Chemical Genomics in Plant Biology - NOPR

Indian Journal of Biochemistry & Biophysics Vol. 49, June 2012, pp. 143-154

Review

Chemical Genomics in Plant Biology Ayan Sadhukhan1, Lingaraj Sahoo1 and Sanjib Kumar Panda2* 1

Department of Biotechnology, Indian Institute of Technology, Guwahati 781039, Assam, India

2

Department of Life Science & Bioinformatics, Assam University, Silchar 788011, Assam, India Received 25 October 2011; revised 08 May 2012

Chemical genomics is a newly emerged and rapidly progressing field in biology, where small chemical molecules bind specifically and reversibly to protein(s) to modulate their function(s), leading to the delineation and subsequent unravelling of biological processes. This approach overcomes problems like lethality and redundancy of classical genetics. Armed with the powerful techniques of combinatorial synthesis, high-throughput screening and target discovery chemical genomics expands its scope to diverse areas in biology. The well-established genetic system of Arabidopsis model allows chemical genomics to enter into the realm of plant biology exploring signaling pathways of growth regulators, endomembrane signaling cascades, plant defense mechanisms and many more events. Keywords: Chemical Genomics, Genetics, Plant biology

Introduction So far, plant biology has evolved along with other branches of life science with the aid of classical genetic and biochemical methods which have their own limitations. The future, however, has to rely on substantial inputs from novel interdisciplinary approaches. Chemical genetics stands out to be one of the most powerful and versatile among them. The advent of the term ‘chemical genetics’ has ushered a new era in biology which marks use of chemicals to understand gene functions. The key idea in chemical genetics is the systematic design and synthesis of chemicals and their subsequent use as probes for —————— *Corresponding author2 E-mail: [email protected] Phone: +919435370608 Abbreviations: ABA, abscissic acid; ABC, ATP-binding cassette; ABCB, B group ABC transporter; ABI, ABA insensitive; ATL, alkyl transferase-like; AUX, auxin; BAK, BRI1 associated receptor kinase; ber, bestatin resistant; BES, bri1-EMS-suppressor; BR, brassinosteroid; BRI1 brassniosteroid insensitive 1; BUM, 2-[4(diethylamino)-2-hydroxybenzoyl] benzoic acid; DAS, 4-amino-3chloro-. 6-(4-chlorophenyl)-5-fluoro-pyridine-2-carboxylic acid; DEX, dexamethasone; FLS2, flagellin sensing 2; GSK, glycogen synthase kinase; GUS, beta-glucuronidase; HNA, 2-hydroxy-1naphthaldehyde; IAA, indole acetic acid; JA, jasmonic acid; moco, molybdopterin; MOD1, mosaic death1; MTX, methotrexate; PAMP, pathogen associated molecular patterns; PIN, PIN-formed; PYL, pyrabactin resistant 1-like; PYR1, pyrabactin resistant 1; QTL, quantitative trait loci; SAR, structure activity relationships; SCF, Skp, Cullin, F-box containing complex; TIR, transport inhibitor response; UDP, uridine diphosphate.

biological processes1-3. Low molecular mass molecules bind directly to proteins and modulate their functions4. They are functionally analogous to mutations in classical genetics and can assist in the unravelling of biological pathways5-7. Moreover, serious limitations of mutation-based classical genetics are overcome by this approach. Apparently seeming to be somewhat unique, actually small chemical molecules have been deployed for long to answer many questions in biology. The first appearance of the term chemical genetics may be traced to a paper by von Euler et al. in 1935, wherein the chlorophyll and gramine content of some barley mutants were reported. Historically, the term has been used in a somewhat different sense to explain difference in chemical constitution between a mutant and a wild type2. The mid 1990s saw chemical genetics in the modern sense of the term when the chemicals referred to were not natural products but made synthetically by chemists9,10. Development of advanced combinatorial chemistry techniques11, algorithms for synthesis of a myriad of molecules close to natural products12 and methods of application of those in in vitro and in vivo screening13 heralded the start of this new era in biology. ‘Chemical genomics’ is an expansion of the term ‘chemical genetics’ targeting entire genomes, possible due to the vast archiving of gene and protein structure-function data and the powerful tools,

