plntgenes3 by Rev Jeremy D

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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 HMGrelated domains. Genes Dev. 13, 1079–1088 9 Siegfried, K.R. (1999) Members of the YABBY gene family specify abaxial cell fate in Arabidopsis. Development 126, 4117–4128 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 Arabidopsis. Development 125, 2935–2942

Masao Tasaka Graduate School of Biological Science, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan. e-mail: m-tasaka@bs.aist-nara.ac.jp

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 http://plants.trends.com

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.

Research Update

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 http://plants.trends.com

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(a) In non-hair cells of root epidermis

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(b) In root hair cells of root epidermis

TTG WER

TTG CPC

TTG GL1

bHLH

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No root hairs

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Trichomes TRENDS in Plant Science

Fig. 1. A model for the action of WER-repressing root hair development in non-hair cells of the root (a,b), modified from 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.

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

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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 Natural Bridge Road, St Louis, MO 63121, USA. e-mail: tkellogg@umsl.edu

Meeting Report

Medicago truncatula, going where no plant has gone before Giles E.D. Oldroyd and René Geurts 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 http://plants.trends.com

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