERRATE gene encodes a

zinc-finger protein required for normal shoot

development. Plant Cell 13, 1263–1279

8 Sawa, S. et al. (1999) FILAMENTOUS FLOWER, a

meristem and organ identity gene of Arabidopsis,

encodes a protein with a zinc finger and HMG-

related domains. Genes Dev. 13, 1079–1088

9 Siegfried, K.R. (1999) Members of the YABBY

gene family specify abaxial cell fate in


10 Bowman, J.L. and Smyth, D.R. (1999) CRABS

CLAW, a gene that regulates carpel and nectary

development in Arabidopsis, encodes a novel

protein with zinc finger and helix–loop–helix

domains. Development 126, 2387–2396

11 Baker, S.C. and Robinson-Beers, K. (1997)

Interactions among genes regulating ovule

development in Arabidopsis thaliana. Genetics

145, 1109–1124

12 McConnell, J.R. et al. (2001) Role of

PHABULOSA and PHAVOLUTA in

determining radial patterning in shoots.

Nature 411, 709–713

13 Kerstetter, R.A. et al. (2001) KANADI regulates

organ polarity in Arabidopsis. Nature 411,

706–709

14 Eshed, Y. et al. (1999) Distinct mechanisms

promote polarity establishment in carpels of

Arabidopsis. Cell 99, 199–209

15 McConnell, J.R. and Barton, M.K. (1998)

Leaf development and meristem formation in


Masao Tasaka

Graduate School of Biological Science, Nara Institute of Science andTechnology, Ikoma, Nara 630-0101, Japan.e-mail: [email protected]

TRENDS in Plant Science Vol.6 No.12 December 2001

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550 Research Update

Root hairs, trichomes and the evolution of duplicate

genes

Elizabeth A. Kellogg

The MYB-class proteins WEREWOLF

and GLABRA1 are functionally

interchangeable, even though one is

normally expressed solely in roots and the

other only in shoots. This shows that their

different functions are the result of the

modification of cis-regulatory sequences

over evolutionary time. The two genes thus

provide an example of morphological

diversification created by gene duplication

and changes in regulation.

How does a plant know to make root hairs

on a root epidermis and trichomes on a

leaf epidermis? Why don’t they ever get it

mixed up? This question is all the more

compelling because so much of the

relevant machinery is the same at the two

ends of the plant. An Arabidopsis protein,

TRANSPARENT TESTA GLABRA (TTG),

regulates trichome production in the leaf

epidermis and root hair production in the

root epidermis, but in opposite directions1.

Mutations in TTG cause loss of trichomes

on the leaf, but proliferation of root hairs.

GLABRA2 (GL2), which operates

downstream of TTG, also has opposite

effects in the shoot and in the root,

producing malformed trichomes on leaves

and ectopic root hairs in the root1,2. The

question is, how can TTG and GL2 have

such different effects in different parts of

the plant?

WEREWOLF and GLABRA1 are

interchangeable proteins

Functional comparisons of the MYB-class

proteins WEREWOLF (WER) and

GLABRA1 (GL1) have deepened the

mystery. GL1 is expressed only in epidermal

cells in the shoot, and is necessary for the

production of trichomes on leaves; it acts at

the same point genetically as TTG and itself

regulates GL2 (Refs 3,4). Conversely, WER

is expressed only in the root and hypocotyls

in a subset of epidermal cells where it

suppresses root hairs (in the root) and

stomatal cells (in the hypocotyl)5. Like GL1,

WER acts at the same point as TTG and

itself regulates GL2. GL1 and WER might

thus be alternate components of a pathway

connecting TTG to GL2 and thence to

determination of epidermal cell identity.

A model for WER action was proposed by

Myeong Min Lee and John Schiefelbein5

(Fig. 1) in which TTG activates an unknown

bHLH protein, which then interacts with

WER to activate GL2, thus blocking

root-hair formation in non-root hair cells. In

cells where WER is not expressed, another

protein with a truncated MYB domain,

CAPRICE (CPC), represses GL2 and

permits root-hair formation. An obvious and

testable corollary is that GL1 could replace

WER in the root.