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including microarray, quantitative trait loci (QTL) mapping, etc. Chemical genomics emerges out of the interplay between chemistry, cheminformatics, biology and bioinformatics14,15. This rapidly progressing field has already found applications in diverse areas of biology including microbiology16, cancer biology17, vertebrate developmental biology18 and neurobiology1, 19. Now it’s time for it to enter into the realm of plant sciences. Bioactive chemicals and plants have been faithful partners for long. Due to the immense importance of plant hormones and secondary metabolites, the plant scientist was already acquainted with studying small molecules of plants20. Foreign molecules have also found their way into plant research and are being used to inhibit and track down different biological components and processes. Herbicides have been extensively used for long as weed control agents and their chemistry and physiology have been studied in detail20. They have begun to be seen as potent probes for intervening plant biosynthetic pathways. Hunt for new herbicides by agrochemical companies has also opened way for a new set of small chemicals like isoxaben and benzothiadiazole21,22. It is worth mentioning

here that plant roots are highly efficient in uptake of small molecules, providing the scope for chemical genetic studies in plants2. The small size of the plant model Arabidopsis makes it easily amenable for phenotyping and when chemical genomics is combined with its well established genomics and proteomics tools, we can efficiently dissect a complex plant gene network affected by chemicals of our interest23. Plant chemical genetics: An edge over classical genetics Study of classical genetics has usually started with causing either directed or random mutations in the genome (Fig. 1). Termed as reverse and forward genetics respectively, both these approaches cause permanent irreversible change in the genetic as well as phenotypic make-up of the organism. The classical approach encounters mainly two problems: i) effect of a gene mutation been virtually masked by the product of related genes (genetic redundancy), and ii) mutation of an indispensible gene, leading to lethality5-7. The problems associated with classical genetics like gene redundancy is more frequent in plants bringing in the necessity of chemical genetics.

Fig. 1—A comparison between classical and chemical genetics [The classical approach to genetics begins with either random or site directed mutagenesis. But a redundant biological pathway may render this effort meaningless. The chemical approach deals with screening with chemical libraries for novel phenotypes. Chemicals specifically and reversibly bind to proteins behaving either as specific agonists or as general antagonists thereby overcoming problems of genetic redundancy]

SADHUKHAN et al: CHEMICAL GENOMICS IN PLANT BIOLOGY

In Arabidopsis, T-DNA inactivation mutants in some cases can either lead to lethality or no phenotype due to redundancies in gene function. The problem can be easily solved by implementing chemical genetics, wherein a small chemical molecule behaves either as a general antagonist and inhibits multiple targets in a redundant biological network (like different members of a protein family), or as a specific agonist the compound activates a specific component of a pathway (Fig. 1). The possible reverse situation would be of no help as specific antagonists analogous to single genetic mutations would not prevent redundant responses and a general agonist would augment too many targets to be identified24. Forward and reverse chemical genomic tools for plant biologists Forward chemical genomics essentially refers to screening of whole organisms or cells with a chemical library for candidates that cause some phenotypic changes. If the results are reproducible the next challenge becomes target identification which is usually done through a biochemical approach25. The small chemical molecule is tagged and incorporated into a matrix for affinity chromatography to identify which protein binds to the molecule26. Targets can also be identified via a yeast-three-hybrid system, where the compound interacts with DEX/MTX binding protein via its methotrexate (MTX) or dexamethasone (DEX) tag27. The DNA-binding domain of a transcription factor is fused to the DEX/MTX binding protein while its activation domain is fused to proteins from a plant cDNA library. As the compound and target protein from library interact successfully, a reporter gene in yeast is trans-activated, leading to visual scoring27. Methods of identifying scarce targets are phage display24 and protein microarray28. In the former, targets are expressed on phage surfaces and captured by interaction with compounds immobilized on a matrix24. In protein microarray, fluorescent- or radiolabelled chemical molecules are screened against protein chips28. But, these methods are insensitive against post-translational modifications. In this regard, it is worth mention of a newly emerged technique for labelling called ‘click chemistry’ at the heart of which is an azide-alkyne cycloaddition reaction29. Membrane-permeable azide (N3) or alkyne (≡) tags are added to the chemical molecule which interacts with the proteins. The complexes are

