A chemical genetics approach to explore anthocyanin ... · Eshan Naik Masters of Science Department...
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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
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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.
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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
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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
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3.14 Chemical compounds attenuate anthocyanin levels irrespective of
inorganic nitrogen source (ammonium vs. nitrate)……………………………………..32
Chapter 3 Results & Discussion (Section 2)
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
Chapter 3 Results & Discussion (Section 3)
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
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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
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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
attenuate anthocyanin levels in seedlings grown under nitrogen deficiency……….65-66
Table 4 Structures of benzoxazole-2 analogue compounds scored for their ability to
attenuate anthocyanin levels in seedlings grown under nitrogen deficiency……….71-73
Table 5 Structures of carbazole analogue compounds scored for their ability to
attenuate anthocyanin levels in seedlings grown under nitrogen deficiency……….77-78
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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
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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
anthocyanin levels in nitrogen-deprived seedlings……………………………………..67
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
anthocyanin levels in nitrogen-deprived seedlings……………………………………..74
Figure 11b Benzoxazole-2-analogue (A-) dose response (25-50 µM) ……………………………..75
Figure 12a Carbazole (parent) and carbazole analogues (A-, B-, etc.) exhibit varying
anthocyanin levels in nitrogen-deprived seedlings……………………………………..79
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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
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Chapter 1
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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.
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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).
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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
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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.
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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
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(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.
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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
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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
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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
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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
Compounds identified Class/Group
1-[3-(1-benzofuran-2-
yl)-1H-pyrazol-
4-yl]-N-(1,3-
benzoxazol-2-
ylmethyl)-N-
methylmethanam
ine
(C21H18N4O2)
25 µM Benzoxazole-2
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
Results & Discussion
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
anthocyanin levels in nitrogen-deprived seedlings
39
Figure 6c. Benzoxazole-2 applied at a range of concentrations proportionally attenuated
anthocyanin levels in nitrogen-deprived seedlings
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
triplicates (n=3). Statistical analysis was performed using ANOVA (Tukey’s B test, p < 0.05). Errors
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
Results & Discussion
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
Compounds identified Class/Group
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
Compounds identified Class/Group
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
triplicates (n=3). Statistical analysis was performed using ANOVA (Tukey’s B test, p < 0.05). Errors
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
anthocyanin levels in seedlings grown under nitrogen deficiency. The analogues have been
categorized as A-Benzoxazole-1, B-Benzoxazole-1, C-Benzoxazole-1 etc.
Chemical
Conc.
Applied
Compounds identified Class/Group
3-(1,3-benzoxazol-2-
yl)-7-(diethylamino)-
2H-chromen-2-one
(C20H18N2O3)
25 µM Benzoxazole-1
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
Compounds identified Class/Group
2-(1,3-benzoxazol-2-
yl)-3H-
benzo[f]chromen-3-
one
(C20H11NO3)
25 µM
C-Benzoxazole-1
3-(1,3-benzoxazol-2-
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
anthocyanin levels in nitrogen-deprived seedlings. DMSO-treated seedlings under high (N+) and low
(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
triplicates (n=3). Statistical analysis was performed using ANOVA (Tukey’s B test, p < 0.05). Errors
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
anthocyanin levels in seedlings grown under nitrogen deficiency. The analogues have been
categorized as A-Benzoxazole-2, B-Benzoxazole-2, C-Benzoxazole-2 etc.
Chemical
Conc.
Applied
Compounds identified Class/Group
1-[3-(1-benzofuran-2-
yl)-1H-pyrazol-4-yl]-
N-(1,3-benzoxazol-2-
ylmethyl)-N-
methylmethanamine
(C21H18N4O2)
25 µM Benzoxazole-2
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
Compounds identified Class/Group
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
1-[3-(1-benzofuran-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
Compounds identified Class/Group
2-[3-(1-benzofuran-2-
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
anthocyanin levels in nitrogen-deprived seedlings. DMSO-treated seedlings under high (N+) and low
(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
ANOVA (Tukey’s B test, p < 0.05). Errors bars indicate SD.
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
anthocyanin levels in seedlings grown under nitrogen deficiency. The analogues have been
categorized as A-Carbazole-2, B-Carbazole-2, C-Carbazole-2 etc.
Chemical
Conc.
Applied
Compounds identified Class/Group
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
Compounds identified Class/Group
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
anthocyanin levels in nitrogen-deprived seedlings. DMSO-treated seedlings under high (N+) and low
(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
was performed using ANOVA (Tukey’s B test, p < 0.05). Errors bars indicate SD.
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
tested in triplicates (n=3). Statistical analysis was performed using ANOVA (Tukey’s B test, p < 0.05).
Errors bars indicate SD.
82
Chapter 4
83
Results & Discussion
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.
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 Benzimidazole
Hig
h N
L
ow
N
88
Figure 14b. Benzimidazole treatment generated a shorter primary root under low N conditions.
DMSO/chemical treatment under high (N+) and low (N-) nitrogen conditions are indicated in green and
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
ANOVA (Tukey’s B test, p < 0.05). Errors bars indicate SD.
89
Figure 15a. Benzoxazole-1 treatment generated a longer primary root irrespective of N regimen.
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 Benzoxazole-1
Hig
h N
L
ow
N
90
Figure 15b. Benzoxazole-1 treatment generated a longer primary root irrespective of N regimen.
DMSO/chemical treatment under high (N+) and low (N-) nitrogen conditions are indicated in green and
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
was performed using ANOVA (Tukey’s B test, p < 0.05). Errors bars indicate SD.
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Figure 16a. Benzoxazole-2 treatment generated a longer primary root irrespective of N regimen.
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 Benzoxazole-2
Hig
h N
L
ow
N
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Figure 16b. Benzoxazole-2 treatment generated a longer primary root irrespective of N regimen.
DMSO/chemical treatment under high (N+) and low (N-) nitrogen conditions are indicated in green and
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
was performed using ANOVA (Tukey’s B test, p < 0.05). Errors bars indicate SD.
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.
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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
tested in triplicates (n=3). Statistical analysis was performed using ANOVA (Tukey’s B test, p < 0.05).
Errors bars indicate SD.
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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).
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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
ANOVA (Tukey’s B test, p < 0.05). Errors bars indicate SD.
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.
99
(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.
100
(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.
103
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