A chemical genetics approach to explore anthocyanin ... · Eshan Naik Masters of Science Department of Cell & Systems Biology University of Toronto 2016 Abstract Plants often experience

A chemical genetics approach to explore anthocyanin regulation in

nitrogen-deprived Arabidopsis seedlings

by

Eshan Naik

A thesis submitted in conformity with the requirements

for the degree of Masters in Science

Department of Cell & Systems Biology

University of Toronto

© Copyright by Eshan Naik 2016

ii

A chemical genetics approach to explore anthocyanin regulation in

nitrogen-deprived Arabidopsis seedlings

Eshan Naik

Masters of Science

Department of Cell & Systems Biology

University of Toronto

2016

Abstract

Plants often experience varying soil nitrogen levels during their life cycle, and adjust growth and

development to accommodate these changes. In the present study, a chemical genetics approach was

implemented to discover molecular components involved in the induction of anthocyanin biosynthesis,

in response to altered nitrogen levels. Using a robust high-throughput approach, a chemical library was

screened to discover compounds capable of attenuating anthocyanin levels in nitrogen-deprived

Arabidopsis seedlings. Chemical screens were successful in identifying four compounds that appear to

mis-regulate anthocyanin accumulation during nitrogen deprivation. A chemical-induced phenotype was

characterized, and the results indicate that chemical application must occur at a specific developmental

stage. Furthermore, low nitrogen-induction of anthocyanins is highly sensitive to chemical application,

and chemicals appear to exert their effect throughout early seedling growth. Future mutational and

genome-wide studies will bridge the gap in understanding the link between anthocyanin biosynthesis

and nitrogen availability.

iii

Acknowledgements

I am grateful to my supervisor Dr. Malcolm M. Campbell for his support and academic guidance. I

began my journey as a young scientist in his lab during the end of my undergraduate studies. I soon

discovered that Malcolm and I share a fascination for the natural sciences, and was convinced to embark

onto graduate studies under his supervision. I would also like to thank my committee members, Dr. Greg

Vanlerberghe and Dr. Sonia Gazzarrini, as well as my external examiner, Dr. Dan Riggs, for their

academic advice and support in successfully completing my thesis work.

Aside from faculty members, I would like to thank my graduate mentor, Dr. Michael Stokes, as well as

my wonderful lab mates Katrina Hiiback, Dr. Katharina Bräutigam, and Dr. Marc Champigny for their

friendship and support. I am grateful for research assistance provided by Joan Ouellette and Dr. Michael

Stokes, as they both played a crucial role in my smooth transition into the research world. I also humbly

acknowledge federal funding and financial support from the Natural Sciences and Engineering Research

Council of Canada (NSERC). Being an NSERC recipient encouraged and compelled me to strive and

pursue academic excellence.

I am highly indebted to my parents, Sonali & Rajat Naik, for their relentless support in pursuing higher

education. It was highly unlikely for me to step into the academic world without their emotional and

financial support. I cannot emphasize enough the countless opportunities bestowed upon me because of

the choices and sacrifices made by my parents. I am also truly grateful to my sister and academic

mentor, Urja Naik, as she guided me throughout my undergraduate and graduate studies. More

importantly, she helped me discover my immense passion and love for the natural sciences; therefore,

my academic and professional accomplishments can be credited to her.

I would like to thank my friends for their love and support. Lastly, I am grateful to Emily Maclean for

her friendship and immense support in helping me remain determined while writing my thesis.

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Table of Contents

Acknowledgements…...............................................................................................................................iii

Table of

Contents....…………………………………………………………………………….............................iv

List of

tables……………………………………………………………………………………………………viii

List of figures……………………………………………………………………………………........ix-xi

Chapter 1 Introduction

1.1 Nitrogen and plants……………………………………………………………......................2

1.2 Adaptability to nitrogen limitation in Arabidopsis thaliana……………………....................2

1.3 Flavonoid metabolism: Anthocyanin biosynthesis…………………………………………..3

1.4 Low nitrogen-induced anthocyanin accumulation:

Biological relevance in Arabidopsis thaliana………………………………………………..5

1.5 Low nitrogen-induced anthocyanin accumulation: Regulation via transcription factors…....6

1.6 Low nitrogen-induced anthocyanin accumulation: Regulation via small metabolites

1.6a Regulation via phytohormones………………………………………………….………8

1.6b Regulation via ubiquitin ligases……………………………………………….………..9

1.7 Low nitrogen-induced anthocyanin accumulation: Carbon-nitrogen interactive effects…...11

v

1.8 Small molecules can dissect part of the nitrogen deprivation response associated with

anthocyanin accumulation………………………………………………………………….12

1.9 Research hypotheses and objectives………………………………………………………..13

Chapter 2 Materials & Methods

2.1 Plant Material and growth conditions……………………………………………………….16

2.2 Optimize growth conditions for nitrogen-deprived seedlings………………………………16

2.3 Chemical library…………………………………………………………………………….16

2.4 Chemical screen: primary and secondary…………………………………………………...17

2.5 Dose-response curves and statistical analysis………………………………………………18

2.6 Chemical analogues (derivatives)…………………………………………………………...18

2.7 Anthocyanin and chlorophyll quantification………………………………………………..18

2.8 Primary root length measurement…………………………………………………………...19

2.9 Optimize growth conditions for phosphate-deprived seedlings…………………………….19

Chapter 3 Results & Discussion (Section 1)

3.11 An introduction to chemical genetics……………………………………………………...21

3.12 Setup a robust assay: Anthocyanins accumulate under a moderately

high C/N treatment………………………………………………………………………....22

3.13 Screen chemical library: Identified small molecules in an anthocyanin inhibition assay…25

vi

3.14 Chemical compounds attenuate anthocyanin levels irrespective of

inorganic nitrogen source (ammonium vs. nitrate)……………………………………..32


3.21 Introduction: Establish biological characteristics of a small molecule………………….....36

3.22 Early chemical treatment generates a dose-dependent anthocyanin inhibition response.....36

3.23 Chemical application is critical during radicle seed emergence...........................................42

3.24 Small molecules attenuate anthocyanin levels during early seedling growth.......................49


3.31 Establish structure-activity relationship……………………………………………………57

3.32 Analogues: Nomenclature and anthocyanin inhibition screen…………………………….57

3.33 2-(3-nitrophenyl)-1H-benzimidazole activity requires a central imidazole

and a nitro side group………………………………………………………………………58

3.34 3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one activity requires

a central oxazole-chromene backbone……………………………………………………..64

3.35 1-[3-(1-benzofuran-2-yl)-1H-pyrazol-4-yl]-N-(1, 3-benzoxazol-2-ylmethyl)-N

methylmethanamine activity requires a pyrazole moiety, along with a central oxazole

backbone and a methanamine side group…………………………………………………70

vii

3.36 2-{4-[(9-ethyl-9H-carbazol-3-yl) methyl]-1-methyl-2-piperazinyl} ethanol activity

requires a central carbazole backbone……………………………………………………..76

Chapter 4 Results & Discussion

4.1 Nitrogen-deprived seedlings have a longer primary root and reduced

chlorophyll leaf content……………………………………………………………………..83

4.2 All four chemical compounds modify primary root length under low N conditions……….83

4.3 Chemical compounds fail to modify leaf chlorophyll content……………………………...93

4.4 Chemical compounds suppress seedling anthocyanin content under

phosphate starved conditions………………………………………………………………..95

Chapter 5.1 Conclusion and Future directions……………………………………………………….98

References……………………………………………………………………………………………...103

viii

List of Tables

Table 1 Structures of candidate compounds in the ChemBridge library scored for their

ability to attenuate anthocyanin levels in seedlings grown under

nitrogen deficiency….................................................................................................30-31

Table 2 Structures of benzimidazole analogue compounds scored for their ability to

attenuate anthocyanin levels in seedlings grown under nitrogen deficiency……….59-60

Table 3 Structures of benzoxazole-1 analogue compounds scored for their ability to


Table 4 Structures of benzoxazole-2 analogue compounds scored for their ability to


Table 5 Structures of carbazole analogue compounds scored for their ability to


ix

List of Figures

Figure 1 The flavonoid biosynthetic pathway, including general phenylpropanoid metabolism…4

Figure 2a Red pigmentation visible in seedlings grown under a moderately high C/N treatment..23

Figure 2b Anthocyanins accumulate under a moderately high C/N treatment……………………24

Figure 3 Chemical library screened in a microtitre plate under low nitrogen growth

conditions (high C/N)…………………………………………………………………..26

Figure 4a Lack of red pigmentation visible on seedlings grown under low nitrogen levels

(high C/N)…………………………………………………………………………...27-28

Figure 4b Nitrogen-deprived seedlings, pre-treated with candidate chemical compounds, failed

to accumulate anthocyanins…………………………………………………………….29

Figure 5a Seedlings pre-treated with chemical compounds accumulate attenuated anthocyanin

levels under low ammonium growth conditions………………………………………..33

Figure 5b Seedlings pre-treated with chemical compounds accumulate attenuated anthocyanin

levels under low nitrate growth conditions……………………………………………..34

Figures 6a-d Chemicals (Benzimidazole, Benzoxazole-1, Benzoxazole-2, Carbazole) applied at

a range of concentrations proportionally attenuates anthocyanin levels in nitrogen-

deprived seedlings…………………………………………………………………..37-41

Figure 7a Illustration of chemical applied at six different time points to identify the critical

period of exposure……………………………………………………………………...43

x

Figures 7b-e Chemical (Benzimidazole, Benzoxazole-1, Benzoxazole-2, Carbazole) application

at six different time points to identify the critical period of exposure……………...44-48

Figures 8a-d Anthocyanins measured during post-germinative growth in DMSO/chemical treated

Arabidopsis seedlings……………………………………………………………….51-55

Figure 9a Benzimidazole (parent) and benzimidazole-analogues (A-, B-, etc.) exhibit varying

anthocyanin levels in nitrogen-deprived seedlings……………………………………..61

Figure 9b Benzimidazole-analogue (B-) failed to strongly attenuate anthocyanins in nitrogen-

deprived seedlings……………………………………………………………………...62

Figure 9c Benzimidazole-analogue (CA-) failed to strongly attenuate anthocyanins in nitrogen-

deprived seedlings……………………………………………………………………...63

Figure 10a Benzoxazole-1 (parent) and benzoxazole-1-analogues (A-, B-, etc.) exhibit varying


Figure 10b Benzoxazole-1-analogue (C-) dose response (25-50 µM) ……………………………..68

Figure 10c Benzoxazole-1-analogue (D-) dose response (25-50 µM) ……………………………..69

Figure 11a Benzoxazole-2 (parent) and benzoxazole-2-analogues (A-, B-, etc.) exhibit varying


Figure 11b Benzoxazole-2-analogue (A-) dose response (25-50 µM) ……………………………..75

Figure 12a Carbazole (parent) and carbazole analogues (A-, B-, etc.) exhibit varying


xi

Figure 12b Carbazole-analogue (D-) dose response (25-50 µM) ………………………………….80

Figure 12c Carbazole-analogue (E-) dose response (25-50 µM) …………………………………..81

Figures 13a-b Carbazole treatment generates a long primary root under low N conditions……….85-86

Figures 14a-b Benzimidazole treatment generates a short primary root under low N conditions…87-88

Figures 15a-b Benzimidazole-1 treatment generates a longer primary root irrespective of

N regimen...................................................................................................................89-90

Figures 16a-b Benzimidazole-2 treatment generates a longer primary root irrespective of

N regimen…………………………………………………………………………...91-92

Figure 17 Chemical compounds do not modify total chlorophyll leaf content………………........94

Figure 18 Seedlings pre-treated with candidate chemical compounds fail to accumulate

anthocyanins under low phosphate levels………………………………………………96

1

Chapter 1

2

Introduction

1.1 Nitrogen and plants

Nutrients are necessary for plants to meet cellular metabolic requirements, in order to sustain growth and

development. One such nutrient is nitrogen, which must be captured from soil, because after water, it is

most limiting to plant survival (Vitousek & Howarth, 1991). Nitrogen (N) plays two key roles crucial for

survival. First, it serves as an important macronutrient, essential for the production of amino acids and

its derivatives (Coruzzi, 2003). Second, nitrogen is an important signalling molecule, mediating

developmental processes such as root morphogenesis as well as regulating primary and secondary

metabolism (Bongue-Bartelsman & Phillips, 1995; Foyer et al., 2003; Malamy & Ryan, 2001).