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identified by coupling to an alkyne- or azide tagged reporter or a matrix for its purification29,30. Targets of cysteine protease inhibitor E-64 in Arabidopsis have been identified by click chemistry31. Quantitative proteomics have also been used in target identification especially to eliminate possible identification of non-specific target proteins. Proteins are either labelled with heavy or light amino acids and only the light population is incubated with excess of the free chemical prior to purification. After purification against the chemical coupled to a matrix, only those complexes due to specific interactions are enriched in proteins with heavy amino acids. The excess small molecules in the proteins with light amino acids compete with those in the matrix and eliminate possible purification of non-specifically interacting complexes32. If the organism possesses a tractable genetic system, finding mutants insensitive or hypersensitive to the compound may lead to target identification in a genetic approach2. Using DNA microarrays33, the effects of the compound on an entire cellular pathway can be studied. Expression profiles of wild type versus specific gene deletion mutants provide information whether the compound is targeting multiple proteins34,35. The wealth of information available on the structure and function of the genome of the model plant Arabidopsis thaliana and variety of methods of genetic and genomic analysis including Arabidopsis whole genome microarrays and a huge collection of mutants create excellent opportunities for plant forward chemical genomics studies23. In reverse chemical genomics, we begin with a known protein target and screen it with the chemical library to identify candidates that modulate protein function in vitro and in vivo – an approach quite similar to site-directed mutagenesis2. When the compound inhibits protein function, we get a loss-offunction. Varying the concentration of chemical may lead to a series of mutants from leaky to null alleles2. Chemicals acting as agonists of protein function may lead to a gain of function phenotype. If a chemical intervenes with the function of all the members of a protein family, the situation becomes analogous to a multiple knock-out25 which is very difficult to construct, particularly in plants. Also, addition and removal of the chemical lead to a situation similar to conditional mutants2. The added advantage of this is that protein functions can be studied at any time in the developmental history of the plant23.

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To appreciate the role of chemical genomics in plant research, we need to have a basic idea about the difference between chemical genomics and classical pharmacology, the two terms being often confused between as well as the corner-stones of chemical biology — chemical libraries. Chemical genomics is born out of pharmacology Chemical genetics should not be confused with its forerunner — classical pharmacology which deals with the effects of synthetic chemicals or purified natural products on the living system. The use of combinatorial chemistry and high-throughput screening was pioneered by the pharmaceutical sciences for the discovery of new drugs. These approaches require a bigger chemical library synthesized around ‘scaffold’ compounds with druglike properties. On the other hand, chemical genomics makes use of highly diversified and unbiased libraries. Although membrane permeability for the chemical is essential to a plant chemical geneticist, but generally the high potencies or metabolic turnover necessary for a drug are not required36. The candidate compounds tested as potent drugs have to meet a more rigorous set of criteria like membrane permeability, mass, low toxicity, lack of side effects and short half-lives in the body as summarized by Lipinski in his ‘rule of five’37. On the other hand, bioactive compounds used in plant chemical genomics screens follow a slightly different set of rules — the ‘Tice’s rule-of-five’ formulated originally in context of some agrochemicals38. Also, reversibility of the compound is not absolutely essential in chemical genomics. The most important difference between drug discovery and chemical genomics lies in the fact that while drug discovery makes it absolutely essential to identify the target of a drug and to understand the drug-target interaction, chemical genomics in many cases is satisfied simply with the knowledge of the pathway involved36. Chemical libraries Chemical genetics or its expanded term chemical genomics uses highly diverse libraries rather than working on libraries designed around defined scaffolds36. Especially in case of plant biology, these unbiased libraries serve as invaluable tools to discover function of the huge number of uncharacterized plant genes. Ideally, a diverse chemical library containing a small molecule for every protein would be immensely

