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Opportunities and challenges in chemical

Glenn R Hicks & Natasha V Raikhel

Chemical biology is beginning to enhance our understanding of diverse cellular processes in , including endomembrane trafficking, hormone transport and wall . To reach its potential requires the development of a -wide infrastructure of technology and expertise. We present some of the opportunities and challenges in this emerging branch of plant biology and offer some suggestions for enhancing the approach to the benefit of the community at large.

Although resources devoted to plant biol- Mutational has been the primary Within the past decade, plant chemical ogy are modest in comparison to biomedical approach toward understanding such net- have begun to make important con- and other areas of research, plant biologists works through the elucidation of individual tributions to our understanding of will play a disproportionate and crucial role gene . However, even in plants such biosynthesis, the , hormone biosyn- in the future of mankind. Basic plant biology as Arabidopsis thaliana where there are well- thesis and signaling, gravitropism, pathogenesis, and its application will be the major vehicle established genetic tools, many networks have purine biosynthesis and endomembrane traf- through which we will improve human health been recalcitrant to classical genetic strategies ficking (reviewed in refs. 3,4). In many cases, in a cost-effective manner on a global scale. owing to a combination of gene redundancy plant phenotype–based chemical screens using Progress toward meeting the challenges fac- (yielding no observable change in phenotype) cell cultures and seedlings have identified small ing the world—improved , increased and gene lethality. targeting these processes (reviewed crop yield, resistance to pests, sustainable bio- Plant can address these in ref. 4). In other cases, important cognate gene fuels, raw materials for industry and serious issues by using bioactive chemicals to affect targets have been identified5–10. Several exam- environmental problems—is being fueled by the active sites of individual targets or, ples below highlight the recent contributions of © All rights reserved. 2009 Inc. America, ongoing technical and intellectual advances. alternatively, whole classes of protein targets in a chemical biology in uncovering new and useful One area of advancement is plant , manner that is very controllable in terms of dose knowledge of broad potential impact. which has resulted in the sequencing of the and treatment time. The application of small- genomes of Arabidopsis thaliana, rice, poplar approaches to plant biology requires Some impacts of plant chemical biology and many other species. More recently, the a new cadre of plant who are using Cellulose is the primary polymeric constitu- introduction of massively parallel next-gen- approaches that straddle the interface of chem- ent of plant cell walls and is thus an important eration sequencing technologies has spawned istry, informatics and biology. They are poised to source of biomass for biofuels and industrial the beginnings of a revolution through meta- make important contributions by addressing the products. Understanding the mechanisms of genomic studies1 and an era of rapid access inherent challenges of multi-cellular land plants cellulose synthesis and deposition are impor- to genotypic variation between individual as biological systems. We will discuss some of tant basic and practical goals. Investigations of genomes2. the enormous opportunities to advance our resistance to the cellulose biosynthesis inhibitor Another critical, but as yet less developed, knowledge of basic plant processes through the isoxaben have provided insight into two genes innovation has been the integration of small- use of small bioactive molecules and some of the now known to encode the cellulose synthases molecule approaches with plant biology. We major hurdles to jump to maximize the poten- CESA3 and CESA6 (refs. 10,11). These see plant chemical biology as the applica- tial of plant chemical biology. We will highlight are two of at least ten CESA cellulose synthases tion of small bioactive chemicals to inter- some investigations of small-molecule that are involved in a macromolecular com- rogate dynamic cellular networks in plants. and the identification of cognate targets, but will plex known as the particle rosette or terminal emphasize the ability of plant-bioactive chemi- complex that coordinates the