1.2 Adaptability to nitrogen limitation in Arabidopsis thaliana

When soil nitrogen becomes limiting, plants must reprogram metabolism to accommodate for any

changes in the environment. Arabidopsis thaliana (Arabidopsis) roots absorb nitrogen predominantly in

its inorganic form as nitrate (NO3-) (Lam et al., 1996). Plants have developed a suite of adaptive

responses towards N-limitation to survive and successfully produce offspring. This adaptability has been

well characterized in Arabidopsis, and includes a reduction in photosynthesis, dramatic increases in root

growth and lateral branching, remobilizing N from older to actively growing tissues, chlorophyll

degradation and the accumulation of secondary metabolites, particularly anthocyanins (Bongue-

Bartelsman & Phillips, 1995; Chalker-scott, 1999; Diaz et al., 2006; Ding et al., 2005; Fritz et al., 2006).

Re-structuring growth and development enables plants to acclimate to their environment (Chalker-scott,

1999; Ono et al., 1996). N-limitation and its control over flavonoid metabolism require further

exploration; therefore, the focus here will be to examine the role of anthocyanins, a special class of

flavonoids, in Arabidopsis under low-N availability.

3

1.3 Flavonoid metabolism: Anthocyanin biosynthesis

Anthocyanins, a subgroup of flavonoids, are water-soluble pigments that accumulate in plant vacuoles in

response to developmental and environmental signals. Flavonoid metabolism has been well

characterized in different plant species including Arabidopsis thaliana (Shirley et al., 1995) (Figure 1).

Anthocyanin biosynthesis begins with L-phenylalanine entering the phenylpropanoid pathway (Lillo et

al., 2008). The phenylpropanoid pathway is split into ‘early’ and ‘late’ biosynthetic steps (blue & purple

respectively in Figure 1). The ‘early’ enzyme, PHENYLALANINE AMMONIA LYASE (PAL), is the

entry point in the phenylpropanoid pathway. PAL catalyzes the de-amination of phenylalanine to

produce cinnamate and 4-coumarate. The conversion of 4-coumarate to 4-coumaroyl-CoA, by 4-

COUMARATE:CoA LIGASE (4CL) creates the direct precursor to the flavonoid pathway. The

flavonoid pathway begins with the production of chalcones, from 4-coumaroyl-CoA and malonyl-CoA,

catalyzed by CHALCONE SYNTHASE (CHS) (red in Figure 1), and the isomerization of chalcones to

flavonones by CHALCONE ISOMERASE (CHI). Subsequently, flavonones are reduced to

dihydroflavonols, dihydrokaempferol and dihydroquercetin, catalyzed by FLAVONONE 3- β-

HYDROXYLASE (F3H) and FLAVONONE 3’- β-HYDROXYLASE (F3’H) respectively. The

production of dihydroflavonols is a bottleneck, as they are necessary to generate flavonols and (pro)

anthocyanidins. The ‘late’ enzymes in the flavonoid pathway, DIHYDROFLAVONOL 4-REDUCTASE

(DFR), ANTHOCYANIDIN SYNTHASE (ANS), UDP-DEPENDENT FLAVONOID 3-O-

GLYCOSYLTRANSFERASE (UF3GT), and GLUTATHIONE S-TRANSFERASE (GST) determine

anthocyanin production and storage, which is strongly influenced by environmental signals (Lillo et al.,

2008; Scheible et al., 2004; Solfanelli et al., 2006).

4

L-phenylalanine

PAL

Cinnamate

C4H

4-Coumarate

4CL

4-Coumaroyl-CoA Monolignols

CHS

Naringenin chalcone

CHI

Naringenin

F3H

Dihydrokaempferol Kaempferol

F3’H

Dihydroquercitin Quercetin

DFR

Leucocyanidin

ANS

Anthocyanidin

UF3GT

Anthocyanins

Figure 1. The flavonoid biosynthetic pathway, including general phenylpropanoid metabolism.

Early phenylpropanoids produced (black) will be used as substrates for products in the flavonoid

pathway (red). ‘Early’ structural enzymes are indicated in blue, while ‘late’ structural enzymes are

indicated in purple.

NH4+

FLS

FLS

3X Malonyl-CoA

5

1.4 Low nitrogen-induced anthocyanin accumulation: Biological relevance in

Arabidopsis thaliana

During low-N growth conditions, anthocyanins have been shown to accumulate in stems and leaves of

seedlings and adult plants (Bongue-Bartelsman & Phillips, 1995). Anthocyanins accumulate during

early seedling growth as well as during senescence, where it precedes chlorophyll breakdown (Diaz et

al., 2006; Field et al., 2001; Lea et al., 2007). During low N-induced early leaf senescence, nitrogen

assimilation is reduced; as a result, plants concomitantly reduce their photosynthetic efficiency to

minimize the production of organic acids which would normally be utilized towards assimilation (Field

et al., 2001; Fritz et al., 2006). Additionally, by reducing photosynthetic output, plants are vulnerable to

excess light-induced oxidative damage. Anthocyanins attenuate the light absorbing capacity of bound

and free chlorophyll molecules, thereby minimizing the production of reactive oxygen species (Chalker-

scott, 1999; Field et al., 2001; Hoch et al., 2003; Jeong et al., 2010). Anthocyanins also facilitate nutrient

recovery to effectively remobilize nitrogen to younger active regions. Thus, plants that fail to

accumulate anthocyanins may undergo abnormal senescence (Aoyama et al., 2014; Peng et al., 2007a).

In the first step of phenylpropanoid metabolism, PAL produces ammonium as a by-product (Fig1),

which can further be assimilated by the GLUTAMINE SYNTHETASE/GLUTAMATE SYNTHASE

(GS/GOGAT) system to produce amino acids and its derivatives (Singh et al., 1998). Thus, the

production of anthocyanins via the phenylpropanoid/flavonoid pathway recycles nitrogen, which enables

nitrogen to be remobilized and re-utilized during low-N plant response. Perhaps, this may partially

explain why nitrogen abundance is inversely proportional to anthocyanin production (Rubin et al.,

2009). In support of this, accumulation of anthocyanins is regarded as a phenotypic stress marker and an

indicator of nutrient imbalance (Chalker-Scott & Scott, 2004; Chalker-scott, 1999; Diaz et al., 2006;

Rubio et al., 2009). Nevertheless, the potential multi-functionality of anthocyanins and its specific

regulation during low-N availability is still unclear.

6

1.5 Low nitrogen-induced anthocyanin accumulation: Regulation by

transcription factors

Nitrogen abundance indirectly represses structural enzymes within the flavonoid pathway. When

nitrogen is available to plants, members of the LATERAL BOUNDARY DOMAIN (LBD) family of

transcription factors are upregulated (Scheible et al., 2004). LBD37 was shown to repress positive

regulators of the anthocyanin biosynthetic pathway, specifically PRODUCTION OF ANTHOCYANIN

PIGMENT 1 (PAP1) and its homologue PAP2, which belong to the MYB family of transcription factors

(Rubin et al., 2009). The LBD37 gene is strongly induced by nitrate and to a lesser extent by ammonium

(Rubin et al., 2009; Scheible et al., 2004). In contrast, when nitrogen becomes limiting, structural

enzymes within the flavonoid pathway are induced. The ‘early’ genes in the pathway induced by

nitrogen depletion include PAL1, 4CL3, CHS, F3H, F3’H and FLS1. The transcription of ‘late’ genes,

DFR and ANS, are most strongly induced by nitrogen withdrawal (Lillo et al., 2008; Scheible et al.,

2004). Besides structural genes that generate products within the flavonoid pathway, regulatory genes

play a role in governing the pathway, consequently regulating the biosynthesis of anthocyanins. In

Arabidopsis, several transcription factors belonging to the MYB, Basic-Helix-Loop-Helix (bHLH) and

WD-40 like classes of proteins have been implicated in governing the flavonoid pathway (Borevitz,

2000; Broun, 2005; Hichri et al., 2011; Nesi et al., 2000). Among the MYB family of proteins, PAP1,

PAP2, GLABROUS 1 (GL1) activate the pathway; while MYB-LIKE 2 (MYBL2) and CAPRICE

(CPC) negatively feedback on the flavonoid pathway (Gonzalez et al., 2008; Matsui et al., 2008). The

bHLH family of transcription factors that regulate anthocyanin biosynthesis include TRANSPARENT

TESTA 8 (TT8), GLABRA 3 (GL3) and ENHANCER OF GLABRA 3 (EGL3). These bHLH proteins

have a MYB-binding domain and their interaction with MYB partners determines activation/repression

of specific genes within the flavonoid pathway. Finally, the WD-40 protein TRANSPARENT TESTA

GLABROUS 1 (TTG1) has been identified in playing the common denominator for controlling

anthocyanin biosynthesis in Arabidopsis, since it acts upstream of bHLH and MYB transcription factors

7

(Broun, 2005). The MYB-bHLH-WD40 transcription factors form a ternary complex, known as the

MBW complex, to tightly govern the flavonoid pathway, especially the ‘late’ genes controlling

anthocyanin biosynthesis (Winkel-Shirley, 2001). During N-limitation, high anthocyanin levels were

strongly correlated to activation of the PAP1/2-GL3-TTG1 complex (Zhou et al., 2012). Interestingly,

while transcription factors within the same family (for instance, bHLH proteins, EGL3 and GL3) may

play redundant roles in regulating the flavonoid pathway, under specific stress conditions, one member

within the family may dominate over the other in controlling anthocyanin biosynthesis. For instance,

under nutrient replete conditions, the EGL3 gene is expressed at relatively higher levels than GL3.

However, under N-limiting conditions, higher transcript abundance levels were seen for GL3, compared

to EGL3 (Lea et al., 2007). Additionally, GL3 was discovered to have a weak binding affinity to

inhibitors (MYBL2), compared to EGL3, which positively correlated with high anthocyanin levels under

N-depletion (Nemie-Feyissa et al., 2014; Nemie-Feyissa et al., 2015). In subsequent studies in support

of this, gl3 mutants accumulated attenuated anthocyanin levels, compared to wild-type (WT) and egl3

mutants (Feyissa et al., 2009). The direct or indirect interaction between nitrogen and GL3 is still

unclear. Similarly, MYB transcription factors PAP1 and PAP2 both appear to respond to soluble sugars

and nitrate (Lea et al., 2007). However, PAP1 transcription is strongly induced by sucrose under N-

replete conditions, while PAP2 transcription is pronounced during N-withdrawal, in both Arabidopsis

seedlings and rosette leaves (Feyissa et al., 2009; Lea et al., 2007; Scheible et al., 2004). Among other

MYB family members, MYBL2 is largely responsible for negatively regulating the flavonoid pathway

under nutrient replete conditions. Upon N-depletion, CPC appeared to play a more significant role in

negative feedback to disrupt the MBW complex (Nemie-Feyissa et al., 2014); thus controlling

anthocyanin biosynthesis in response to changing nutritional cues. Together, N-deprivation stabilizes the

anthocyanin pool mediated through positive and negative transcriptional regulators.

8

1.6 Low nitrogen-induced anthocyanin accumulation: Regulation by small

metabolites

1.6a Regulation by phytohormones

Plant hormones have previously shown to mediate sucrose-specific induction of anthocyanins in

Arabidopsis seedlings (Das et al., 2012; Jeong et al., 2010; Kwon et al., 2011; Loreti et al., 2008). Light

and a high C/N ratio served as pre-requisites for anthocyanin production; and phytohormones have

shown to either positively (cytokinin, abscisic acid and jasmonate) or negatively (ethylene, gibberellin)

regulate PAP1 transcript abundance, consistent with enhanced and attenuated anthocyanin levels

respectively. By contrast, little is known about the interplay between hormone signalling and low N-

induction of anthocyanins. Ethylene has recently been implicated in suppressing anthocyanin production

under severe N-limiting conditions (Wang et al., 2015). A Root hair defective 3 (rhd3) loss-of-function

mutant was found to over-accumulate anthocyanins under N-deficiency alone, manifested in purple leaf

pigmentation. RHD3 was previously characterized as a transmembrane protein localized to the ER with

GTPase activity, possibly mediating endomembrane trafficking between ER and the Golgi. Interestingly,

ethylene insensitive mutants’ etr1, ein2, and ein3/eil1 had a similar phenotype (enhanced purple

pigmentation) under N-starvation. This suggests that ethylene signalling may negatively feedback to

regulate anthocyanin levels under N-deprivation, and RHD3 may play a partial role in the ethylene

suppression of anthocyanin biosynthesis. Microarray analysis revealed a higher transcript abundance of

GL3 in rhd3 seedlings, compared to WT (Wang et al., 2015). GL3 was previously shown to play a

positive role in N-induced anthocyanin accumulation, over its bHLH competitor EGL3 (Feyissa et al.,

2009; 2014). Furthermore, rhd3 mutants showed lower abundance of ethylene response transcripts,

ERF2 & ERF5. ETHYLENE RESPONSE FACTORS (ERFs) are transcription factors known to activate

ethylene response genes (Solano et al., 1998). In order to strengthen the association between RHD3 and

ethylene response, Arabidopsis seedlings were fed with ethylene precursor, 1-

AMINOCYCLOPROPANE-1-CARBOXYLIC ACID (ACC). Interestingly, ACC managed to diminish

9

purple pigmentation in WT, but not in rhd3 seedlings. This reinforces the assertion that ethylene

suppresses N-induced anthocyanin biosynthesis. Further research is necessary to explore the role of

RHD3 in mediating an ethylene response during N-starvation. Likewise, auxin and cytokinins are

known to regulate the N-starvation response (Kiba et al., 2011); thus, it will be of interest to explore

overlapping hormonal signalling pathways (ethylene ��auxin��cytokinin) that help mediate

nitrogen control over the anthocyanin biosynthetic pathway.