In a carefully controlled set of

experiments, Lee and Schiefelbein6 have

now shown that the transcriptional units of

GL1 and WER are interchangeable, and

that specificity is conferred by their

upstream and downstream regulatory

sequences. A pair of constructs was created

in which the transcriptional unit of each

gene was connected to the regulatory

sequences (both 5′ and 3′) of the other gene.

When the WER (regulatory)–GL1 (gene)

construct was introduced into a wer

mutant, the plants produced wild-type

numbers of root hairs and stomata in roots

and hypocotyls, respectively, and trichomes

remained unaffected. Conversely, the GL1

(regulatory)–WER (gene) construct was

able to rescue a gl1 mutation, and produced

wild-type numbers of trichomes in the

leaves. The constructs each regulated a

GL2::GUS reporter gene in a pattern

appropriate for each tissue.

Differences between WER and GL1

expression are because of cis-regulatory

sequences

The functional equivalence of the proteins

might not have been predicted from their

sequences alone. WER and GL1 both

contain MYB domains with two related

helix–turn–helix motifs (known as R2

and R3). Although the MYB domains are

91% identical, there is little similarity

outside that region. Based on Lee and

Schiefelbein’s6 alignment, only six of the

15 residues of the MYB domains are

identical at the N-terminal, and only 23%

are identical at the C-terminal. The

extensive differences suggest that WER

and GL1 are not recent duplicates.

Lee and Schiefelbein considered the

possibility that the interchangeability of

WER and GL1 might indicate functional

equivalence of all R2R3–MYBs, so they

created two other constructs linking the

regulatory regions of WER and then of

GL1 to the transcriptional unit of a

distantly related MYB class gene,

AtMYB2. This construct was unable to

rescue either mutant. Thus, equivalence

of WER and GL1 does not extend to all

R2R3–MYB proteins. The MYB repeat

region of AtMYB2 is different from that

of WER and GL1 (58% and 57% identity,

respectively), suggesting that the

functional differences could be because

of variation between the MYB domains

and/or between the highly divergent

C-terminal regions.

Gene duplications in evolution

Changes in gene function over

evolutionary time frequently occur by

modification of cis-regulatory

sequences7–9. Comparisons of WER and

GL1 provide additional evidence for this

hypothesis, and show that the changes in

the proteins themselves, although

considerable, are not responsible for

their divergent developmental roles.

Duplication of genes can permit

diversification of function, and lead to

developmental complexity. When a gene

duplicates, one copy retains its ancestral

function whereas the other copy is free to

accumulate mutations10. Frequently

these mutations are thought to be

deleterious and lead to the formation of

a pseudogene. In other cases, the new

mutations are selectively favored and

the gene acquires a new function. One

recent suggestion is that duplicate genes

might partition the expression patterns

of their single ancestral gene so that

each copy retains a distinct subset of

the ancestral regulatory sequences11.

This duplication–degeneration–

complementation (DDC) hypothesis

could be tested with a pair of genes such

as WER and GL1. The DDC hypothesis

postulates that the ancestor of WER and

GL1 had separate promoter elements,

some of which specified root expression

and some of which specified shoot

expression. After duplication, the

shoot-specification elements of GL1 were

preserved while the root-specification

elements accumulated mutations, and

the converse occurred with WER.

A corollary hypothesis is that TTG and

GL2, which remain single genes, should

have cis-regulatory sequences made up of

both root and shoot elements, perhaps the

same as or similar to those in the WER

and GL1 ancestral sequence. Knowledge

of the regulatory sequences might tell us

whether gene duplication and divergence

provide a simple evolutionary alternative

to modification and proliferation of

cis-regulators. It is conceivable that different

genes are constrained or predisposed –

by structure, function or genome position

– to acquire new functions by duplication,

whereas others are more likely to diversify

by changes in their regulatory sequences.

Testing the DDC model for WER and

GL1 requires a better gene phylogeny

than is currently available. WER and GL1

are only two of the many R2R3–MYB class

genes in land plants. Arabidopsis has

>100 R2R3–MYBs in its genome12,13, and

maize expresses at least 82 (Ref. 14). The

maize genes have been analyzed

phylogenetically with a small set of

Arabidopsis genes, and a larger set of

Arabidopsis genes have been included in

an analysis of R2R3–MYBs from multiple

species12. Because WER was cloned only

recently, neither analysis included it

along with GL1. The number of

differences between the two genes

suggests that their duplication might be

old, although their relative age can be

tested only by searching for WER and GL1

orthologs in other major groups of plants.