vast23 as number of possible organic compounds of molecular mass less than 1000 is more than 1060. Combinatorial synthesis now makes it possible to synthesize a large library. These libraries also called ‘Rule of Five Libraries’ include derivatives for structure-activity relationships (SAR) studies, but are somewhat biased to water-soluble, heterocyclic compounds36. These libraries have yielded valuable compounds like gravacin39, morlin40, hypostatin41, bikinin42 and pyrabactin42,43. Natural product libraries are also diverse and rich in bioactive chemicals, but are limited by the difficulty in organic synthesis of natural compounds36. ‘Diversity Oriented Synthesis Libraries’ fall in between ‘Rule of Five’ and ‘Natural Product libraries, as they contain synthetic compounds mimicking natural products and have high potential for yet uncovered targets44. These libraries are available from commercial suppliers in 96- or 384-well plates. In addition, there are the ‘NIH Molecular Libraries’ as well as ‘ChemMine’45 developed by the Centre for Plant Cell Biology, University of California, Riverside. Also, sometimes it is necessary to assemble an optimal library from chemicals of other libraries according to need46. Focused libraries19 find use at a later stage in chemical genomics only when initial screening has yielded at least some informative chemicals. Being small, these libraries have a high yield rate for targets47,48. After primary screening once a certain compound is identified, possible sites of it have to be modified to reduce the structural complexity and for desirable features for transport and so on. This requires extensive study of SAR. Synthesis of derivatives of a compound after SAR studies may be useful in uncoupling different phenotypic outcomes caused by the sensitivity of different derivatives even to members of the same biochemical pathway. Also, SAR studies lead to the discovery of antagonists and non-functional analogs of the chemical molecule which are valuable as experimental controls36. In addition, SAR studies critically judge the role of important functional groups on the bioactive chemical molecule, as they may lead to the non-selective binding to off-target proteins, leading to false-positives49. Plant specialized chemical libraries have also been created. Special mention can be made of the ‘Library of AcTive Compounds in Arabidopsis’ (LATCA) developed by Sean Cutler and colleagues (http://cutlerlab.blogspot.com/2008/05/latca.html)24.


Having overviewed most of our chemical genomic weapons, we shall now see how they have been used in actual warfare. We shall discuss in the following sections a few landmarks in plant chemical genomics screens. Cell wall biosynthesis Cell wall synthesis in plants which depends on cellulose biosynthesis has been subject of forward chemical genomic screens. The DIVERSet library of Chem Bridge Company has been searched for new chemical inhibitors of cellulose synthesis40. A candidate morlin from the library causes swollen roots in Arabidopsis due to interaction with cellulose synthase, resulting in disordered microfibril orientation in the cell40. So, morlin is a good candidate for studying the poorly understood process of cellulose biosynthesis. Several other libraries including LATCA have been screened on tobacco BY-2 cells, resulting in a chemical cobtorin, causing a swollen phenotype affecting parallel alignment of cortical microtubules with cellulose microfibrils50. It can be speculated that the yet undiscovered target connects microtubules to the cell wall biosynthetic machinery in some manner36. Cellulose biosynthesis inhibitor herbicide isoxaben targets have been characterized by isoxabeninsensitive mutant analysis. The insensitive loci have been found to code for cellulose synthases21,22. Effects of isoxaben on other proteins of cellulose synthesis machinery have also been reported51,52. Screens of 4800 chemicals have identified ID 620780 as a novel inhibitor of plasma membrane and golgi localized glucosyltransferases — the major enzymes in cell wall biosynthesis; the compound actually inhibits transfer of glucose from UDP glucose in golgi membranes and activates callose synthase in plasma membrane as well53. Several plant cell wall (xeg) mutants have been identified in a more recent and novel forward chemical genetic screen using an enzyme xyloglucanase as the small molecule probe in a population of mutagenized Arabidopsis54. Twenty-three loci involved in making plant cell walls have been discovered in this approach54. One glycosyltransferase (GT77) mutant Xeg-113 with more elongated hypocotyls has its cell wall extensins (glycoprotein) under-arabinosylated54. This provides evidence for the first time that arabinosylation of extensin is important for cell elongation in plants54.