1.6b Regulation by ubiquitin ligases

In Arabidopsis plants, an early response to low-N availability includes cytoplasm-related protein

degradation to facilitate nutrient remobilization (Wang et al., 2012). Cellular protein degradation

controls the stability of structural and signalling components essential in central metabolism. In a

previous study (Peng et al., 2007a), a maladaptive response to low-N conditions was identified using a

forward genetics screen, where the nitrogen limitation adaptation (nla) mutant failed to accumulate

anthocyanins, and plant senescence was accelerated. Once genetically mapped, the nla loss-of-function

mutant corresponded to the NLA gene, which encodes a RING-type E3 ubiquitin ligase. In a genome-

wide study, WT and nla plants were found to differentially regulate 1272 genes under low-N availability

(Peng et al., 2007b). These included genes influencing transcription, protein degradation, redox status,

energy, primary and secondary metabolism, cell wall modification, and signal transduction. During low-

N growth conditions, the failure of nla plants to accumulate anthocyanins was consistent with low

expression profiles of structural genes (CHS, CHI, F3H, DFR, & ANS) in the flavonoid pathway. The

failure to produce abundant anthocyanins may have increased the nla plant’s susceptibility to photo-

damage, which could partially explain accelerated senescence observed in these mutants (Peng et al.,

2007b; Peng et al., 2008). Additionally, anthocyanins facilitate the recovery of nitrogen and other

nutrients during senescence. In the nla mutant, nitrate transport and the transport of other nutrients

(potassium, phosphate, sulfate, and calcium) were differentially regulated compared to WT under low-N

conditions. The poor management of nutrient remobilization may partly explain accelerated leaf

10

senescence in nla plants. It may be that NLA positively regulates the flavonoid pathway by controlling

the stability of proteins that negatively regulate this stress response. In a follow up study (Peng et al.,

2008), suppression of low N-induced anthocyanins in the nla mutant was relieved under combined

nitrogen and phosphate (N-, P-) limiting conditions, similar to WT. So, NLA could possibly play a

unique role in mediating low N-induced anthocyanin production. This suggests that different molecular

components may be activated under varied stressful environments to regulate the flavonoid pathway.

NLA, as a regulator of the flavonoid pathway, and its interaction with nitrogen is yet to be explored. The

Arabidopsis ATL31 gene is another ubiquitin ligase implicated as an essential molecular player in

controlling anthocyanin metabolism during low-N availability. A high C/N (high C/low N) supply can

inhibit post-germinative growth and produce purple pigmentation in Arabidopsis seedlings. A

CARBON/NITROGEN INSENSITIVE 1-DOMINANT (CNI1-D) is an ATL over-expressor line, identified

in a screen for suppressing post-germinative growth inhibition under a high C/low N growth regimen,

and displayed expanded green cotyledons (Sato et al., 2009). Chlorophyll levels must decline to unmask

abundant anthocyanins in the plant vacuole (Chalker-scott, 1999). The failure of cni1-D seedlings to

display purple pigmentation is in support of expressing photosynthetic markers, CHLOROPHYLL A/B-

BINDING PROTEIN (CAB) and RUBISCO (RBCS1-A), along with reduced CHS transcript

accumulation. This was further supported in atl31 loss-of-function seedlings which displayed enhanced

purple pigmentation under a high C/low N treatment. The ATL31 gene encodes a putative RING-type E3

ubiquitin ligase. During seedling establishment under a high C/low N nutritional supply, ATL31 appears

to negatively regulate flavonoid metabolism. It will be of interest to compare ATL31 to NLA during the

low-N response; however to do so, ATL31 transcript mis-regulation must be explored during low N-

induced early leaf senescence. Similar to the first study, ATL31 ox (over-expressor) adult plants failed to

accumulate anthocyanins, compared to WT (Sato et al., 2011). On the other hand, atl31 loss-of-function

line over-accumulated abundant anthocyanins, and similar to nla plants, senescence appeared to be

accelerated. The transcription of ATL31 in WT plants was higher during low N-induced early

senescence, and coincided with high transcript abundance of senescence markers, WRKY53

11

transcription factor and SAG12 (Senescence-associated gene 12). Furthermore, over-expression of

WRKY53 led to increased transcript abundance of ATL31, suggesting a direct relationship between these

two molecular players (Aoyama et al., 2014; Sato et al., 2009). It seems that both NLA and ATL31

regulate early senescence initiated during low-N availability; however unlike NLA, ATL31 appears to

negatively modulate anthocyanin metabolism. Finally, anthocyanin accumulation precedes chlorophyll

breakdown during senescence (Diaz et al., 2006). In contrast to WT, both nla and atl31 ox plants

exhibited a higher chlorophyll content with a concomitant reduction in anthocyanins. These findings

suggest that anthocyanin accumulation and chlorophyll turnover may be tightly regulated during the

low-N stress response.

1.7 Low nitrogen-induced anthocyanin accumulation: Carbon-nitrogen

interactive effects

Numerous studies have demonstrated the induction of anthocyanins by elevated sucrose or N-limitation

alone (Bongue-Bartelsman & Phillips, 1995; Solfanelli et al., 2006). A significant portion of low-N

studies have explored anthocyanin biosynthesis, while disregarding the influence of how the plant C/N

ratio modulates anthocyanin levels, and not C or N alone. Few studies have managed to focus on the

interactive effects of carbon and nitrogen, and emphasize their combined influence on the flavonoid

biosynthetic pathway. Low-N treatment alone is not sufficient to produce anthocyanins in Arabidopsis

seedlings; therefore, sucrose-fed plans must be deprived of nitrogen for proper induction of

anthocyanins in Arabidopsis seedlings (Feyissa et al., 2009; Lea et al., 2007; Martin et al., 2002). At the

transcriptional level, high C/N treatment induced the transcript accumulation of structural enzymes in

the flavonoid pathway including CHS, DFR, ANS, and UF3GT (Feyissa et al., 2009; Martin et al., 2002).

The induction of these structural enzymes correlated with increased transcript abundance for

PAP1/PAP2 and GL3. The carbon-nitrogen interactive effect was also recently explored in crabapple

(Malus sp.), where MYB10, under the control of a high C/N growth treatment, appeared to positively

regulate anthocyanin structural genes (Wan et al., 2015). In this study, a higher C/N ratio additively

12

increased anthocyanin levels in different cultivars of crabapple leaves as well as callus cultures. A

cellular C/N imbalance, particularly high C/low N, was proposed to strongly induce anthocyanin

accumulation. A putative methyltransferase loss-of-function mutant, over-sensitive to sugar 1 (osu1),

accumulated higher levels of anthocyanins in Arabidopsis under high C/low N treatment compared to

WT (Gao et al., 2008). However, under low C/high N treatment, both osu1 and WT Arabidopsis

seedlings failed to accumulate anthocyanins. OSU1 gene is suggested to play an essential role in sensing

cellular C/N balance and signal plant nutrient status. According to Gao et al., OSU1 transcript levels in

WT seedlings were similar during normal growth conditions (high C/high N) as well as under a high

C/low N treatment. Perhaps, post-transcriptional and post-translational regulation could explain the

hypersensitivity of osu1 seedlings to a C/N imbalance (Zheng, 2009).

1.8 Small molecules can dissect part of the nitrogen deprivation response

associated with anthocyanin accumulation

Nitrogen can regulate anthocyanin biosynthesis, and this regulation is mediated by transcription factors

and small metabolites; however, their interaction with nitrogen is still unclear. There still remain gaps in

our knowledge regarding the molecular pathways responsible for inducing anthocyanin biosynthesis in

response to N-deprivation, including local and systemic signalling, transcriptional changes, and post-

transcriptional and post-translational regulation. To name a few, these may include receptors, hormones,

kinases, phosphatases, transcription factors, non-coding RNAs, and metabolic intermediates, which

could play an essential role in regulating flavonoid metabolism in response to altered nitrogen levels.

Classical mutational studies have documented transcription factors and small metabolites in mediating

N-deprivation-induced anthocyanin biosynthesis. An alternate approach can make use of small

molecules to probe molecular pathways by disrupting protein function; consequently affecting

downstream signalling and the accumulation of anthocyanins. Small molecules offer flexibility with

respect to temporal and spatial manipulation of protein targets (Robert et al., 2009). Chemical

application is reversible, and can be applied at lower doses to prevent biological side effects, in order to

13

prevent lethality. Furthermore, small molecules may act as specific agonists or general antagonists to

overcome genetic redundancy (Tóth & van der Hoorn, 2009). The chemical genetics approach, along

with classical genetics, can help dissect the molecular underpinnings of low-N associated anthocyanin

accumulation. The chemical genetics approach has previously been employed to gear plant growth for

improved survival under various biotic and abiotic stresses (Jakab et al., 2012; Schreiber et al., 2008).

Recently, there has been an interest in the use of small molecules to explore nutrient starvation

responses in Arabidopsis (Arnaud et al., 2014; Bonnot et al., 2016).

1.9 Research hypotheses and objectives

This thesis emphasizes the use of small molecules to perturb the accumulation of anthocyanins induced

by N-deprivation. The research conducted aims to provide evidence to address five major hypotheses,

presented in turn below.

(1) A chemical screen will identify small molecules with the capacity to attenuate leaf anthocyanin

content in nitrogen-deprived seedlings.

(2) Chemicals applied at higher concentrations will display a dose-dependent response.

(3) Chemicals will exert their effect during a certain timeframe, at a specific developmental stage.

(4) Chemicals will possess reactive moieties which lend them the capacity to reduce leaf

anthocyanin levels.

(5) Chemical compounds will modify additional N-adaptive responses besides anthocyanin

accumulation.

In order to test the hypotheses stated above, the first objective was to screen a chemical library using an

optimized anthocyanin-inhibition assay. Secondly, candidate chemical compounds were applied at a

range of concentrations, as well as applied at multiple time intervals. Thirdly, chemical analogues were

14

used to reduce structural complexity. Finally, alterations to additional morphological and biochemical

changes associated with N-deprivation were assessed.

15

Chapter 2

16

Materials & Methods

2.1 Plant material and growth conditions

Wild-type (WT) Arabidopsis thaliana (Arabidopsis, ecotype Columbia) seeds were obtained from the

Nottingham Arabidopsis Stock Centre. Surface-sterilized seeds (6-8) were distributed in 96-well

microtitre plates containing 200 µL of liquid growth media. Liquid growth medium consisted of

modified N-free ½ strength MS (Murashige & Skoog, 1962), available as MSP21

(http://www.caissonlabs.com/product.php?id=617). Additionally medium contained 0.05% MES buffer

{2-((N-morpholino) ethanesulfonic acid)}, 10 mM sucrose (3.42 g/L), 0.1% Gamborg’s Vitamins (myo-

inositol, nicotinic acid, pyridoxine hydrochloride, thiamine hydrochloride), adjusted to pH 5.7. All plant

materials were grown in Conviron growth cabinets at 21°C in a 16-h/8-h photoperiod (130 µmol m-2 s-1).

2.2 Optimize growth conditions for nitrogen-deprived seedlings

In order to deprive seedlings of nitrogen, growth conditions in microtitre plates were optimized. A range

of ammonium nitrate concentrations (0-10 mM) in liquid media were tested to limit seedling growth and

stimulate primary root growth, chlorophyll breakdown and anthocyanin biosynthesis. Seedlings grown

in 1 mM ammonium nitrate (NH4NO3) for a period of seven days exhibited all of the symptoms

mentioned above, without severely limiting plant growth and development, and preventing excessive

bleaching (chlorosis). On this basis, a moderately high C/N ratio of 5:1(10 mM sucrose: 2 mM nitrogen)

served as the N-deprivation treatment during subsequent chemical screens.