incorporation of Glenn R. Hicks and Natasha V. Raikhel are at cals to provide a network-level view of complex glucan chains into cellulose microfibrils of the the Center for Plant Cell Biology, Institute for dynamic processes because this is one of the nascent cell wall12 (Fig. 1). The selectivity of Integrative Genome Biology and Department great opportunities in plant chemical biology. isoxaben for two CESA proteins was essential of and Plant Sciences, University of We also offer some suggestions for choosing for overcoming gene redundancy, which is a California, Riverside, California, USA. resources likely to benefit the plant biology common feature in plants including Arabidopsis. e-mail: [email protected] community most. The chemicals morlin13, which affects cortical

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, and 2,6-dichlorobenzonitrile14, differentiated organs derived from the shoot whose precise mode of action is unknown, both ab and root apical meristems. The uniqueness of inhibit rosette motility. This and other evidence plant development is obvious during embryo- indicates that CESA-containing rosettes interact genesis, which is achieved without cell migra- with cortical microtubules as part of a potential tion as is necessary in . As more plant guidance mechanism that determines the orien- genomes are sequenced, other evolutionary tation of cellulose microfibrils12. This orienta- features of plants have been highlighted that tion is crucial in determining the directionality present both inherent challenges to unravel- of and ultimately plant morphol- ing the biology of these interesting species and ogy. Thus, these small bioactive molecules important opportunities for chemical biologists have contributed to the functional dissection to significantly contribute to the field. Figure 1 Cellulose microfibrils are one of the of CESA complexes as well as their interac- primary components of plant cell walls and are tion with another macromolecular complex, composed of 36 hydrogen-bonded β-1,4-linked Lethality and redundancy limits the useful- microtubules. Given the selectivity of isoxaben, glucose residues, each produced from a single ness of mutational genetics. The high degree it is probable that other chemicals could prove CESA subunit. (a) Six CESA subunits assemble of redundancy among plant genes is well estab- valuable in further defining the roles of the CES into a CESA complex. (b) Each CESA complex lished17. As a result of this redundancy, muta- gene family in development. is in turn assembled in the Golgi into a particle tions resulting in loss of function frequently 6 rosette composed of six CESA complexes, which Another recent example is gravacin , iden- is targeted to the plasma membrane. Each of the yield phenotypes not observably different from tified in a screen of a diverse chemical library 36 CESA subunits contributes to the nascent wild type, which limits the effectiveness of the as an inhibitor of root and shoot gravitropism cellulose microfibril. For clarity, cellulose (black existing strategies. In other instances, loss-of- and protein targeting to the tonoplast (that is, lines) is shown emerging from only a single function in essential genes result in membrane) in Arabidopsis seedlings. CESA complex of a particle rosette in the plasma lethality. Both redundancy and lethality have Gravitropism is the bending either toward membrane. There is a family of ten CESA proteins been found among genes encoding compo- in Arabidopsis, of which five appear to be involved (roots) or away from (stems) the gravitational nents of endomembrane trafficking in plants22. in the synthesis of primary cell-wall cellulose force; the orientation is sensed by root tips and microfibrils. Among these genes, mutations in These issues are being addressed effectively by shoots and requires the cell-to-cell transport CESA3 and CESA6 confer resistance to isoxaben. chemical genomics. Given the enormous struc- of the growth-modulating hormone auxin. Thus, a bioactive small molecule was used to tural variations possible among small molecules, This transport is strongly directional and con- overcome gene redundancy. they can be used to perturb protein function in a trolled by a family of plasma membrane highly specific manner, as in the case of essential transporters known as the PINS15, along with genes. Furthermore, because chemical dosages transporters of the large ATP-binding cassette model systems, especially Arabidopsis, which can be modulated, the products of essential sub-family