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Brassinosteroid signalling Cytochrome P450 inhibitors — trizoles have been traditionally used as fungicides and herbicides20. They are also found to inhibit key components of the biosynthesis pathways of important plant hormones known as brassinosteroids (BRs), e.g., brassinolide20. Screening in rice (Oryza sativa) and cress (Lepidium sativum) with a small library of 10 synthetic triazole derivatives for cytochrome P450s inhibitors involved in brassinosteroid biosynthesis has yielded brassinazole as an initial candidate compound55. Screens for BR deficient-like phenotypes in Arabidopsis validate the effects of brassinazole56-59. It is found to bind to an enzyme in BR biosynthesis pathway DWF4, a cytochrome P450 monooxygenase, with high specificity creating a dwarf phenotype55,60. This phenotype is rescued by treatment with BR55,60. Microarray analysis validates that brassinazole has an antagonistic role against BR to the transcriptome61. Analysis of brassinazole-insensitive mutants has revealed a locus brz1 coding a novel transcriptional repressor specific to plants which provides the explanation for dwarf phenotype rescue with BR treatment, suggesting a feedback regulation of the end product BR on DWF462. One more brassinazole-insensitive mutant brz4 has been isolated by QTL mapping63. Another chemical bikinin having similar effects on the transcriptome as with BR has been identified to function in BR signalling64. BR signalling mutants have been screened with bikinin and ultimately the target of bikinin is found to be BIN2- a GSK3 like kinase working on two transcription factors bri1-EMSsuppressor 1 (BES1) and brassinazole resistant 1 (BZR1)64. Bikinin acts as a general antagonist of multiple GSK3 kinases and completely removes phosphorylation of the transcription factors, which is not achieved even by a classical genetic triple-mutant containing residual phosphorylation64. So, the BR signalling pathway is elucidated as follows: BR binds to receptors brassinosteroid insensitive 1 (BRI1) and BRI associated receptor kinase 1 (BAK1) resulting in a sequence of events, leading to activation by phosphorylation of transcription factors bri1-EMSsuppressor 1 (BES1) and brassinazole resistant (BZR1), bringing about transcription of BR-responsive genes24 (Fig. 2). Chemical genomics is also helpful in intervening interactions between biological pathways in plants. In a hypocotyl elongation experiment, a phenotype of

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Fig. 2—Chemical genomics in brassinosteroid signalling [Brassinosteroid signalling components have been elucidated by chemical genomics. Brassinazole binds to an enzyme in brassinosteroid (BR) biosynthesis pathway DWF4 creating a dwarf phenotype which is rescued by treatment with BR. A locus brz1 codes for a novel transcriptional repressor acting somewhere in the feedback regulation pathway of the end product BR on DWF4. Bikinin targets as a general antagonist multiple GSK3 kinases working on two transcription factors bri1-EMS-Suppressor 1(BES1) and brassinazole resistant (BZR1) completely removing their phosphorylation. BR binds to receptors brassinosteroid insensitive 1 (BRI1) and BRI 1 associated receptor kinase (BAK1) leading to a sequence of events leading to activation by phosphorylation of transcription factors BZR1 and BES1 bringing about transcription of BR-responsive genes]

inhibited hypocotyl length has been observed by treatment with a compound brassinopride from a library of 10,000 compounds65. The phenotype is rescued by brassinolide co-treatment, indicating inhibition of brassinosteroid biosynthesis by brassinopride. Brassinopride also increases size of apical hooks — a phenotype also caused by ethylene treatment and reversed by ethylene inhibitors or observed in ethylene insensitive mutants, suggesting the role of brassinopride in ethylene signalling65. In SAR studies, it is found that only one of twelve derivatives of brassinopride acts most specifically on ethylene signalling and least on BR biosynthesis suggesting that the two pathways are separate65. Auxin signalling Auxin is such an important growth regulator in a plant that mutagenesis aimed at component genes in its signalling pathway affects the plant severely20. This paves the way for chemical genomics. Initially some complex spiro-ketal products of fermentation from the soil microorganism Streptomyces diastatochromogenes screened in Arabidopsis with an