2.3 Chemical library

ChemBridge is a diverse-oriented library provided by the ChemBridge corporation in San Diego, CA,

United States (http://www.chembridge.com/screening_libraries/diversity_libraries/index.php). The

ChemBridge chemical stock plates were setup by the Centre for the Analysis of Genome Evolution

17

& Function (CAGEF) at the University of Toronto. Chemicals were dissolved in dimethylsulfoxide

(DMSO) to a stock concentration of 2.5 mM (Katrina Hiiback, PhD student, Campbell lab, University of

Guelph).

2.4 Chemical screen: primary and secondary

During the initial chemical screen, chemicals were distributed in individual micro-wells within a 96-well

microtitre plate; therefore, each micro-well would represent a unique chemical environment. Chemicals

at 2.5 mM in DMSO were diluted in liquid media to a final concentration of 25 µM (4µL in 400µL of

liquid media), following protocols from previous studies (Schreiber et al., 2008; Stokes et al., 2013). For

control conditions, an aliquot of 1% DMSO (4µL in 400µL of liquid media), equal to the volume of the

compound, was added to the liquid medium. Medium was used to germinate 6-8 Arabidopsis seeds in

each micro-well of a 96-well microtitre plate. In order to release seed dormancy, seeds in microtitre

plates were stratified at 4°C in the dark for four days. Following seed stratification, microtitre plates

were placed under growth conditions (light, temperature) to accelerate seed germination. After two days

of growth under germination-promoting conditions, germinated seeds were rinsed repeatedly to remove

any trace amounts of chemicals, subsequently seedlings were placed in treatment medium (control or N-

depleted) for seven days. The developmental stage at which germinated seeds were rinsed corresponds

to radicle emergence. Following seven days of post-germinative growth in N-deprived conditions,

seedlings were assessed phenotypically. The entire chemical library was screened twice (2X ~4183) to

identify chemicals that appeared to relieve N-deprivation symptoms, such as anthocyanin accumulation

and chlorosis, in Arabidopsis seedlings. A chemical-induced phenotype was validated in secondary

screens, resulting in four chemical compounds discovered (Results, Table 1). All subsequent tests

performed on the four candidate chemical compounds were conducted in 24-well microtitre plates to

avoid nutrient depletion and excessive plant over-crowding.

18

2.5 Dose-response curves and statistical analysis

Four candidate chemical compounds (Results, Table 1) and their respective analogues (Results, Tables

2-5) were tested at an increasing range of concentrations (0, 0.05, 1, 2, 3, 4, 5, 10, 25, 50, 75, 100 µM).

The compounds were applied at different concentrations in separate micro-wells in triplicates (3 micro-

wells designated per compound per concentration). Similar to a primary screen, chemical-treated

seedlings were rinsed after six days, followed by seven days of growth under N-deprivation. Dose-

response curves and statistical analysis were generated using GraphPad Prism 6 (Graphing & Statistical

software). One-way and two-way ANOVA (Tukey’s B test, p < 0.05) tests were used to analyze all

scientific data.

2.6 Chemical analogues (derivatives)

Analogues for all four chemical compounds were selected by substituting atoms within substructures.

Chemical structure modifying tools on ChemMine (http://chemmine.ucr.edu/myCompounds/) and

Hit2lead (http://www.hit2lead.com/search.asp) helped select chemical analogues according to their

availability.

2.7 Anthocyanin and chlorophyll quantification

Plant tissue, with a fresh weight of 18-30 mg, was harvested and stored in -80°C. Subsequently, frozen

tissue was ground in liquid nitrogen using a mortar and pestle. An anthocyanin extraction protocol

(modified by Martin et al., 2002) was adopted, using methanol in 1% HCl as the extraction solvent.

Extraction solvent and distilled water were added at a ratio of 3:2, to form a methanol-water solution.

Finally, chloroform was added to the methanol-water solution in a 1:1 ratio; followed by mixing and

centrifuging at 15,000 rpm for 5 minutes. The supernatant was used for measuring anthocyanin

absorbance at 530 nm and 657 nm using a NanoDrop 1000 Spectrophotometer. Relative anthocyanin

concentrations were calculated as A530 minus A657 to eliminate chlorophyll absorbance. Values were

19

normalized according to fresh weight. Chlorophyll quantification required frozen ground tissue to be

dissolved in 96% ethanol and centrifuged. The supernatant was used to measure total chlorophyll

absorbance at A665 and A649 using modified calculations by Lichtenthaler & Wellburn, 1983.

2.8 Primary root length measurement

Following seven days of post-germinative growth under N-deprivation, or under control conditions,

seedlings were vertically plated and photographed. ImageJ software (US National Institutes of Health,

Bethesda, MD, USA) was used to measure primary root length for DMSO and chemical-treated

seedlings. Average primary root length was calculated from three independent replicates on 18 plantlets.

Lateral root length and number were not examined due to difficulties in accurately splitting lateral roots

from the main primary root of drenched-seedlings (grown in liquid media).

2.9 Optimize growth conditions for phosphate-deprived seedlings

In order to deprive seedlings of phosphorous, growth conditions in microtitre plates were optimized. A

range of dipotassium phosphate concentrations (0-0.5 mM) in liquid media were tested to limit seedling

growth and stimulate anthocyanin biosynthesis. Seedlings completely deprived of phosphate (0 mM

K2HPO4) for a period of seven days displayed abundant anthocyanins.

20

Chapter 3

Section 1

21

Results & Discussion

3.11 An introduction to the fundamental principles of chemical genetics

The aim was to expand our scientific understanding of anthocyanin induction by N-deprivation in

Arabidopsis seedlings. In order to dissect the molecular underpinnings of low N-induced anthocyanin

biosynthesis by implementing a chemical genetics approach, certain principles must be considered

(Robert et al., 2009; Tóth & van der Hoorn, 2009). The first phase of a chemical genetics project

maintains that a robust screening assay must be established, prior to screening a chemical library. Once a

screening assay for anthocyanin inhibition has been optimized, the second phase of a chemical genetics

project, the chemical screen, must follow. Candidate chemical compounds identified and subsequently

verified in secondary screens enter the third phase of chemical genetics project, to examine and

characterize a chemical-induced phenotype. The final phase revolves around identifying protein targets

as well as downstream consequences of chemical perturbation, eventually resulting in attenuated

anthocyanin levels.

22

3.12 Setup a robust screening assay: Anthocyanins accumulated under a

moderately high C/N growth treatment

Growth conditions must be optimized for anthocyanins to accumulate during early seedling growth and

development. Arabidopsis (Col0) seeds were plated in liquid growth medium (half-strength modified

Murashige & Skoog) for ease of manipulating nitrogen concentrations. Seeds in individual wells were

plated in 24 and 96-well microtitre plates for the second phase of chemical genetics, which entails high-

throughput chemical screening. Analogous to several published studies which investigated anthocyanin

accumulation in Arabidopsis seedlings under N-deficiency (Aoyama et al., 2014; Feyissa et al., 2009;

Gao et al., 2008), young seedlings were allowed to develop in the presence and absence of sucrose under

low-N conditions (Figure 2a). It is interesting to observe that anthocyanins only accumulated under low-

N growth conditions when co-treated with exogenous sucrose (Figure 2a-b). Furthermore, this assay

established that exogenous sucrose treatment alone is not sufficient to induce anthocyanin production,

rather a moderately high carbon: nitrogen (C/N) growth treatment must be maintained for anthocyanins

to accumulate. As expected, exogenous sucrose treatment increased shoot and root biomass under both

nitrogen treatments (data not shown).

23

High N Low N

Figure 2a. Red pigmentation visible in seedlings grown under a moderately high C/N treatment

High nitrogen and low nitrogen conditions are indicated vertically, while absence/presence of exogenous

sucrose treatment is indicated horizontally. Each well contains 6-8 seedlings grown in liquid medium,

within a 24/96-well microtitre plate. Presence of exogenous sucrose treatment produced red

pigmentation only when nitrogen was limited (high C/N).

Sucrose

Without

Sucrose

24

Figure 2b. Anthocyanins accumulated under a moderately high C/N treatment

High (N+) and low nitrogen (N-) conditions are indicated as green and red respectively. Absence of

sucrose treatment failed to induce anthocyanins under high and low nitrogen conditions. Presence of

exogenous sucrose treatment induced anthocyanin accumulation only when nitrogen was limited (high

C/N). All samples were tested in triplicates (n=3). Statistical analysis was performed using ANOVA

(Tukey’s B test, p < 0.05). Errors bars indicate standard deviation (SD).

25

3.13 Screen chemical library: Identified small molecules in an anthocyanin-

inhibition assay

In this study, the ChemBridge library was screened for small molecules in an anthocyanin-inhibition

assay during early seedling development. ChemBridge is a diverse-oriented library, consisting of

thousands of natural and synthetic compounds, created using combinatorial chemistry techniques

(Robert et al., 2009). In a 96-well microtitre plate, each micro-well contained Arabidopsis seeds, plated

in liquid media, and imbibed in a specific chemical compound. Following seed radicle protrusion, which

marks the completion of germination, seeds were rinsed repeatedly to remove any trace amounts of

chemical left behind, and a subsequent high C/N (5:1) growth treatment was applied for a period of

seven days. Following this protocol, chemical compounds were scored positive based on the lack of red

pigmentation (anthocyanin-inhibition) visible on seedling cotyledon and upper hypocotyl region

(Figures 3, 4a). Four chemical compounds tested positive for their ability to attenuate anthocyanin levels

in N-deprived Arabidopsis seedlings. The chemical compounds appeared to significantly reduce

anthocyanin levels during low-N availability (Figure 4b). These were verified in a secondary screen

conducted as outlined above. Table 1 lists each of the four candidate compounds categorized into three

classes/groups (benzimidazole, benzoxazole and carbazole), according to their structural backbone.

26

Figure 3. Chemical library screened in a microtitre plate under low nitrogen growth conditions

(high C/N)

Seedlings pre-treated with DMSO and subsequently grown under high (N+) or low (N-) nitrogen growth

conditions are indicated vertically at each end of the microtitre plate. The rest of the microtitre plate

contains seedlings in each micro-well, pre-treated with a different chemical compound. Each micro-well

contains 6-8 seedlings grown in liquid medium, where seeds were initially imbibed in a unique chemical

environment. Chemical-treated seedlings were scored positive for their lack of ability to accumulate

anthocyanins under nitrogen deprivation (red square). Seeds were imbibed in DMSO/chemical for 6

days, subsequently germinated seeds and young seedlings were grown under low-N for a period of 7

days.

DM

SO

p

re-t

reat

men

t

Hig

h N

(N

+)

Seedlings pre-treated with each chemical compound

under low N levels (N-) DM

SO

p

re-t

reat

men

t

Lo

w N

(N

-)

27

DMSO

High N (C:N, 1:2)

DMSO

Low N (C:N, 5:1)

Post-chemical

treatment

Low N (C:N, 5:1)

A

B

C

28

Figure 4a. Lack of red pigmentation visible on seedlings grown under low nitrogen levels (high

C/N)

High nitrogen conditions represent a C:N ratio of 1:2 (10 mM sucrose: 20 mM nitrogen). In contrast, a

low nitrogen growth condition represents a C:N ratio of 5:1 (10 mM sucrose: 2 mM nitrogen). Seedlings

grown under high N conditions failed to display anthocyanin pigmentation (A). Seedlings grown under

low nitrogen conditions exhibited anthocyanin (red) pigmentation on the cotyledon as well as in the

upper hypocotyl region (B). Finally, seedlings pre-treated with a candidate compound failed to

accumulate anthocyanins, in support of lack of red pigmentation (C).

29

Figure 4b. Nitrogen-deprived seedlings, pre-treated with candidate chemical compounds, failed to

accumulate anthocyanins

High (N+) and low (N-) nitrogen conditions are indicated in green and red respectively, to represent

absence and presence of anthocyanin pigmentation. DMSO pre-treated seedlings exhibited an abundance

of anthocyanins under low nitrogen levels. In contrast, seedlings pre-treated with any of the four

candidate chemical compounds (Benzimidazole, Benzoxazole-1, Benzoxazole-2, Carbazole) failed to

display red anthocyanin pigmentation during low nitrogen availability. All samples were tested in

triplicates (n=3). Statistical analysis was performed using ANOVA (Tukey’s B test, p < 0.05). Errors

bars indicate SD.

30

Table 1. Structures of candidate compounds in the ChemBridge library scored for their ability

to attenuate anthocyanin levels in seedlings grown under nitrogen deficiency. The table

lists chemical name, chemical formula, structure, and concentration at which the

compound was applied during early screening of the library. The candidate compounds

have been categorized into three classes/groups, according to their structural backbone.

