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.
SADHUKHAN et al: CHEMICAL GENOMICS IN PLANT BIOLOGY
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).
SADHUKHAN et al: CHEMICAL GENOMICS IN PLANT BIOLOGY
149
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.
SADHUKHAN et al: CHEMICAL GENOMICS IN PLANT BIOLOGY
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,
151
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.
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