B (ABCB)/P-glycoprotein (PGP) has a small, fully sequenced genome. These genes can be studied under nonlethal condi- family. The ABCBs, or multidrug-resistance tools include (i) well-developed reverse genet- tions. Redundancy can be addressed through transporters, may function by stabilizing PIN ics, including the availability of extensive collec- the use of chemicals that are more broadly localization at distinct domains in the plasma tions of T-DNA insertion and activation-tagged targeted to protein families sharing common membrane16. The demonstration that ABCB19/ lines17, (ii) molecular markers that facilitate the features or activities that can be perturbed. The PGP19 is one of the cognate targets of grava- fine mapping of mutations18, (iii) structurally cellulose synthases and ABCBs underscore the © All rights reserved. 2009 Inc. Nature America, cin6 indicates not only gravacin’s utility as a diverse chemical collections (reviewed in ref. ability of small bioactive chemicals to identify chemical probe for ABCB function, but sug- 4), which in some cases were pre-screened for functionally one or a small number of members gests that it could help in the search for drugs plant active compounds7,19, and (iv) structural of redundant families. to enhance the effectiveness of therapeu- databases of chemicals incorporating plant phe- tics. Interestingly, whereas the pgp19 mutant notype data20,21. Combined, these features make In vivo approaches are needed to dissect cel- displayed resistance to the inhibitory effects of plants a valuable research platform for funda- lular networks. Plant stem cells differentiate gravacin on gravitropism, the mutant was not mental discovery. into a wide range of cell types and organs, and resistant to a second chemically induced phe- plant are composed of distinct layers notype, which resulted in the mistargeting of Challenges faced by plant biologists of cells, each with its own identity. Because of tonoplast proteins to the endoplasmic reticu- Of course, there is little value in developing this high degree of complexity, it is difficult, lum6. Because endomembrane trafficking relies such an extensive genomics infrastructure with few exceptions, to isolate and culture plant on different machinery than gravitropism, this in plants, and at least the foundations of a cells derived exclusively from a single or suggests that there is a genetically distinct sec- community-wide infrastructure in chemical cell lineage and, if isolated, to maintain normal ond target site for the compound in a separate genomics, without the goal of insightful, inter- cell function under the conditions necessary to pathway (Fig. 2a). This kind of cross-talk can be esting biology and the development of useful establish the cultures. Thus, despite the appar- viewed as positive in uncovering new pathways, applications. In this regard, plants are poised ent ease and scalability of obtaining non-pure or as a hindrance in genetic screens for resis- to excel. Their are characterized by relative cultured plant cells for in vitro chemical screens, tance mutations. Bioactive compounds, such as immobility, resulting in the of adap- the approach should be considered cautiously endosidin 1, discussed below, can also help us to tive responses to environmental challenges that in terms of biological discovery. An in vitro define points of interaction within a network of are highly flexible, permitting plants to survive approach may work for basic cell-autonomous pathways (Fig. 2b). extensive drought, flood and biotic extremes. At processes perhaps, but will be of limited value The contributions of these and other chemi- the cellular level, lack of mobility has resulted in studying development, responses cal biology studies rely on the availability of in vegetative development that is depen- to environment or even cellular processes, such community-wide tools established in a few dent upon specific stem cells that give rise to as the establishment of cellular polarity. Thus,

