CELLULAR AND MOLECULAR ASPECTS OF THE TRANSPORT AND SEQUESTRATION OF ANTHOCYANINS IN MAIZE AND ARABIDOPSIS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the
Graduate School of The Ohio State University
By
Niloufer G. Irani
* * * * *
The Ohio State University
2006
Dissertation Committee:
Professor Erich Grotewold, Adviser
Professor Gregory Armstrong Approved by
Professor Iris Meier
Professor Venkat Gopalan ______
Professor Brenda S. J. Winkel Adviser Graduate Program in Plant Biology ABSTRACT
Not much is known about the efficient trafficking of potentially toxic
phytochemicals from their site of synthesis to their correct intracellular destinations.
Anthocyanins, the colored products of the well characterized flavonoid pathway, were used as a convenient model system to understand the cellular, biochemical and molecular processes involved in its transport from the cytoplasmic face of the ER to the central vacuole. Cell biological observations of maize Black Mexican Sweet (BMS) cells in culture, constitutively expressing the MYB (C1) and bHLH (R) transcription factors, show these cells to accumulate anthocyanins in multiple vacuoles with anthocyanic vacuolar inclusions (AVIs). Exposure to light caused calli to darken. This was attributed to the fusion of the vacuoles and ‘spread’ of anthocyanins from the AVIs into the sap but not due to changes in transcripts of the enzymes or the accumulation of pigments.
Formation of vacuoles from dynamic pigmented compartments was observed in maize tassel glumes. These observations indicated an alternate vesicular route of transport of anthocyanins to the central vacuole. In Arabidopsis, I exploited the transparent testa mutant tt5, deficient in the enzyme chalcone isomerase (CHI) and its anthocyanin complementation with naringenin to develop an easily manipulatable seedling model system. Naringenin-treated tt5 and wt seedlings accumulated a new anthocyanin peak
ii identified as cyanidin 3-glucoside. Global transcriptome changes were monitored using
microarrays to identify potential transferases and transporters involved in either the
detoxification of naringenin or the transport of anthocyanins. The induction of signaling
components, jasmonic acid biosynthetic genes and defense-related stress response genes,
suggested additional roles of flavonoids in several cellular processes. Lastly, the need for
the catalytic function of CHI was investigated in driving flux into the flavonoid pathway.
The catalytic mutants Y104F, T46A and R34A were generated in maize CHI (ZmCHI).
In vitro assays demonstrated ZmCHIY104F to retain 20% of the ZmCHIwt activity while
ZmCHIT46A and ZmCHIR34A were catalytically inactive. Only ZmCHIY104F and, surprisingly, the in vitro catalytically inactive ZmCHIT46A complemented Arabidopsis
tt5 mutants. These findings revealed additional roles of CHI in the flavonoid pathway.
Taken together, these observations and results provided significant insights in
understanding processes involved in phytochemical trafficking.
iii
for mum and dad
iv
Nature has all the answers. We simply have to observe and understand.
v ACKNOWLEDGMENTS
“In the realm of life, people enrich its fabric.”
No words can express my heartfelt gratitude for my adviser, Dr Erich Grotewold, for being my guide and a good friend through this journey. Thank you sir, for your guidance, patience and tolerance, and opening my mind to wide, wide world of science.
I am grateful to my committee members, Dr Iris Meier, Dr Greg Armstrong, Dr
Venkat Gopalan, Dr Brenda S.J. Winkel for taking the time to attend all the meetings, giving invaluable feedback on the science and this thesis. My appreciation goes to all the
PCMB faculty members who have been my invaluable teachers with their doors ever open to offer help, support and advice.
I would like to thank Dr Herbert Auer and Dr Karl Kornacker for the extensive help with the microarray analysis, Dr Edward Behrman for helping me synthesize chalcone and teaching me that chemisty is like cooking, Dr Biao Ding for providing the use of the fantastic Nikon microscopes, Dr JC Jang for the use of his stereomicroscope imaging station and Dr Steven Schwartz for use of the reflectometer and the analysis of anthocyanins using LC-MS.
vi To all the members of the Grotewold lab, past and present, post docs, graduate
students and undergrads, thank you for all the scientific discussions, teaching me
experimental procedures and making the lab a lively place to work in – Ed Braun, Lila
Pooma, Xiaoyung Dong, Anusha Dias, Robert Lockwood, Yuhua Lu, Vinod Malik,
Kenogo Morohasi, Frantisek Poutska, Marcela Hernandez, George Heine, Antje Feller,
Zidian Xie, Laura Martz, Matteo Citarelli, Carlos Molina, George Wang, Jared Dominik,
Mike Ribaudo, Linda Diec, Gabriella Camone, David Ciarlariello, Carl Mayernick, Todd
Matulnik, Manjusha Kulkarni, Joseph, Margarita Barros, Alejandra Acuna, Tatiyana,
Sarat Subramaniam and Patricia Norambuena.