Chemical

Conc.

Applied

Compounds identified Class/Group

2-(3-nitrophenyl)-1H-

benzimidazole

(C13H9N3O2)

25 µM

Benzimidazole

3-(1,3-benzoxazol-2-

yl)-7-

(diethylamino)-

2H-chromen-2-

one

(C20H18N2O3)

25 µM Benzoxazole-1

31

Table 1. continued

Chemical

Conc.

Applied


1-[3-(1-benzofuran-2-

yl)-1H-pyrazol-

4-yl]-N-(1,3-

benzoxazol-2-

ylmethyl)-N-

methylmethanam

ine

(C21H18N4O2)


2-{4-[(9-ethyl-9H-

carbazol-3-

yl)methyl]-1-

methyl-2-

piperazinyl}etha

nol

(C22H29N2O)

25 µM Carbazole

32

3.14 Chemical compounds attenuated anthocyanin levels irrespective of

inorganic nitrogen source (ammonium vs. nitrate)

Low nitrate induction of anthocyanins, and conversely, nitrate repression of anthocyanins in Arabidopsis

have received attention in the past (Lea et al., 2007; Lillo et al., 2008; Nemie-Feyissa et al., 2014; Rubin

et al., 2009; Scheible et al., 2004). In contrast, fewer Arabidopsis studies have explored the regulation of

anthocyanin biosynthesis by varying ammonium levels in the environment (Rubin et al., 2009; Zhou et

al., 2012). In the present study, all four chemical compounds (Table 1) were tested for their ability to

reduce anthocyanin levels under two separate N-deprivation treatments, nitrate and/or ammonium.

Arabidopsis seeds, pre-treated with each of the four compounds, developed into young seedlings under

different low-N growth conditions (ammonium chloride or potassium nitrate). Strikingly, all four

compounds significantly attenuated anthocyanin levels under low-ammonium or low-nitrate availability

(Figures 5a-b). This finding ruled out the possibility of compounds exerting an effect under a

preferential nitrogen source (nitrate or ammonium). Furthermore, during low-N availability (N-), when

comparing anthocyanin abundance in DMSO-treated seedlings under a single nitrogen source, to

seedlings under combined nitrogen sources (ammonium nitrate), it was interesting to observe that

anthocyanin accumulation was heightened when one source of nitrogen was removed (Figures 5a-b, red

bar vs checkered/diagonal red bar). In other words, the presence of ammonium and nitrate combined

during N-withdrawal appeared to additively repress anthocyanin production in Arabidopsis seedlings.

This effect was not achieved with abundant nitrogen (N+), possibly due to high levels of either nitrogen

source fully capable of suppressing anthocyanin production. The effect was only visible during N-

withdrawal because seedlings are highly sensitive to nitrogen, particularly when it becomes limiting.

Nevertheless, the molecular mechanism underlying the ammonium regulation of anthocyanin

biosynthesis in Arabidopsis seedlings and adult plants should receive further attention in the future.

33

Figure 5a. Seedlings pre-treated with chemical compounds accumulate attenuated anthocyanin

levels under low ammonium growth conditions

DMSO treated seedlings under high (N+) or low (N-) ammonium nitrate growth conditions are indicated

in green and red respectively. DMSO treated seedlings under high (N+) and low (N-) ammonium

chloride (control) growth conditions are indicated in checkered green and red bars respectively. DMSO

pre-treated seedlings exhibited an abundance of anthocyanins under low ammonium nitrate levels.

Furthermore, anthocyanin production was heightened under low nitrogen conditions, particularly when

ammonium was the only nitrogen source available (checkered red). In contrast, ammonium-deprived

seedlings, pre-treated with any of the four chemical compounds (Benzimidazole, Benzoxazole-1,

Benzoxazole-2, Carbazole), displayed significantly attenuated anthocyanin levels. All samples were

tested in triplicates (n=3). Statistical analysis was performed using ANOVA (Tukey’s B test, p < 0.05).

Errors bars indicate SD.

34

Figure 5b. Seedlings pre-treated with chemical compounds accumulate attenuated anthocyanin

levels under low nitrate growth conditions

DMSO treated seedlings under high (N+) or low (N-) ammonium nitrate growth conditions are indicated

in green and red respectively. DMSO treated seedlings under high (N+) and low (N-) potassium nitrate

(control) growth conditions are indicated in diagonal green and red bars respectively. DMSO pre-treated

seedlings exhibited an abundance of anthocyanins under low ammonium nitrate levels. Furthermore,

anthocyanin production was heightened under low nitrogen conditions, particularly when nitrate was the

only nitrogen source available (diagonal red). In contrast, nitrate-deprived seedlings, pre-treated with

any of the four chemical compounds (Benzimidazole, Benzoxazole-1, Benzoxazole-2, Carbazole),

displayed significantly attenuated anthocyanin levels. All samples were tested in triplicates (n=3).

Statistical analysis was performed using ANOVA (Tukey’s B test, p < 0.05). Errors bars indicate SD.

35

Chapter 3

Section 2

36


3.21 Introduction: Establish biological characteristics of a small molecule

Small bioactive molecules have the capacity to perturb biological processes by disrupting a well

characterized phenotype (Robert et al., 2009). Such compounds are likely to vary in bioactivity with

respect to the time frame in which the chemical is applied, and the developmental stage at which this

application occurs. Compounds may also disrupt a phenotype for a certain period of time, and under a

range of tested concentrations; therefore, testing such parameters define characteristics for each

chemical compound. The third stage of a chemical genetics project includes characterizing a compound-

induced phenotype (Tóth & van der Hoorn, 2009).

3.22 Early chemical treatment generated a dose-dependent anthocyanin

inhibition response

During initial screening of the ChemBridge library, all compounds were tested at a baseline

concentration of 25 μM, from which four chemical compounds were identified (Table 1). These four

chemicals were individually tested at a range of concentrations to establish the optimal concentration at

which the chemical generates the strongest inhibition of anthocyanins. According to Figures 6a-d, all

four chemical compounds exerted an effect in a dose-dependent manner, by proportionally decreasing

seedling anthocyanin content with gradual increases in compound concentration. It is interesting to

observe that all four compounds demonstrated potency at much lower concentrations than initially

screened at 25 μM. These results suggest that anthocyanin regulation is very sensitive to compound

application in young Arabidopsis seedlings. Compound effect (benzimidazole, benzoxazole-2, and

carbazole) appeared to plateau around 50 μM, with no further suppression of anthocyanins; except for

benzoxazole-1 response which demonstrated highest potency at 25 μM itself.

37

Figure 6a. Benzimidazole applied at a range of concentrations proportionally attenuated

anthocyanin levels in nitrogen-deprived seedlings

38

Figure 6b. Benzoxazole-1 applied at a range of concentrations proportionally attenuated


39

Figure 6c. Benzoxazole-2 applied at a range of concentrations proportionally attenuated


40

Figure 6d. Carbazole applied at a range of concentrations proportionally attenuated anthocyanin

levels in nitrogen-deprived seedlings

41

Figure 6a-d. Compound (benzimidazole, benzoxazole-1, benzoxazole-2, or carbazole) applied at

increasing concentrations proportionally decreased anthocyanin levels in nitrogen deprived

seedlings

DMSO-treated seedlings under low nitrogen conditions (N-) exhibited an abundance of anthocyanins

(equivalent to no chemical applied). In contrast, nitrogen-deprived seedlings, pre-treated with increasing

concentrations of compound (benzimidazole, benzoxazole-1, benzoxazole-2, or carbazole),

proportionally attenuated anthocyanin levels. Additionally, compound appeared to be potent at much

lower concentrations, compared to the initial concentration at which it was applied (25 μM). Finally, the

optimal chemical concentration appeared to be around 50 μM after which the response plateaued, except

benzoxazole-1 which appeared to plateau after 25 μM itself. All samples were tested in triplicates (n=3).

Data represent mean values +/- SD.

42

3.23 Chemical application was critical during seed radicle emergence

During initial screening of the chemical library, small molecules were applied for four days of

stratification (dark), followed by two days of germination (light), a total period of six days (see

Materials & Methods). Chemical application was restricted to six days, as prolonged application would

generate significant reductions in plant growth, displaying stunted shoot and root growth (data not

shown). In this period of six days, it was necessary to identify the critical “time window” of chemical

application responsible for subsequently attenuating anthocyanin levels during low-N availability.

Simply put, the critical period of chemical exposure within the six day period must be identified to

understand the developmental timeframe in which the chemical exerts its effect. Chemicals were applied

at six different time intervals (Figure 7a). These time intervals included exposure during stratification

alone (-4:0), exposure during germination alone (-2:-1, -1:0), as well as exposure during the

stratification-germination overlap (-6:0, -5:0). In this experimental setup, the original time interval (-6:0)

served as a reference point. Except for benzimidazole treatment, all three compounds (benzoxazole-1,

benzoxazole-2, and carbazole) attenuated anthocyanin levels at each of the six time intervals.

Benzimidazole treatment must absolutely occur during seed stratification-germination overlap (-5:0), or

during the second day of seed germination alone (-1:0), to have an impact on seedling anthocyanin

content under N-deprivation (Figure 7b). All four compounds applied on the second day of seed

germination (-1:0) strongly attenuated anthocyanin levels in N-deprived seedlings, comparable to

control time point (-6:0) (Figures 7b-e). The second day of seed germination under optimal growth

conditions (light, temperature) corresponds to radicle emergence, a developmental stage highly sensitive

to environmental cues. Thus, one day of chemical exposure during seed radicle protrusion was sufficient

to strongly attenuate anthocyanin levels during subsequent low-N growth conditions.

43

Figure 7a. Illustration of chemical applied at six different time points to identify the critical period

of exposure

Seeds were pre-treated with chemicals at six different time intervals; some of which include

stratification alone (-4:0), germination alone (-2:-1, -1:0), or stratification-germination overlap (-6:0, -

5:0). Here, the original time interval (-6:0) served as a control time point.

44

Figure 7b. Benzimidazole application at six different time points to identify the critical period of

exposure

45

Figure 7c. Benzoxazole-1 application at six different time points to identify the critical period of

exposure

46

Figure 7d. Benzoxazole-2 application at six different time points to identify the critical period of

exposure

47

Figure 7e. Carbazole application at six different time points to identify the critical period of

exposure

48

Figure 7b-e. Chemical application at six different time points to identify the critical period of

exposure

Seeds pre-treated with DMSO and compound (benzimidazole, benzoxazole-1, benzoxazole-2, or

carbazole) were tested at six different time intervals. These time intervals include stratification alone (-

4:0), germination alone (-2:-1, -1:0), or stratification-germination overlap (-6:0, -5:0). Here, the original

time interval (-6:0) served as a control, indicated to the left (checkered). It is apparent that one day of

chemical exposure during germination (-1:0) was sufficient to reduce anthocyanin levels during low

nitrogen availability. Furthermore, during seed germination, it is the second day of chemical exposure (-

1:0), and not the first (-2:-1), which generated a low anthocyanin phenotype (indicated by green arrow).

This developmental time point (-1:0) corresponds to radicle protrusion. All samples were tested in


bars indicate SD.

49

3.24 Small molecules attenuated anthocyanin levels during early seedling

growth

Nitrate-mediated regulation of anthocyanin biosynthesis in Arabidopsis seedlings has been well

documented (Nemie-Feyissa et al., 2015; Aoyama et al., 2014; Feyissa et al., 2009; Rubin et al., 2009;

Sato et al., 2009; Lea et al., 2007; Scheible et al., 2004). Many studies vary in their measurement of

anthocyanin levels in Arabidopsis seedlings. While some studies have examined anthocyanin content

immediately following germination (1-3 days), others did so after the first true leaves had emerged (1-2

weeks post-germination). In the present study, low N-induction of anthocyanins was suppressed in

chemical-treated seedlings. Seedling anthocyanin content was measured after seven days of growth

under N-deprivation, which corresponds to day 7 post-germination. It was of interest to determine

whether anthocyanin levels were attenuated in N-deprived seedlings throughout early seedling growth,

or whether this phenotype was visible on day 7 alone. Seedling anthocyanin content was measured

during 11 days of N-limiting growth conditions. The experiment could not be conducted beyond 11 days

due to seedling overcrowding in the microtitre plates. DMSO-treated seedlings under low-N conditions

exhibited high anthocyanin levels relatively early, at day 3 of N-deprivation, and persisted throughout

the duration of the experiment, up until day 11 (Figures 8a-d). In contrast, a significant reduction in

anthocyanin levels was observed early on during post-germinative growth in chemical treated seedlings.