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in the absence of pure cell cultures, plants must secondary effects that include the disruption be studied in vivo. a of endocytosis and exocytosis and their associ- An in vivo approach utilized the endocytosis ated compartments such as . In the inhibitor endosidin 1 (ES1)19 to define endo- case of BFA and ES1, which is more specific some/trans-Golgi network compartments for endosomes26, their value as reagents resides involved in the sorting of plasma membrane in their well-characterized cellular effects. For proteins, which are either recycled between example, starting with ES1, one could envision endosomes and the plasma membrane or are that a suite of chemicals having different speci- sent to the vacuole for turnover. The compound ficities for markers of dynamic cycling plasma was found by means of a high-throughput in membrane would permit the definition of a vitro screen for chemical inhibitors of pollen network of functionally overlapping compart- germination. Among other factors, pollen ger- ments necessary for endocytosis (Fig. 4). Such a mination and pollen tube elongation are depen- Gravitropism ER-tonoplast systems-based approach toward the trafficking dent upon proper endocytosis and exocytosis and application of chemicals would take maxi- at the tip of the growing pollen tube, which is a mum advantage of the rapid and conditional single cell displaying highly polarized tip growth. b nature of bioactive chemicals, especially for the In vivo studies were done in Arabidopsis roots direct study of highly dynamic processes. This expressing cycling plasma membrane mark- is not to reduce the importance of identifying ers including the auxin transporters PIN2 and cognate targets, but rather to acknowledge AUX1 and the plant steroid hormone receptor the conceptual complementarity of thinking brassinosteroid-insensitive1 (BRI1). ES1 was more broadly than targets as the predominant discovered to block a step in endocytosis at an endpoint of chemical biology to dissect net- early /trans-Golgi network compart- works27. In other words, although targets are ment containing the syntaxin SYP61, which is important, we should not fixate on them to involved in the endocytosis specifically of PIN2, the exclusion of the highly informative biol- AUX1 and BRI1, and allowed visualization of Endocytosis ogy that is achievable with chemicals that have the colocalization of these proteins with SYP61 well-characterized effects. (Fig. 3). ES1 also perturbs steroid signaling by Identifying cognate targets efficiently is also BRI1, providing important support for previous Figure 2 Bioactive chemicals can perturb distinct important for discovering mechanistic details suggestions that BRI1 resides in-part in an endo- or intersecting biological pathways. of new gene functions and networks. At this (a) Two independent pathways leading to distinct some compartment from which it is involved in responses can be perturbed by a single chemical. point, the main approach to target identifica- signal transduction leading to the regulation of This could occur via conserved functional domains tion is forward genetic screening using ethane steroid-responsive genes23. shared by the two protein targets or by interaction methyl sulfonate for chemical resistance and of each target site with a distinct of the hypersensitivity. This requires the establish- Many cellular processes occur rapidly in vivo. small molecule. For example, the auxin transporter ment of large mapping to define a Many cellular processes are highly dynamic ABCB19 (×) is the protein target of gravacin within physical region within reach of Sanger sequenc- the pathway for gravitropic response (blue). The in nature. Endocytosis is known to occur in a ing methods. However, with the advent of the

© All rights reserved. 2009 Inc. Nature America, means by which gravacin causes defects in ER-to- matter of minutes. Whereas mutants are clearly tonoplast protein targeting (orange) is unknown. new generation of massively parallel sequencing valuable in the study of such processes, unless (b) Bioactive chemicals can also identify points platforms, it is becoming feasible to sequence the mutations are conditional, one has to accept where multiple pathways intersect. For example, individual genomes to identify mutations2,28 that the state of the cell is at equilibrium with the chemical endosidin 1 (ES1), whose target associated with microscopy-based intracellular the . In the case of ES1, the compound is not known, identified a SYP61 endocytic phenotypes that might otherwise be too difficult compartment used by a subset of plasma is added and the plant cells respond in less than and impractical to score and map using conven- membrane proteins involved in distinct response an hour. When the chemical is removed from pathways in plants (see text). Pathways depicted tional fine-mapping approaches. As scientists the growth medium, the cells return to the state are hypothetical. become more familiar with the complexities before treatment within a