The duplication of WER and GL1 could

correlate with any of several major

morphological transitions. The tidy lines

of root-hair cells alternating with lines of

non-hair cells in Arabidopsis form a

characteristic striped pattern that is not

widespread in angiosperms15,16. The

pattern apparently originated within the

Brassicales, and independently in other

lineages of angiosperms15,17 (P.F. Stevens,

unpublished; http://www.mobot.org/

MOBOT/research/Apweb). The WER and

GL1 duplication might correlate with the

origin of stripes of root hairs, although the

considerable divergence of the C-terminal

domains suggests that differentiation of

WER and GL1 is much older. It seems

more likely that the production of stripes

on root hairs is caused by modifications in

expression of WER and CPC, a testable

prediction made by Liam Dolan and

Silvia Costa17.

Roots that look morphologically

distinct from shoots appear early in

land-plant evolution, and plants with

clear bipolar embryos have evolved more

than once18. If the last common ancestor

of WER and GL1 is sufficiently old, the

duplication might be placed at the origin

of roots in seed plants. The earliest


http://plants.trends.com

551Research Update

TRENDS in Plant Science

In non-hair cells ofroot epidermis

TTGWER

bHLH

GL2

No root hairs

(a) In root hair cells ofroot epidermis

TTGCPC

bHLH

GL2

Root hairs

(b) In trichome cells ofshoot epidermis

TTGGL1

GL3

GL2

Trichomes

(c)

Fig. 1. A model for the action of WER-repressing root hair development in non-hair cells of the root (a,b), modifiedfrom Ref. 2. (c) One of several possible models for the action of GL1-activating trichome development in the shoot19.Proteins containing a MYB domain are shown in indigo, bHLH proteins are in yellow, TTG (a WD40 protein) is in red,and GL2 (a homeodomain protein) is in green.

Meeting Report

Medicago truncatula, going where no plant has gone

before

Giles E.D. Oldroyd and René Geurts

diverging angiosperms produce root hairs

in no apparent pattern, and they appear to

lack specialized hair-producing cells

(trichoblasts)15,16 (P.F. Stevens, unpublished;

http://www.mobot.org/MOBOT/research/

Apweb). If the WER and GL1 duplication

occurred after the origin of seed plants,

but early in angiosperm evolution, it

might correlate with the origin of

trichoblasts. Whatever the history of the

two genes, elucidating that history could

give us some clues about the genetic basis

of morphological elaboration.

Acknowledgements

Thanks to John Schiefelbein and an

anonymous reviewer for helpful

comments on the manuscript.

References

1 Marks, M.D. (1997) Molecular genetic analysis of

trichome development in Arabidopsis. Annu. Rev.

Plant Physiol. Plant Mol. Biol. 48, 137–163

2 Schiefelbein, J.W. (2000) Constructing a plant

cell. The genetic control of root hair development.

Plant Physiol. 124, 1525–1531

3 Larkin, J.C. et al. (1996) The control of trichome

spacing and number in Arabidopsis. Development

122, 997–1001

4 Oppenheimer, D.G. et al. (1991) A myb gene

required for leaf trichome differentiation in

Arabidopsis is expressed in stipules. Cell

67, 483–493

5 Lee, M.M. and Schiefelbein, J. (1999)

WEREWOLF, a MYB-related protein in

Arabidopsis is a position-dependent regulator of

epidermal cell patterning. Cell 99, 473–483

6 Lee, M.M. and Schiefelbein, J. (2001)

Developmentally distinct MYB genes encode

functionally equivalent proteins in Arabidopsis.