This was followed by a slight increase in anthocyanin levels after day 7 up until day 11; however

anthocyanin content continued to remain attenuated during this period. Notably, benzoxazole-2

demonstrated a special case, as anthocyanin suppression was relieved on days 8-9 of N-deprivation,

displaying high anthocyanin content similar to DMSO-treated seedlings (Figure 8c). However, this spike

in anthocyanin content was transient, as benzoxazole-2 treated seedlings continued to mildly suppress

anthocyanin levels on days 10-11 of N-deprivation. Thus, early chemical exposure reduced anthocyanin

content in N-deprived seedlings, and this altered phenotype persisted throughout early seedling growth.

As anthocyanins play a crucial role during early senescence (Diaz et al., 2006), it will be interesting to

50

examine leaf anthocyanin content of chemical-treated plants at later stages of development, and the

consequences of low anthocyanin levels on nutrient recovery.

51

Figure 8a. Anthocyanins measured during post-germinative growth in DMSO/benzimidazole

treated seedlings

52

Figure 8b. Anthocyanins measured during post-germinative growth in DMSO/benzoxazole-1

treated seedlings

53

Figure 8c. Anthocyanins measured during post-germinative growth in DMSO/benzoxazole-2

treated seedlings

54

Figure 8d. Anthocyanins measured during post-germinative growth in DMSO/carbazole treated

seedlings

55

Figure 8a-d. Attenuated anthocyanin levels persisted during early seedling growth in Arabidopsis

seedlings

Seeds pre-treated with DMSO under high (N+) or low (N-) conditions were analyzed for anthocyanin

content during early seedling growth, indicated in green and red respectively. DMSO-treated seedlings

(red) not only maintained high anthocyanin levels on day 7, but did so throughout post-germinative

growth, until day 11 of nitrogen deprivation. In contrast, chemical suppression of anthocyanins was

observed earlier in post-germinative growth, as early as day 3, with a slight increase seen after day 7, up

until day 11. Benzoxazle-2 represents a special case: it transiently lost its effect from days 8-9 of

nitrogen deprivation, and regained it from days 10-11. Therefore, it was apparent that early chemical

exposure can attenuate anthocyanin levels in nitrogen-deprived seedlings throughout early seedling

growth (1-1.5 weeks post-germination). All samples were tested in triplicates (n=3). Statistical analysis

was performed using ANOVA (Tukey’s B test, p < 0.05). Errors bars indicate SD.

56

Chapter 3

Section 3

57


3.31 Establish structure-activity relationship

The last phase of a chemical genetics project focusses on identifying a protein target (Robert et al.,

2009; Tóth & van der Hoorn, 2009). In order to identify a protein target and fill gaps in our current

knowledge of signalling networks, it is important to develop a deeper understanding of the structural

properties of a chemical compound, and the manner in which a chemical may interact with its target(s).

That is, it is essential to identify structural moieties responsible for exerting the chemical-suppression of

anthocyanins. Active and inactive analogues help define and characterize chemical substructures,

thereby reducing structural complexity. Ultimately, chemical derivatives help bridge the gap between

compound structure and function (Robert et al., 2009; Tóth & van der Hoorn, 2009).

3.32 Analogues: Nomenclature and anthocyanin inhibition screen

Numerous analogues (ChemBridge) were tested for their influence on anthocyanin accumulation in N-

deprived seedlings. The compound derivatives were designated as: letter-parent compound (A-

benzimidazole, etc.), listed in Tables 2-5. Analogues were selected by substituting central atoms in the

compound backbone, as well as replacing or repositioning substituent R groups. Arabidopsis seeds, pre-

treated with each chemical analogue at 25 µM, were subsequently subjected to N-limiting growth

conditions. Chemical analogues that appeared to mildly suppress anthocyanin levels were selected for

re-testing at higher concentrations (50-100 µM), to observe whether an analogue could strongly suppress

anthocyanin levels with the same potency as the parent compound. Finally, seedling anthocyanin

inhibition (or lack of) helped determine the status of each chemical analogue, as “active” or “inactive”.

58

3.33 2-(3-nitrophenyl)-1H-benzimidazole activity required a central

imidazole and a nitro side group to strongly attenuate anthocyanins

Six benzimidazole derivatives (Table 2, A-, B-, CA-, DA-, E-, and F-) had varied anthocyanin

responses in N-deprived seedlings (Figure 9a). A-benzimidazole contains an additional methyl group,

and had higher potency over parent compound, benzimidazole. Repositioning of the nitro group in B-

benzimidazole mildly relieved anthocyanin suppression. CA- and DA-benzimidazole are chemical

analogues of the parent compound and A-benzimidazole, as they retain a methyl group. Similar to B-

benzimidazole, repositioning the nitro group in CA-benzimidazole resulted in weaker suppression of

anthocyanins, reinforcing the spatial significance of the nitro group, and the importance of nitrophenyl

in strongly suppressing anthocyanins. Furthermore, B- and CA-benzimidazole applied at higher doses

(50-100 µM) failed to lower anthocyanin levels to the same extent as the parent compound (figures 9b-

c). Finally, replacing the nitro group with aniline resulted in complete loss of anthocyanin suppression,

where F-benzimidazole displayed high anthocyanin levels comparable to DMSO control. F-

benzimidazole activity suggests that the presence of a nitro group takes precedence over its spatial

position, as repositioning the nitro group managed to weakly suppress anthocyanins. DA-and E-

benzimidazole substitute the central N atom with an oxygen atom, converting benzimidazole to a

benzoxazole. Interestingly, this modification in the central backbone of the compound resulted in

complete loss of phenotype, failing to attenuate seedling anthocyanin content. Thus, active (A-, B-, CA)

and inactive (DA-, E-, F-) analogues demonstrated the importance of a central imidazole backbone and a

nitro side group in strongly suppressing seedling anthocyanin content under low-N availability.

59

Table 2. Structures of benzimidazole analogue compounds scored for their ability to attenuate

anthocyanin levels in seedlings grown under nitrogen deficiency. The analogues have been

categorized as A-Benzimidazole, B-Benzimidazole, C-Benzimidazole etc.

Chemical

Conc.

Applied


2-(3-nitrophenyl)-1H-

benzimidazole

(C13H9N3O2)

25 µM

Benzimidazole

Parent compound

5-methyl-2-(3-

nitrophenyl)-1H-

benzimidazole

(C14H11N3O2)

25 µM A-Benzimidazole

5-nitro-2-phenyl-1H-

benzimidazole

(C13H9N3O2)

25 µM

B-Benzimidazole

60

Table 2. Continued

Chemical

Conc.

Applied


5-methyl-2-(4-

nitrophenyl)-1H-

benzimidazole

(C14H11N3O2)

25 µM CA-Benzimidazole

5-methyl-2-(4-

nitrophenyl)-1,3-

benzoxazole

(C14H10N2O3)

25 µM DA-Benzimidazole

2-(4-nitrophenyl)-1,3-

benzoxazole

(C13H8N2O3)

25 µM

E-Benzimidazole

3-(1H-benzimidazol-

2-yl)aniline

(C13H11N3)

25 µM

F-Benzimidazole

61

Figure 9a. Benzimidazole (parent) and benzimidazole-analogues (A-, B-, etc.) exhibited varying

anthocyanin levels in nitrogen-deprived seedlings. DMSO-treated seedlings under high (N+) and low

(N-) nitrogen conditions are indicated in green and red respectively. Benzimidazole analogues are

alongside their parent compound (benzimidazole) to compare anthocyanin attenuation capacity.

Analogue (A-) continued to strongly reduce anthocyanin levels under low nitrogen conditions, while

derivatives (B-and CA-) mildly suppressed anthocyanin levels. All samples were tested in triplicates

(n=3). Statistical analysis was performed using ANOVA (Tukey’s B test, p < 0.05). Errors bars indicate

SD.

62

Figure 9b. Benzimidazole-analogue (B-) failed to strongly attenuate anthocyanins. Benzimidazole

analogues which mildly suppressed anthocyanin levels in a primary screen were selected for re-

screening under higher concentrations. Under higher concentrations (50-100 µM), B-analogue failed to

strongly suppress anthocyanin levels comparable to parent compound. All samples were tested in


bars indicate SD.

63

Figure 9c. Benzimidazole-analogue (CA-) failed to strongly attenuate anthocyanins. Benzimidazole

analogues which mildly suppressed anthocyanin levels in a primary screen were selected for re-

screening under higher concentrations. Under higher concentrations (50-100 µM), CA-analogue failed

to strongly suppress anthocyanin levels comparable to parent compounds, Benzimidazole and A-

benzimidazole. All samples were tested in triplicates (n=3). Statistical analysis was performed using

ANOVA (Tukey’s B test, p < 0.05). Errors bars indicate SD.

64

3.34 3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one activity

required a central oxazole-chromene backbone to strongly attenuate

anthocyanins

Four benzoxazole-1 derivatives (Table3, A-, B-, C, and D-) had varied anthocyanin responses in N-

deprived seedlings (Figure 10a). A- and B-benzoxazole-1 analogues have substituted oxygen with

nitrogen and a sulfur atom, respectively. This conversion of benzoxazole to benzimidazole (A-) and

benzothiazole (B-) resulted in complete loss of anthocyanin suppression (Figure 10a), emphasizing the

importance of a central oxazole backbone in the chemical inhibition of anthocyanins.

C- and D-benzoxazole-1 derivatives contain a benzene ring and a nitro side group, respectively, in place

of a diethylamino side group. Replacing the diethylamino group still managed to weakly suppress

anthocyanin levels in N-deprived seedlings. Notably, both C- and D- analogues strongly suppressed

anthocyanins in a dose-dependent manner (Figures 10b-c). Both active (C-, D-) and inactive (A-, B-)

analogues signify the importance of an oxazole central backbone. Furthermore, while the diethylamino

group can be dispensed with, it appears to enhance the potency of the parent compound, benzoxazole-1,

by strongly suppressing anthocyanins at a low concentration of 25 µM.

65

Table 3. Structures of benzoxazole-1 analogue compounds scored for their ability to attenuate


categorized as A-Benzoxazole-1, B-Benzoxazole-1, C-Benzoxazole-1 etc.

Chemical

Conc.

Applied



yl)-7-(diethylamino)-

2H-chromen-2-one

(C20H18N2O3)


Parent compound

7-(diethylamino)-3-

(1-methyl-1H-

benzimidazol-2-yl)-

2H-chromen-2-one

(C21H21N3O2)

25 µM A-Benzoxazole-1

3-(1,3-benzothiazol-2-

yl)-7-(diethylamino)-

2H-chromen-2-one

(C20H18N2O2S)

25 µM B-Benzoxazole-1

66

Table 3. Continued

Chemical

Conc.

Applied



yl)-3H-

benzo[f]chromen-3-

one

(C20H11NO3)

25 µM

C-Benzoxazole-1


yl)-6-nitro-2H-

chromen-2-one

(C16H8N2O5)

25 µM

D-Benzoxazole-1

67

Figure 10a. Benzoxazole-1 (parent) and benzoxazole-1-analogues (A-, B-, etc.) exhibited varying


(N-) nitrogen conditions are indicated in green and red respectively. Benzoxazole-1 analogues are

alongside their parent compound (benzoxazole-1) to compare anthocyanin attenuation capacity.

Derivatives (C-, and D-) appeared to mildly suppress anthocyanin levels. All samples were tested in


bars indicate SD.

68

Figure 10b. Benzoxazole-1-analogue (C) dose-response (25-100 µM). Benzoxazole-1 analogues

which mildly suppressed anthocyanin levels in a primary screen were selected for re-screening under

higher concentrations. Under higher concentrations (50-100 µM), C-analogue strongly suppressed

anthocyanin levels in a dose-dependent manner; however not to the same extent as the parent compound.

All samples were tested in triplicates (n=3). Statistical analysis was performed using ANOVA (Tukey’s

B test, p < 0.05). Errors bars indicate SD.

69

Figure 10c. Benzoxazole-1-analogues (D) dose-response (25-100 µM). Benzoxazole-1 analogues

which mildly suppressed anthocyanin levels in a primary screen were selected for re-screening under

higher concentrations. Under higher concentrations (50-100 µM), D-analogue strongly suppressed

anthocyanin levels in a dose-dependent manner; however not to the same extent as the parent compound.

All samples were tested in triplicates (n=3). Statistical analysis was performed using ANOVA (Tukey’s

B test, p < 0.05). Errors bars indicate SD.