few hours. In other of plant cells, -based approaches words, the addition of bioactive chemicals this is helpful in forward genetic screens to to identify protein targets, such as specialized permits the study of dynamic processes on a identify resistance genes, it does not negate the tagged libraries that permit the direct identifica- biological time scale. value of compounds with well-characterized tion of protein targets29, will complement the cellular effects, which as discussed, can provide genetic tools developed thus far. Chemical biology presents inherent many biological insights. An excellent example challenges of this concept is the toxin Brefeldin A (BFA)24, How do we take full advantage of plant Although chemical biology addresses many of which is now known to target the SEC7 family systems? the inherent difficulties of plants as biological of ARF/GEFs (ADP-ribosylation factor/GDP/ To more fully capitalize on the potential of systems, the discipline has yet to gain broad GTP exchange factor) of which the best known chemical biology to uncover new pathways acceptance among plant scientists and biolo- in plants is GNOM25. Even before its target and networks there are several key areas that gists at large. For example, biologists often find sites were known, BFA was the most widely need attention. it bothersome when a bioactive chemical is pro- used drug for studying the endomembrane miscuous, as the overriding feeling is that, to be system in plants and still is today. The impact Automation. There is an overall need for informative, chemicals should display strong of BFA on its multiple targets results in the increased automation to save time and labor. activity against a single cognate target. Although disruption of Golgi-based and has Typical plant-based screens utilize seedlings

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Figure 3 Bioactive small molecules can dissect rapid cellular processes. (a) PIN2 (shown in abPIN2 PIN2 blue), and other endocytosed proteins, transit through a continuum of compartments known as endosomes or the trans-Golgi network (TGN) in plants where they are sorted for recycling back to the plasma membrane or turnover in SYP61 the vacuole. During early endocytosis, PIN2 transits through a compartment containing the syntaxin SYP61; however, due to the rapidity SYP61 of endocytosis, fluorescent protein markers for PIN2 and SYP61 co-localize only rarely. PIN2 PIN2 (b) Endosidin 1 (ES1) inhibits an early step TGN TGN in endocytosis resulting in the accumulation of PIN2 and other specific cargo proteins as ES1 well as SYP61 into highly specific endosome agglomerations known as endosidin bodies21, demonstrating that PIN2 transits through the SYP61 compartment. Thus, bioactive chemicals can perturb dynamic processes and begin to define, in this example, endosome compartments and pathways associated with specific plasma membrane proteins.

germinated in medium containing chemicals support to develop other phenotyping tools needed to reduce the data to meaningful of interest. Much of the effort is manual and would greatly benefit plant biologists. quantitative measurements. can be laborious. The development of auto- In addition to examining deep layers mated plate preparation, seed plating and Phenotyping tools at the cellular level. One cell by cell, the opposite tact is also critical. imaging are critical to screen large, diverse, of the most powerful applications of chemi- Microscopy for the most part is still done chemical libraries effectively. This need for cal biology is the dissection of events at the sample-by-sample rather than by automa- automation is particularly acute in determin- cellular level that underlie plant develop- tion, and the automated microscopes that are ing the phenotypes resulting from chemi- ment and morphological responses to biotic available were mostly designed to image ani- cal treatment. What may seem as simple as and abiotic cues. The ability to detect small mal cells that are grown as monolayers rather recording root length and curvature becomes molecule–induced changes in cellular mor- than the complex multi-layers in plant tissues. a daunting task when attempted on a large phology will provide new insights. But this High-throughput chemical screens demand scale. Hardware and software solutions for requires improvements in microscopy and the ability to image thousands of samples image-based plant phenotyping are required probes. Owing to the complexity of plant rapidly at the cellular level and to analyze that can simultaneously collect large statisti- organs, it is critical to develop new micros- this enormous volume of image data to detect cally significant data on copy approaches to examine cells in vivo at subcellular phenotypes of interest. Imaging and responses to stress or environmental chal- cell layers that are deeper than the uppermost in great detail and at great depth and imaging lenges (Fig. 5). Some imaging tools are being layers now accessible. This would allow the thousands of samples in an automated and © All rights reserved. 