auxin responsive gus reporter for candidates inhibiting auxin-signaling66,67 yielded two potent compounds yokonolides A and B. Later studies have shown the role of yokolonide B upstream in auxin signalling, as it prevents auxin-induced degradation of the transcription factors AUX/IAA, but does not inhibit the proteasome68. But, the difficulties in synthesis as well as isolation of these complex natural products limit their further use2. A small library of 57 biaryl-derived molecules screened for their effects on germination in Arabidopsis has yielded (P)-4k, which stunts development, causes pigmentation loss and ultimately death69. A combinatorial library of 10000 compounds has also been used to screen for inhibitors of auxinmediated proteolysis of AUX/IAA transcription factors and has reported 30 potent compounds70. Sirtinol, originally discovered in yeast as an inhibitor of the Sirtuin family of NAD-dependent deacetylases, is found to affect Arabidopsis root and vascular tissue development, hence is speculated to act in auxin signalling71. Forward chemical screens72 for compounds altering expression patterns/levels of DR5-GUS, an auxin reporter line73 and an auxin overproducing mutant of Arabidopsis, yucca74 have also yielded sirtinol. Other evidences, including activation by sirtinol of auxin-inducible genes, promotion of auxin-related phenotypes, resistance of auxin-signaling mutants to sirtinol and sirtinol-induced degradation of auxin/indole-3-acetic acid (AUX/IAA) proteins — a characteristic of auxin signalling suggest the agonistic role of sirtinol in auxin signalling2. Sir mutants like sir1 is sirtinol-resistant, but is hypersensitive to auxin in contrast to auxin mutants (axr) which are sensitive to both auxin and sirtinol, suggesting role of SIR1 upstream of axr mutants72. After cloning, sir1 gene is found to encode a protein containing an ubiquitin-activiating enzyme E1 domain, as well as a Rhodanese-like domain homologous to that of a prolyl isomerase72. Other sir genes sir3, sir4 and sir5 have been found to encode enzymes for molybdopterin co-factor (moco) biosynthesis, while other moco biosynthesis mutants are sirtinol insensitive. The work on sirtinol derivatives has shown that sir genes are involved in metabolizing sirtinol to an active auxin 2-hydroxy-1naphthoic acid. The oxidation of an aldehyde intermediate, 2-hydroxy-1-naphthaldehyde (HNA) in the process requires an aldehyde oxidase which utilizes the co-factor moco75 (Fig. 3).


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a novel auxin transport inhibitor 2-[4-(diethylamino)2-hydroxybenzoyl] benzoic acid (BUM) which strongly antagonizes auxin-related development and physiology. Biochemical and physiological studies show B group ATP-binding cassette (ABC) transporters (ABCBs) to be the targets for BUM. While ABCBs and pin-formed (PIN) families of proteins both control the cellular efflux leading to the basipetal polar transport of auxins, BUM does not affect PIN proteins. BUM may emerge as an efficient tool in understanding auxin transport and at the same time uncoupling ABCB- and PIN-mediated efflux systems81.

Fig. 3—Chemical genomics in auxin signalling [Chemical genomics has helped unravelling components of the auxin signalling pathway. The chemical Sirtinol is metabolized into 2-hydroxy-1-naphthaldehyde (HNA) which undergoes oxidation by an aldehyde oxidase to 2-hydroxy-1-naphthoic acid, an active auxin. The molybdopterin cofactor (moco) of the aldehyde oxidase is biosynthesized by products of sirtinol resistant (sir) genes. Other products of sir genes biosynthesize ubiquitin activating enzyme E1 which leads to ubiquitination for eventual proteasome mediated degradation of AUX/IAA repressors – a hallmark of auxin signalling. The ubiquitin ligase SCF complex is assembled with F-box proteins- products of tir (transport inhibitor response) genes, the recruitment of F-box proteins being facilitated by auixns]

Earlier screens for mutants resistant to auxin transport inhibitors have resulted in isolation of the transport inhibitor response 1 (tir1)76. Tir1 is an F-box protein and a component of the E3 ubiquitin ligase, Skp, Cullin, F-box containing complex (SCFTIR complex) which marks the AUX/IAA repressors for proteasome mediated degradation. TIR1 has been shown to be a bonafide auxin receptor77,78. The interaction between TIR1 and AUX/IAA repressor proteins is promoted by auxin. By introducing alkyl chains to the α-position of auxin IAA, several derivatives have been generated and screened for inhibitors of F-box protein recruitment by the SCF complex (Fig. 3) and the whole picture has been visualized by X-ray crystallography79. Another member of the auxin signalling F-box protein family — ABF5 has been discovered in a screen using DAS534, an herbicide triggering auxin responses. This is possible only due to the high specificity exhibited by the chemical (DAS534) to a member (ABF5) of an otherwise redundant gene family80. Recently, a screen with a Korean chemical library of 6,500 small organic compounds has yielded