70

3.35 1-[3-(1-benzofuran-2-yl)-1H-pyrazol-4-yl]-N-(1, 3-benzoxazol-2-

ylmethyl)-N-methylmethanamine activity required a pyrazole moiety,

along with a central oxazole backbone and a methanamine side group to

strongly attenuate anthocyanins

Six benzoxazole-2 derivatives (Table 4, A-, B-, C-, D-, E-, and F-) had varied anthocyanin responses in

N-deprived seedlings (Figure 11a). A-benzoxazole-2 contains a substituted benzofuran with a

dimethoxyphenyl side group, and yet managed to strongly suppress anthocyanin levels. Furthermore, A-

benzoxazole-2 demonstrated dose-dependent inhibition of anthocyanins, attenuating anthocyanins to the

same degree as the parent compound (Figure 11b). E-benzoxazole-2 contains a benzofuran structural

backbone in place of benzoxazole, and failed to reduce anthocyanin levels, accumulating anthocyanins

comparable to DMSO control. This supports the expendable quality of benzofuran in the benzoxazole-2

suppression of anthocyanins. The modification (B-benzoxazole-2) and elimination (C-benzoxazole-2) of

pyrazole resulted in the complete loss of anthocyanin suppression, and further emphasized the

significance of pyrazole towards benzoxazole-2 activity. D-benzoxazole-2 replaces the methanamine

side group with a piperidine, while E-benzoxazole-2 eliminates the N atom in benzoxazole, generating

an additional benzofuran. D- and E-benzoxazole-2 treatment failed to attenuate anthocyanin levels in N-

deprived seedlings (Figure 11a). This suggests that methanamine and oxazole moieties must partially

contribute to benzoxazole-2 function. More analogues must be tested to determine their specific role in

benzoxazole-2 suppression of anthocyanins. Notably, F-benzoxazole-2 strongly inhibited anthocyanins.

This demonstrates that in the presence of a central imidazole backbone, oxazole and methanamine can

be replaced, and still manage to strongly inhibit seedling anthocyanin content.

71

Table 4. Structures of benzoxazole-2 analogue compounds scored for their ability to attenuate


categorized as A-Benzoxazole-2, B-Benzoxazole-2, C-Benzoxazole-2 etc.

Chemical

Conc.

Applied



yl)-1H-pyrazol-4-yl]-

N-(1,3-benzoxazol-2-

ylmethyl)-N-

methylmethanamine

(C21H18N4O2)


Parent compound

(1,3-benzoxazol-2-

ylmethyl){[3-

(2,4-

dimethoxyphenyl

)-1H-pyrazol-4-

yl]methyl}methy

lamine

(C21H22N4O3)

25 µM

A-Benzoxazole-2

(1,3-benzoxazol-2-

ylmethyl){[2-(2,3-

dimethoxyphenyl)-5-

methyl-1,3-oxazol-4-

yl]methyl}methylami

ne

(C22H23N3O4)

25 µM B-Benzoxazole-2

72

Table 4. Continued

Chemical

Conc.

Applied


N-(1,3-benzoxazol-2-

ylmethyl)-N-methyl-

1-benzofuran-5-

carboxamide

(C18H14N2O3)

25 µM C-Benzoxazole-2

2-(1-{[3-(1-

benzofuran-2-

yl)-1H-pyrazol-

4-yl]methyl}-4-

piperidinyl)-1,3-

benzoxazole

(C24H22N4O2)

25 µM D-Benzoxazole-2


yl)-1H-pyrazol-

4-yl]-N-(2,3-

dihydro-1-

benzofuran-2-

ylmethyl)-N-

methylmethanam

ine

(C22H21N3O2)

25 µM E-Benzoxazole-2

73

Table 4. Continued

Chemical

Conc.

Applied



yl)-1H-pyrazol-4-yl]-

1H-benzimidazole

(C18H12N4O)

25 µM F-Benzoxazole-2

74

Figure 11a. Benzoxazole-2 (parent) and benzoxazole-2-analogues (A-, B-, etc.) exhibited varying


(N-) nitrogen conditions are indicated in green and red respectively. Benzoxazole-2 analogues are

alongside their parent compound (benzoxazole-2) to compare anthocyanin attenuation capacity. Both A-

and F-benzoxazole-2 appeared to strongly attenuate anthocyanin levels, comparable to parent

compound. All samples were tested in triplicates (n=3). Statistical analysis was performed using


75

Figure 11b. Benzoxazole-2-analogue (A-) dose-response. Benzoxazole-2 analogue strongly attenuated

anthocyanin levels in a dose-dependent fashion (50-100 µM). All samples were tested in triplicates

(n=3). Statistical analysis was performed using ANOVA (Tukey’s B test, p < 0.05). Errors bars indicate

SD.

76

3.36 2-{4-[(9-ethyl-9H-carbazol-3-yl) methyl]-1-methyl-2-piperazinyl}

ethanol activity required a central carbazole backbone to strongly

attenuate anthocyanins

Five carbazole derivatives (A-, B-, C-, D-, and E-) had varied anthocyanin responses in N-deprived

seedlings (Figure 12a). The A-carbazole derivative eliminates the methyl group and ethanol side chain

on piperazine, yet managed to strongly attenuate anthocyanin levels comparable to the parent compound.

C-, D-, and E-carbazole analogues are similar, as they all modify the piperazine ring by repositioning a

single nitrogen atom (C-), replacing it with an oxygen atom (E-), or completely eliminating it (D-). C-

carbazole strongly suppressed anthocyanins to the same degree as the parent compound, while E-

carbazole managed to do so at higher concentrations in a dose-dependent fashion (Figure 12c). In

contrast, D-carbazole managed to reduce anthocyanins at higher concentrations; however not to the

same extent as the parent compound. So it appears that replacing N with an O atom in piperazine, which

converts piperazine to a morpholine substituent, can retain chemical function. Finally, B-carbazole is the

only derivative which completely failed to reduce anthocyanin levels in N-deprived seedlings. B-

carbazole lacks a N atom in the central carbazole backbone, substituted with an oxygen atom, in the

process generating a dibenzofuran. Thus, it appears that the central carbazole backbone played a

dominant role in the carbazole-attenuation of anthocyanin accumulation. Additionally, the nitrogen atom

in piperazine can be substituted for an oxygen atom, while piperazinyl side groups, methyl and ethanol,

are completely dispensable.

77

Table 5. Structures of carbazole analogue compounds scored for their ability to attenuate


categorized as A-Carbazole-2, B-Carbazole-2, C-Carbazole-2 etc.

Chemical

Conc.

Applied


2-{4-[(9-ethyl-9H-

carbazol-3-

yl)methyl]-1-

methyl-2-

piperazinyl}etha

nol

(C22H29N3O)

25 µM Carbazole

Parent compound

9-ethyl-3-(1-

piperazinylmethyl)-

9H-carbazole

(C19H23N3)

25 µM A-Carbazole

2-[4-

(dibenzo[b,d]furan-4-

ylmethyl)-1-methyl-2-

piperazinyl]ethanol

(C20H24N2O2)

25 µM B-Carbazole

78

Table 5. Continued

Chemical

Conc.

Applied


N-[(9-ethyl-9H-

carbazol-3-

yl)methyl]-N,1-

dimethyl-4-

piperidinamine

(C22H29N3)

25 µM C-Carbazole

9-ethyl-3-[(4-methyl-

1-piperidinyl)methyl]-

9H-carbazole

(C21H26N2)

25 µM

D-Carbazole

9-ethyl-3-(4-

morpholinylmethyl)-

9H-carbazole

(C19H22N2O)

25 µM

E-Carbazole

79

Figure 12a. Carbazole (parent) and carbazole-analogues (A-, B-, etc.) exhibited varying


(N-) nitrogen conditions are indicated in green and red respectively. Carbazole analogues are alongside

their parent compound (carbazole) to compare anthocyanin attenuation capacity. Both A- and C-

benzoxazole-2 derivatives appeared to strongly attenuate anthocyanin levels; while D- and E-

benzoxazole-2 appear to do so mildly. All samples were tested in triplicates (n=3). Statistical analysis


80

Figure 12b. Carbazole-analogue (D) dose-response (25-100 µM). Carbazole analogues which mildly

suppressed anthocyanin levels in a primary screen were selected for re-screening under higher

concentrations. Under higher concentrations (50-100 µM), D-analogue strongly suppressed anthocyanin

levels in a dose-dependent manner; however not to the same extent as the parent compound. All samples

were tested in triplicates (n=3). Statistical analysis was performed using ANOVA (Tukey’s B test, p <

0.05). Errors bars indicate SD.

81

Figure 12c. Carbazole-analogue (E) dose-response (25-100 µM). Carbazole analogues which mildly

suppressed anthocyanin levels in a primary screen were selected for re-screening under higher

concentrations. Under higher concentrations (50-100 µM), E-analogue strongly suppressed anthocyanin

levels in a dose-dependent manner, and to the same extent as the parent compound. All samples were



82

Chapter 4

83


4.1 Nitrogen-deprived seedlings display a longer primary root and reduced

chlorophyll leaf content

Besides anthocyanins, N-deprived plants reconfigure root architecture as well as chlorophyll

metabolism. During N-deficiency, Arabidopsis plants develop longer primary roots and undergo

chlorophyll turnover (Diaz et al., 2006; Linkohr et al., 2002; Smart, 1994). In the present study, root

architectural modifications and chlorophyll breakdown were measured in N-deprived Arabidopsis

seedlings. Under seven days of N-limiting conditions, Arabidopsis seedlings generated chlorotic aerial

tissue along with longer primary roots (data not shown). Using this well-defined phenotype, it was

interesting to identify whether early chemical exposure possibly mis-regulated root architectural changes

and chlorophyll breakdown under N-deprivation.

4.2 All four chemical compounds modified primary root length under low N

conditions

Primary root length (PRL) of young Arabidopsis seedlings, treated with DMSO/chemical (Table 1), was

measured after seven days of growth in a low-N environment. DMSO-treated seedlings had longer

primary roots under low-N conditions as expected (Figures 13-16). Notably, all four chemical

compounds appeared to alter root architectural changes. Carbazole treatment appeared to enhance

primary root length under low-N conditions (Figures 13a-b), possibly increasing root surface area to

optimize nitrogen scavenging. In contrast, benzimidazole treatment generated shorter primary roots

under low-N conditions (Figures 14a-b). This suggests a maladaptive response demonstrated by a

concomitant inhibition in anthocyanin accumulation and primary root growth. Interestingly, both

benzoxazoles enhanced primary root growth under high and low-N conditions (Figures 15, 16, a-b);

therefore, it appears that benzoxazole-induction of root growth is independent of nitrogen availability.

84

Under N-deficiency, a direct association between root architectural reprogramming and anthocyanin

metabolism is missing. Thus, by implementing a chemical genetics approach, a link between root

structural alterations and leaf biochemical changes can further be investigated.

85

Figure 13a. Carbazole treatment generated a longer primary root under low N conditions.

DMSO/chemical treatment are indicted vertically, while nitrogen treatment is indicated horizontally.

Following seven days of post-germinative growth under nitrogen deprivation, or under control

conditions, seedlings were vertically plated and photographed. ImageJ software (US National Institutes

of Health, Bethesda, MD, USA) was used to measure primary root length for DMSO and chemical-

treated seedlings. Average primary root length was calculated from three independent replicates on 18

plantlets.

DMSO Carbazole

Hig

h N

L

ow

N

86

Figure 13b. Carbazole treatment generated a longer primary root under low N conditions.

DMSO/chemical treatment under high (N+) and low (N-) nitrogen conditions are indicated in green and

red respectively. Carbazole-treatment generated a longer primary root under low N conditions. All

samples were tested in triplicates (n=3). Statistical analysis was performed using ANOVA (Tukey’s B

test, p < 0.05). Errors bars indicate SD.

87

Figure 14a. Benzimidazole treatment generated a shorter primary root under low N conditions.






plantlets.

DMSO Benzimidazole

Hig

h N

L

ow

N

88

Figure 14b. Benzimidazole treatment generated a shorter primary root under low N conditions.


red respectively. Benzimidazole-treatment generated a shorter primary root in seedlings under low N

conditions. All samples were tested in triplicates (n=3). Statistical analysis was performed using


89

Figure 15a. Benzoxazole-1 treatment generated a longer primary root irrespective of N regimen.






plantlets.

DMSO Benzoxazole-1

Hig

h N

L

ow

N

90

Figure 15b. Benzoxazole-1 treatment generated a longer primary root irrespective of N regimen.


red respectively. Benzoxazole-1-treatment generated a longer primary root under high and low N

conditions, irrespective of N availability. All samples were tested in triplicates (n=3). Statistical analysis


91

Figure 16a. Benzoxazole-2 treatment generated a longer primary root irrespective of N regimen.






plantlets.