2009 Inc. Nature America, developed to address these issues, for example, collection of data at multiple scales and sites. quantifiable manner are necessary to provide by measuring curvature angles dynamically New instrumentation also should incorpo- the range of scales required to truly merge during gravitropism30. However, additional rate the analytical and computational tools chemical biology with cell and . In all cases, there is a clear need for the application of image recognition, pro- Figure 4 Small molecules can help to define cessing and analysis to analyze posi- complex cellular networks. Depicted is a Protein 1 tion, diameter, velocity and morphology in a conceptual model of interconnected and distinct manner that is meaningful and quantifiable. pathways of the endocytosis of plasma membrane Endosomes/TGN proteins in Arabidopsis. The model shows Such data must be compiled and made avail- four hypothetical proteins that traffic through Protein 2 able to the scientific community through the endosomes/trans-Golgi network (TGN) during development of databases and web portals. endocytosis. Spheres (green) represent distinct Image analysis tools are becoming available endosome/TGN endomembrane compartments Protein 3 SYP61 to cell biologists, but as with existing auto- involved in endocytosis. Endocytosis may mated microscopes, they are designed mostly involve a network of pathways, each pathway Protein 4 being shared by one of more plasma membrane with cultured cells in mind and not proteins (proteins 1, 2, 3). However, as for PIN2, targeted toward the unique challenges faced AUX1 and BRI, the pathways may share specific Plasma by plant chemical biologists. Addressing the endosome compartments (in this case marked membrane challenges of microscopy and image process- as SYP61 to provide clarity). Alternatively, the ing that plant chemical biologists face will endocytic pathway may be completely distinct benefit the plant community as a whole. (protein 4). The targeting of proteins to the plasma membrane is not part of the model but can be expected to show equivalent complexity. Conceptually and using ES1 as an example, a suite of Investment in infrastructure. Thus far, signifi- compounds affecting the endocytosis of diverse plasma membrane proteins could be used to dissect cant discoveries have been made by the efforts the complex network of dynamic compartments underlying protein cycling at the plasma membrane. of a relatively small number of pioneering

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laboratories that have worked to establish chemical biology in plants. But the field is b Automated now at the point where it is crucial to develop phenotyping a shared infrastructure to permit wider acces- sibility. This infrastructure needs to include not only the technologies already discussed but also new chemical collections that are pre-screened for bioactivity. This will permit nonexpert laboratories to bypass some of the most laborious initial screening a Automated that is necessary to find chemicals of biological Quantitative 4,7 preparation c interest. Few such libraries exist and should data be expanded greatly. The housing of such resources should, at least in part, be within Image recognition one or a few centralized facilities that can offer Plant level and processing instrumentation (fluids robotics, automated microscopy), informatics (database manage- ment, image analysis) and technical expertise (plant-based screens, library curation, analyti- Chemical media cal and synthetic ). This arrangement seed plating will facilitate the rapid adoption of chemical biology by the plant community. Of course, such a focused effort requires resources beyond that of individual laborato- ries that have endeavored to establish chemical Cellular level biology within the plant community. Now that some of the fundamentals are in place, broader Figure 5 Enhanced microscopy, increased automation and quantification of seedling and cellular-level funding is