Development 128, 1539–1546

7 Carroll, S.B. et al. (2001) From DNA to Diversity,

Blackwell Science

8 Wang, R-L. et al. (1999) The limits of selection

during maize domestication. Nature

398, 236–239

9 Quattrocchio, F. et al. (1998) Analysis of bHLH

and MYB domain proteins: species-specific

regulatory differences are caused by divergent

evolution of target anthocyanin genes. Plant

J. 13, 475–488

10 Ohno, S. (1970) Evolution by Gene Duplication,

Springer-Verlag

11 Force, A. et al. (1999) Preservation of duplicate

genes by complementary, degenerative

mutations. Genetics 151, 1531–1545

12 Romero, I. et al. (1998) More than 80 R2R3-MYB

regulatory genes in the genome of Arabidopsis

thaliana. Plant J. 14, 273–284

13 Reichmann, J.L. et al. (2000) Arabidopsis

transcription factors: genome-wide comparative

analysis among eukaryotes. Science 290, 2105–2110

14 Rabinowicz, P.D. et al. (1999) Maize R2R3 Myb

genes: sequence analysis reveals amplification in

the higher plants. Genetics 153, 427–444

15 Pemberton, L.M.S. et al. (2001) Epidermal

patterning of seedling roots of eudicotyledons.

Ann. Bot. 87, 649–654

16 Clowes, F.A.L. (2000) Pattern in root meristem

development in angiosperms. New Phytol.146, 83–94

17 Dolan, L. and Costa, S. (2001) Evolution and

genetics of root hair stripes in the root epidermis.

J. Exp. Bot. 52, 413–417

18 Kenrick, P. and Crane, P.R. (1997) The Origin and

Early Diversification of Land Plants, Smithsonian

Institution Press, Washington, DC, USA

19 Payne, C.T. et al. (2000) GL3 encodes a bHLH

protein that regulates trichome development in

Arabidopsis through interaction with GL1 and

TTG1. Genetics 156, 1349–1362

Elizabeth A. Kellogg

University of Missouri-St Louis, 8001 NaturalBridge Road, St Louis, MO 63121, USA.e-mail: [email protected]


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552 Research Update

4th Workshop on Medicago truncatula,

7–10 July 2001, Madison WI, USA.

Legumes have generated much interest in

the plant scientific community, not only

because they are important crop plants,

but also because of their interactions with

microbial symbionts. Several years ago,

Medicago truncatula, a close relative of

alfalfa, was identified as being a suitable

model legume because of its small diploid

genome, autogamous genetics and ease of

transformation1. It was apparent at the

4th Workshop on Medicago truncatula that

the combined effort of several researchers

has pushed this model plant to the

forefront of legume biology. Genomic and

genetic tools are rapidly evolving and the

scope of work performed in M. truncatula

is expanding and diversifying.

Generating the tools

One of the most striking new genomic

initiatives at this meeting came from

Bruce Roe (University of Oklahoma,

Norman, OK, USA), who has initiated

whole-genome shotgun sequencing of

M. truncatula. The goal of this

preliminary project is to sequence to a

genome depth of approximately onefold

coverage. To date, 25 000 shotgun

sub-clone end-sequence reads have been

generated and are available at

http://www.genome.ou.edu/medicago.html.

Numerous contiguous sequence regions

have been assembled, of which the

majority represents repeated sequences.

Cytogenetic analysis of M. truncatula

pachytene chromosomes performed in

the group of Ton Bisseling (Wageningen

University, The Netherlands) in

collaboration with Doug Cook (University

of California, Davis, CA, USA), indicates

that 80% of the genome is clustered as

heterochromatin in the pericentromeric

region2. Because it is generally thought

that heterochromatin contains mainly

repeat sequences, the majority of the

newly identified repetitive sequences are

thought to be located in the pericentromeric

regions of the chromosome. Olga Kulikova

(Wageningen University, The Netherlands)

has characterized two repeat sequences

that make up 1.8% and 4.9% of the

genome content and are located in

the pericentromeric regions.

Functional genomics: expansion and

application

To date, >125 000 M. truncatula expressed

sequence tags (ESTs), generated from

>30 different cDNA libraries, are publicly

available, and the work is still ongoing.

Kate Vandenbosch (University of

Minnesota, St Paul, MN, USA) described

how the available ESTs can be assembled

into a uni-gene set of ~30 000 sequences.

Detailed information about the ESTs is

available at http://www.medicago.org.

Vandenbosch and Helge Küster (Bielefeld

University, Germany) discussed the

next goal for functional genomics in

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