DMSO Benzoxazole-2

Hig

h N

L

ow

N

92

Figure 16b. Benzoxazole-2 treatment generated a longer primary root irrespective of N regimen.


red respectively. Benzoxazole-2-treatment generated a longer primary root under high and low N

conditions, irrespective of N availability. All samples were tested in triplicates (n=3). Statistical analysis


93

4.3 Chemical compounds failed to modify leaf chlorophyll content

Similar to anthocyanin accumulation, young Arabidopsis seedlings undergo additional biochemical

changes, including chlorophyll turnover. Chlorophyll breakdown is necessary to prevent photo-oxidative

damage, as well as remobilize nitrogen to younger tissues (Diaz et al., 2006). Chlorophyll content of

DMSO/chemical treated seedlings was measured after seven days of growth in a low-N environment. As

expected, DMSO-treated seedlings displayed a significant reduction in leaf chlorophyll content under

low-N conditions (Figure 17). Under N-deficiency, reduced leaf chlorophyll content was not altered by

chemical treatment, suggesting that chemical application does not appear to interfere with chlorophyll

metabolism.

94

Figure 17. Chemical compounds failed to modify total chlorophyll content. DMSO/chemical treated

seedlings under high (N+) and low (N-) nitrogen conditions are indicated in green and yellow (chlorosis)

respectively. Chlorophyll levels declined under nitrogen deficiency, and early chemical treatment

appeared to have no effect on chlorophyll levels during low nitrogen availability. All samples were



95

4.4 Chemical compounds suppressed seedling anthocyanin content under

phosphate starved conditions

Phosphorous (P), like nitrogen, is another major macronutrient necessary to plant survival. Phosphate-

starved Arabidopsis plants exhibit adaptive responses similar to N-deprivation, demonstrating enhanced

lateral root growth and a surge in anthocyanin production. Given this, it was important to establish

whether chemical compounds identified in the present study were capable of attenuating anthocyanin

levels strictly under N-deprivation alone, or also in response to P-deprivation. Following the

optimization of phosphate-starved growth conditions, all four chemical compounds (Table 1) were

tested for their ability to significantly suppress seedling anthocyanin content. Chemical pre-treated

Arabidopsis seedlings displayed significant reductions in total seedling anthocyanin content under P-

starvation (Figure 18). A 2-3 fold attenuation in seedling anthocyanin content was previously observed

under N-deprived conditions (Figure 4b). Therefore, it appeared that all four chemical compounds must

target protein(s) with overlapping functions in nitrogen and phosphate signalling pathways. Transcript

abundance (RNAseq) for previously characterized genes with overlapping functions in nitrogen and

phosphate signalling pathways might be usefully examined in the future (Bonnot et al., 2016; Peng et al.,

2007b; Scheible et al., 2004).

96

Figure 18. Seedlings pre-treated with candidate chemical compounds failed to accumulate

anthocyanins under low phosphate levels

High nitrogen (P+) and low nitrogen (P-) conditions are indicated as green and red respectively. DMSO

pre-treated seedlings exhibited an abundance of anthocyanins under low phosphate levels. In contrast,

seedlings pre-treated with any of the four candidate chemical compounds (Benzimidazole, Benzoxazole-

1, Benzoxazole-2, Carbazole) failed to display red anthocyanin pigmentation during phosphate

starvation. All samples were tested in triplicates (n=3). Statistical analysis was performed using


97

Chapter 5

98

5. 1 Conclusions and Future directions

The major focus of this thesis was to use a novel approach towards developing a deeper understanding

of the complex interaction between nitrogen availability and anthocyanin biosynthesis. The study

presented here relied on the fundamental principles of chemical genetics to characterize anthocyanin

accumulation in N-deprived Arabidopsis seedlings. The major research hypotheses and the supporting

findings are considered in turn below:

(1) Small bioactive molecules will have the capacity to attenuate leaf anthocyanin levels in

nitrogen-deprived seedlings.

A robust anthocyanin-inhibition screen was established, where nitrogen-deprived seedlings could be

tested for their inability to accumulate anthocyanins following chemical treatment. Primary and

secondary chemical screens identified four chemical compounds (benzimidazole, benzoxazole-1/2, and

carbazole) with the ability to significantly reduce seedling anthocyanin content during low-N

availability. Anthocyanins attenuate the absorption capacity of chlorophyll molecules; consequently

preventing photo-oxidative damage while facilitating nutrient recovery (Feild et al., 2001). It will be

interesting to explore the biological ramifications of chemical-suppressed anthocyanins accumulation in

N-deprived seedlings, with little or no anthocyanins present. Next-generation sequencing tools such as

RNAseq can provide valuable data on the transcriptome of anthocyanin-less plants (Peng et al., 2007b).

For instance, transcript abundance of oxidative response genes (superoxide dismutase, catalase) in

response to attenuated anthocyanin levels can be examined, and to observe whether alternate oxidative

mechanisms over-compensate to facilitate nitrogen remobilization. Additionally, the nitrogen content

(nitrate, ammonium, amino acids) of young developing tissues can be measured using a calorimeter, to

examine the impact of reduced anthocyanin abundance on nutrient recovery (Peng et al., 2007b).

Furthermore, test the impact of perturbed nutrient remobilization on growth parameters such as

leaf/silique size and biomass. Doing so will emphasize the essential role anthocyanins in developmental

and stress responses.

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(2) Chemicals applied at higher concentrations will display a dose-dependent response

Chemical compounds applied at a range of concentrations proportionally reduced anthocyanin levels in

a dose-dependent manner. It reinforced the assumption that anthocyanin levels respond to chemical

treatment. Furthermore, chemicals appeared to be potent at much lower concentrations than initially

screened at 25 µM. These afford major advantages to future experiments, where chemicals can be

applied at very low concentrations to limit the possibility of pleiotropic effects on plant development.

(3) Chemicals will exert their effect at a particular developmental stage

Chemical compounds applied at six different time points during seed stratification and germination

demonstrated highest potency during radicle protrusion. Radicle emergence is a highly sensitive process,

as it is the last phase of germination during which the seed commits to seedling growth; thus, it is tightly

controlled by developmental (ABA-GA) and environmental (nutrients) signals (Bewley, 1997; Costa et

al., 2015; Finkelstein, 2000). It is an essential developmental checkpoint, to either complete

germination, or initiate a developmental arrest until conditions are favourable. During germination, it is

also necessary to establish whether the chemical was absorbed, or whether it triggers signalling from the

external environment. If the chemical is absorbed, once it becomes bioavailable, perhaps it must be

chemically conjugated or cleaved to become bioactive. This can be examined using experimental

techniques such as GC-MS to help determine the absorption and spatial distribution of chemical

compounds and analogues (Bonnot et al., 2016). Additionally, this could help narrow down putative

protein targets in a specific organ, tissue, or cell. Defining the critical period for chemical exposure also

provides experimental flexibility to examine chemical effect on adult plants. For instance, post-chemical

treatment, germinated seeds can be transplanted onto soil, or hydroponically grown, to assess

biochemical changes (anthocyanins) in adult tissues of N-deprived Arabidopsis plants. Chemical

compounds perturb biological processes due to their inherent nature as ‘perturbagens’; therefore,

limiting chemical exposure to one day of germination could help prevent any possibilities of growth

impairments during later stages of plant development.

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(4) Specific structural motifs will lend compounds with anthocyanin attenuation capacity.

The use of active and inactive analogues placed emphasis on the importance of reactive moieties in

conferring biological activity. ‘Derivatives’ of all four chemical compounds helped identify essential

substructures partially or completely responsible for attenuating anthocyanin levels in N-deprived

seedlings. Notably, benzimidazole analogues demonstrated that the presence of a reactive moiety as well

as its spatial position lends biological function to a compound. Furthermore, benzoxazole-1 and

carbazole analogues uncoupled the possibility that nitrogen atoms within each compound could

potentially act as a nitrogen source in attenuating anthocyanins. It is the presence of a specific nitrogen

moiety, rather than the number of nitrogen atoms, which afforded each chemical compound with the

ability to attenuate anthocyanin levels. Future studies must make use of additional structural analogues

to complete structure-activity relationships for each of the chemical compounds. A recent study

identified a chemical compound, ‘Phostin’, that mimicked phosphate starvation responses in

Arabidopsis seedlings (Bonnot et al., 2016). Phostin and phostin-analogues were screened to examine

structural moieties responsible for inducing a phosphate starvation response. The study discovered

active and inactive analogues, which led them to propose a certain reactive moiety as biologically active.

The study used LC-UV to measure root absorption of active analogues over inactive analogues, and

subsequently quantified active analogues in tissue extracts using LC-MS. Furthermore, to identify

putative protein target(s), the study screened for phostin-resistant mutants using previously characterized

mutants affected in the phosphate starvation response (phr1, pho1). This approach helped identify

putative proteins targets of phostin, or targets acting downstream of phostin-induced signalling. In a

similar approach, GC-MS can be used to confirm the tissue-specific absorption of active analogues

discussed in Chapter 3, Section 3. Similarly, loss-of-function mutants previously characterized in the N-

deprivation response, P-starvation response, as well as affected in anthocyanin biosynthesis, can be used

to screen for compound-resistant lines. If a loss-of-function mutant exhibited enhanced anthocyanin

pigmentation, and was resistant to chemical treatment, it would be a candidate for a potential target, or

possibly an indirect target which acts downstream of chemical-induced signalling. By contrast, if the

101

same mutant displayed sensitivity to chemical application, the chemical must exert its effect via an

alternate signalling mechanism. Overall, this process helps identify novel functions for previously

identified proteins and unidentified signalling pathways.

(5) Chemical compounds will modify other N-adaptive responses besides anthocyanin

accumulation.

Early chemical treatment failed to modify leaf chlorophyll levels; however, compounds managed to

influence root architectural changes under altered nitrogen levels. Nitrogen-deprived Arabidopsis

seedlings develop a longer primary root, to optimize scavenging for soil nitrogen deposits. During low-

N availability, carbazole and benzimidazole treatment appeared to increase and decrease primary root

length (PRL) respectively. Carbazole-induction of primary root growth may increase nitrogen uptake;

therefore, this must be investigated by examining nitrogen uptake and total nitrogen seedling content. In

contrast, benzimidazole-suppression of root growth and anthocyanin accumulation suggests an overall

inhibition of N-adaptive responses. Notably, both benzoxazoles enhanced root growth under high and

low-N conditions; as a result, appear to play a role in root growth independent of N-availability. Few

studies have explored an association between anthocyanin biosynthesis and root architectural changes

during N-deprivation. Recently, a root-hair-defective-3 (rhd3) loss-of-function mutant, previously

characterized for its role in root hair development (Schiefelbein & Somerville, 1990), was identified to

hyper-accumulate anthocyanins under nitrogen or phosphate deficiency (Wang et al., 2015). RHD3

functions to control flavonoid metabolism as well as root hair development, which suggests an overlap

between the two signalling pathways. Small molecules described in this thesis could examine the

association between root growth and anthocyanin biosynthesis under N-deprived conditions.

Transcriptomic analysis (RNAseq) can provide valuable information on mis-regulated gene expression

of candidate genes with possible overlapping functions in the two signalling pathways (Peng et al.,

2007b; Scheible et al., 2004). Subsequently, loss-of-function mutations of candidate genes can be

screened to identify lines resistant to chemical-suppression of anthocyanin accumulation and chemical-

102

induced root architectural changes (Arnaud et al., 2014). Additionally, GC-MS data can demonstrate the

root-shoot distribution of chemical compounds, to inspect the tissue/organ in which the compound exerts

its effect (Bonnot et al., 2016). This could help determine whether the compound is distributed in aerial

tissues as well as the root, or perhaps induces signalling in one organ with downstream effects on both

organs.

Flavonoids play an essential role in plant development and stress responses. Nitrogen-deprived

Arabidopsis plants lacking anthocyanins have shown to undergo abnormal senescence (Aoyama et al.,

2014; Peng et al., 2008). Chemical genetics is a powerful approach documented for successfully

exploring metabolic pathways in plants, and has received much attention in plant biology in the last

decade. In this study, a robust screening assay facilitated thousands of chemicals to be tested in a high-

throughput manner. Combining chemical genomics with conventional genetic methods will shed light

on the chemical perturbation of anthocyanin accumulation under the strict control of nutrient

availability. It is important to develop an understanding of how internal (developmental) and external

(environmental) signals are integrated to sustain plant growth and development. Expanding scientific

knowledge of developmental and adaptive responses in planta will translate into efficient and

environmentally safer agricultural practices.

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A chemical genetics approach to explore anthocyanin ... · Eshan Naik Masters of Science Department of Cell & Systems Biology University of Toronto 2016 Abstract Plants often experience