needed to develop existing resources phenotypic data will drive plant chemical biology toward a more sophisticated, network-oriented view of development by providing access to more quantifiable phenotypes. (a) Robotics can greatly speed to their full potential. The approaches and not only the preparation of chemical media but the rate of seed sterilization and physical placement instrumentation used by plant cell biologists on media plates. (b) Automated phenotyping of leaf area, stem angle or root hair number should be provides much of the foundation necessary for combined with automated microscopy of complex intracellular phenotypes such as organelle diameter, chemical biology, and increased funding of basic number or velocity in many cell layers to facilitate analysis. (c) The raw image data would then be research in plant cell biology will act synergisti- processed using image recognition and analysis software resulting in data that are quantified to the cally with more focused efforts at infrastructure maximum extent possible. Quantifiable phenotypes, especially at the cellular level, will effectively expand the range of networks that can be dissected by chemical biologists. development. In the current economic climate, funding for new initiatives will be another chal- lenge faced by plant biologists. © All rights reserved. 2009 Inc. Nature America, Overall, plant chemical biology has moved 1. Cokus, S.J. et al. Nature 452, 215–219 (2008). 15. Feraru, E. & Friml, J. Plant Physiol. 147, 1553–1559 rapidly in the past decade and is beginning to 2. Ossowski, S. et al. Genome Res. 18, 2024–2033 (2008). (2008). 16. Titapiwatanakun, B. et al. Plant J. 57, 27–44 (2009). make genuine contributions to basic knowl- 3. Blackwell, H.E. & Zhao, Y. Plant Physiol. 133, 448–455 17. Borevitz, J.O. & Ecker, J.R. Annu. Rev. Genomics Hum. edge. Further advancement will require us to (2003). Genet. 5, 443–477 (2004). 4. Robert, S., Raikhel, N.V. & Hicks, G.R. in The 18. Lukowitz, W., Gillmor, C.S. & Scheible, W.R. Plant take advantage of bioactive small molecules as Arabidopsis Book (American Society of Plant Biologists, Physiol. 123, 795–806 (2000). system-oriented network dissection tools as Rockville, Maryland, USA, 2009). 19. Robert, S. et al. Proc. Natl. Acad. Sci. USA 105, 8464– well as a means to a target. It will also require 5. Dai, X., Hayashi, K., Nozaki, H., Cheng, Y. & Zhao, Y. 8469 (2008). Proc. Natl. Acad. Sci. USA 102, 3129–3134 (2005). 20. Girke, T., Cheng, L.C. & Raikhel, N. Plant Physiol. 138, us to take a more focused tact to overcome 6. Rojas-Pierce, M. et al. Chem. Biol. 14, 1366–1376 573–577 (2005). technical problems and to fund these efforts (2007). 21. Cao, Y. et al. 24, 1733–1734 (2008). on behalf of the plant community. The ben- 7. Zhao, Y. et al. Nat. Chem. Biol. 3, 716–721 (2007). 22. Surpin, M. & Raikhel, N. Nat. Rev. Mol. Cell Biol. 5, 8. Walsh, T.A. et al. Plant Physiol. 144, 1292–1304 100–109 (2004). efit technically will be new tools to delve more (2007). 23. Geldner, N., Hyman, D.L., Wang, X., Schumacher, K. & deeply into the gene networks than current 9. Walsh, T.A. et al. Plant Physiol. 142, 542–552 Chory, J. Genes Dev. 21, 1598–1602 (2007). (2006). 24. Nebenfuhr, A., Ritzenthaler, C. & Robinson, D.G. Plant approaches permit. Ultimately, our fellow citi- 10. Desprez, T. et al. Plant Physiol. 128, 482–490 Physiol. 130, 1102–1108 (2002). zens will benefit through improved health and (2002). 25. Geldner, N. et al. Cell 112, 219–230 (2003). environment. Attaining these goals will be well 11. Scheible, W.R., Eshed, R., Richmond, T., Delmer, D. & 26. Drakakaki, G., Robert, S., Raikhel, N.V. & Hicks, G.R. Somerville, C. Proc. Natl. Acad. Sci. USA 98, 10079– Plant Signal. Behav. 4, 57–62 (2009). worth the effort. 10084 (2001). 27. Peterson, R.T. Nat. Chem. Biol. 4, 635–638 (2008). 12. Somerville, C. Annu. Rev. Cell Dev. Biol. 22, 53–78 28. Sarin, S., Prabhu, S., O’Meara, M.M., Pe’er, I. & Hobert, ACKNOWLEDGMENTS (2006). O. Nat. Methods 5, 865–867 (2008). 13. DeBolt, S. et al. Proc. Natl. Acad. Sci. USA 104, 5854– N.V.R. and G.R.H. are grateful to the US National 29. Mitsopoulos, G., Walsh, D.P. & Chang, Y.T. Curr. Opin. 5859 (2007). Chem. Biol. 8, 26–32 (2004). Science Foundation (MCB-0520325 and MCB- 14. DeBolt, S., Gutierrez, R., Ehrhardt, D.W. & Somerville, 30. Lewis, D.R., Miller, N.D., Splitt, B.L., Wu, G. & Spalding, 0817916) for support. C. Plant Physiol. 145, 334–338 (2007). E.P. Plant Cell 19, 1838–1850 (2007).

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