Abscisic acid signalling Abscisic acid (ABA) is an important plant hormone involved in drought resistance. The triazole library yielding BR biosynthesis inhibitors has given two compounds uniconazole and diniconazole which inhibit Cyp707A3, a cytochrome P450 hydrolyzing abscisic acid. Treatment of Arabidopsis with uniconazole and diniconazole increases drought tolerance owing to greater accumulation of ABA82,83. But the most noteworthy event in the ABA story is the unravelling of the long-sought after ABA receptor by chemical genomics. A chemical genomics screen for germination inhibitors has identified a selective ABA agonist pyrabactin41. The effect of ABA and pyrabactin on seed transcriptome is significantly similar43 placing pyrabactin target(s) in the ABA signaling pathway. Pyrabactin-insensitive mutants have revealed the locus pyrabactin resistance 1 (pyr1)25. Subsequent genetic analyses have also indicated the necessity of PYR1 in vivo, but loss-of-function alleles are uninformative about ABA signalling. This is due to the genetic redundancy of PYR1 — the effects of its 13 other relatives — pyrabactin resistance 1-like (PYL) proteins25. A yeast two-hybrid screen has been performed to identify which proteins bind to PYR1 in presence of pyrabactin in the growth medium43. The results show that in presence of pyrabactin PYR1 interacts with and inhibits ABA insensitive (ABI) 1, ABI2 and hypersensitive to ABA 1 (HAB1) — group A protein phosphatases (PP2Cs) which negatively regulate ABA responses43. This has established PYR1 as a bonafide ABA receptor and has formed an excellent example of a specific agonist selectively activating a critical component of a signalling pathway leading to its discovery.

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Endomembrane trafficking and gravitropism Endomembrane trafficking in plants, itself a complex process is again linked to another phenomenon – gravitropism84. In an attempt to develop chemical probes for the process, a screen has been performed in yeast81 for compounds causing secretion of the yeast vacuolar marker protein carboxypeptidase Y (CPY). Of 14 resultant candidates tested on Arabidopsis, only sortin-1 and -2 partially fragment vacuolar membrane and retard root development36. Sortin-1 successfully triggers CPY secretion in Arabidopsis cell cultures85. It also causes mislocalization of a tonoplast marker and affects vacuole biogenesis, but does not affect other endomembrane compartments85. Identification of several sortin-1 hypersensitive and flavonoid-defective mutants has shown that sortin-1 alters vacuolar accumulation of flavonoids, possibly blocking their transport via ABC transporters localized in vacuoles86. Extensive SAR studies carried out with analogs and sub-structures of sortin-1 from the ChemMine database45 have identified key features of the molecule for bioactivity and also uncoupled flavonoid accumulations and biogenesis defects of vacuoles86. Yeast mutants hypersensitive to sortin-2 help in identification of genome loci, encoding components of endosome compartments involved in vacuole trafficking. SAR studies in this case also threw light on the structural determinants of sortin-2, as well as on the nature of its target87. The 10000-compound library used for screening the inhibitors of auxin signalling71 has also been screened on Arabidopsis for candidates affecting gravitropism84. Of the 34 confirmed inhibitors/ enhancers of gravitropism, 4 result in abnormal endomembrane morphologies. The compound 5403629 is a structural analogue of the synthetic auxin2, 4-dichlorophenoxy acetate, while compound 5850247 decreases auxin responsiveness of roots84. These results suggest some link between gravitropism, endomembrane trafficking and auxin signalling. As a part of the screen, gravacin is identified as a strong inhibitor of gravitropism, auxin responsiveness and protein trafficking to the tonoplast84 (Fig. 4). The target for gravacin is found to be P-glycoprotein19 (PGP19) – an ABC transporter of auxin through a mutation in the protein, resulting in the reduced binding of gravacin to Hela cell microsomal fractions39. The advantage with gravacin for further studies on PGP19 remains that it does not inhibit

Fig. 4—Chemical genomics in endomembrane trafficking and gravitropism [Chemical probes interrogate the complex and related phenomena of Endomembrane Trafficking & Gravitropism. Gravacin, a potent inhibitor of gravitropism responses targets PGP19, an ABC transporter of auxin. Endosidin1 on the other hand slows down vesicular trafficking of another auxin transporter PIN]

other transporters of auxin-like PIN proteins of the plant plasma membrane (PM) which are known to interact with PGP1936. Other than targeting gravitropism, gravacin also causes mis-targeting of a tonoplast marker ‘δ-tonoplast intrinsic protein fused with green fluorescent protein (δ-TIP-GFP)’ from the vacuole to the endoplasmic reticulum84, suggesting it may have some other targets yet to be explored. Compounds intervening with vesicular trafficking in plants have been a goal of some recent chemical genomic screens using several PM localized markers. Examples of such markers are the PIN proteins which directionally transport auxins88-90 and get endocytosed to be either recycled to the PM or sent to vacuoles for degradation90,91 (Fig. 4) and the BR receptor BRI192 which localizes both to the PM and endosomal compartments. BRI 1 initiates a signalling pathway from an endosome compartment93, leading to accumulation of unphosphorylated transcription factors in the nucleus81,94. Pollen tube growth which depends on endomembrane trafficking has been used as an experimental material in a high-throughput chemical genetic screen95,96 in microplates to be visualized by confocal microsopy. A compound endosidin-1 (ES1) stops pollen tube growth by specifically slowing the trafficking of SYP61 and VHA-a1 containing endosomes with PM markers PIN2, BRI1 and AUX1 in Arabidopsis, resulting in ‘endosidin bodies’ containing these proteins95.


Plant defence responses Plant immune responses are triggered by different pathogen-associated molecular patterns (PAMPs) like cellulysin and flagellin20. Cellulysin and flagellin induce the ATL2 promoter — an early PAMP-responsive gene20. A chemical library of 120 bioactive small molecules has been screened for candidates interfering PAMP associated activation of an ATL2 promoter-driven reporter gene in submerged Arabidopsis seedlings48. Of the hits, oxytriazine itself induces ATL2 without PAMPs. Four others (triclosan, fluazinam, cantharidin and fenpiclonil) interfere with PAMP-induced ATL2 expression48. A closer monitoring shows that triclosan and fluazinam interferes with the accumulation of reactive oxygen species and endocytosis of the flagellin sensing-2 (FLS2) immune receptor48. Triclosan derivatives and enzyme inhibition assays have identified Arabidopsis mosaic death-1 (MOD1) enoyl-acyl carrier protein reductase, a subunit of the fatty-acid synthase type II complex as a probable target of triclosan48. The general antagonistic role of triclosan for all tested elicitor-triggered early immune responses suggests role of signaling lipids in PAMP-induced plant immunity48. Zheng et al.97 have employed bestatin, previously shown to be a inducer of wound response genes in tomato in a chemical genetic screen in Arabidopsis to dissect jasmonic acid (JA) signaling. The screen has yielded several bestatin-resistant (ber) mutants in which bestatin have no roles on root elongation97. Further study has resulted in three types of ber mutants — JA-insensitive, JA-hypersensitive, and those showing normal response to JA97. Analyses of these ber mutants have lead to the identification of several novel loci involved in JA signaling97. In a search for novel peptides eliciting plant defence responses, a cell-based high-throughput assay has been designed98. A combinatorial peptide library based on flagellin sequence has been prepared and immobilized in agarose gel via a photo-cleavable linker98. These have been overlaid on tobacco cells. H2O2 generated as plant response is detected using a H2O2 indicator dye98. Plant germination Bassel et al.47 screened a small molecule library against the Arabidopsis seed germination transcriptome identifying three inhibitors of germination — methotrexate, 2, 4-dinitrophenol and cycloheximide. A number of proteins — gibberellic acid insensitive,

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resistant to gibberellic acid, resistance gene like-3, ABA insensitive-4, 5, 8, fiery1 and gibberellic acid insensitive dwarf 1A have shown significantly changed transcript abundance in presence of the inhibitors at a range of doses with respect to control47. Future studies may lead to the establishment of these responsive genes as important regulators of the process. Conclusion Chemical genomics thus offers a helping hand to plant biology by providing a novel means to address serious issues like overlapping gene function, lethality and tissue/development-specific expression. Plant chemical genomics is gradually gaining momentum with results of newer screens aimed at different biological processes and signalling components of plants being published each year. Though plant systems have been quite efficient in taking up the foreign chemical molecules one of the prime concern remains their metabolism by plant enzymes like cytochrome P450 monooxygenases and transferases to inactive products. While constructing substituted, oxidation resistant groups23 in the molecules may help, a better strategy could be synthesis of compounds in such a way that they would be activated inside the plant after metabolism and that activated product would probe the process of interest. A more widespread application of chemical genomics in plant biology should be looked up by researchers as genomic information explodes beyond the Arabidopsis model to many crop plants. Acknowledgements The authors are grateful to Indian Institute of Technology Guwahati and Assam University Silchar for financial support. References 1 2 3

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