Rightmire Hall has been a home to me. My appreciation to all the labs in Plant
Biotech Center, the Meier, Ding, Verma, Bisaro, Somers, Jang and Scholl Labs, ABRC
and the Neurobiotech Center labs, who have seen my face at the oddest hours asking for advice, borrowing equipment or chemicals.
I am indebted to the behind the scenes people who keep the department running
smoothly with ever ready help - Jill Hartman and Denise Blackburn Smith, Joan Leonard,
Debra Gamble, Laurel Shannon (Aranoff Labs) and Kelly Williams, and Diane Furtney,
Melinda Parker, Jan Zianich, Dave Long, Scott Hines and Joe Takayama (Rightmire
Hall).
I would like to acknowledge my friends who have always been there providing
moral and emotional support – Shalaka, Anusha, Kenn, Priya, Antje, Olli, Roopa,
Anahita, Suchi, Padma, Prady, Chaitanya, Sandi, Anupam, Annkatrin, Asuka, Jelena.
Special thanks to Priya, for all the care and support in these final days of thesis writing.
vii The Ohio State University has been a wonderful nuturing ground, right from the beautiful environs to the wide departmental interests. I thank my teachers in art for making me see a different aspect of science through art, especially Dr Richard Harned for encouraging me to follow my heart, all my field hockey mates, and my students who reciprocated the joy in learning as it was in teaching.
My extended family back home in both in Pune and Mumbai and here in
Columbus, who are always there for me and form a safety web and haven- thank you. To my brother Marzban, sisters Kayanoosh and Farah, their genetic leftovers little sister will always have an indebted love and gratitude for all the tutelage and invaluable advice during schooling and life. And finally, to my parents Gillan and Gulshan, without whom my chromosomes would never have been put together, whose support, encouragement and freedom has gotten me to where I am today - there are no words to say how much I love and cherish you.
viii VITA
25th October, 1976………………………………... Born – Mumbai, India 1997………………………………………………. B.Sc. in Botany (with Zoology and Geology as subsidiaries), Department of Botany, Fergusson College, University of Pune, Pune, India 1999………………………………………………. M.Sc. in Botany (with Plant Biotechnology as a specialization), Department of Botany, University of Pune, Pune, India 2000- present……………………………………... Graduate Teaching/Research Associate, Department of Plant Cellular and Molecular Biology, The Ohio State University, Columbus, Ohio, USA
PUBLICATIONS
Lu Y., Irani N.G., Grotewold E. (2005) Covalent attachment of the plant natural product naringenin to small glass and ceramic beads. BMC Chem Biol. 5:3
Irani, N.G., Grotewold, E. (2005) Light-induced vacuolar morphological alteration in anthocyanin- accumulating maize cells. BMC Plant Biol. 5:7.
Hernandez, J., Heine, G., Irani, N.G., Feller, A., Kim, M.-G., Matulnik, T., Chandler, V.L., and Grotewold, E. (2004) Different mechanisms participate in the R-dependent activity of the R2R3 MYB transcription factor C. J. Biol. Chem. 279: 48205-48213.
Lin, Y., Irani, N.G., and Grotewold, E. (2003) Sub-cellular trafficking of phytochemicals explored using auto-fluorescent compounds in maize cells. BMC Plant Biology 5:3.
ix Irani, N., Hernandez, J.M., and Grotewold, E. (2003) Regulation of anthocyanin pigmentation. Recent Adv. Phytochem . 37: 59-78. (Review Article)
FIELDS OF STUDY
Major Field: Plant Biology
Department of Plant Cellular and Molecular Biology
x TABLE OF CONTENTS
Page
Abstract………………………………………………………………………….. ii
Dedication……………………………………………………………………….. iv
Acknowledgements……………………………………………………………… vi
Vita………………………………………………………………………………. ix
List of Figures…………………………………………………………………… xvi
List of Tables………………………………………………………………...... xx
Abbreviations……………………………………………………………………. xxii
Chapters:
1 Light-Induced Morphological Alteration in Anthocyanin-Accumulating Vacuoles of Maize Cells…………………………………………………...… 1
1.1 Introduction…………………………………………………….……… 1 1.2 Materials and Methods……………………………………….………... 5 1.2.1 Growth, maintenance and treatment of BMS cells………….... 5 1.2.2 Transient expression experiments…………………………….. 5 1.2.3 Reflectance analysis…………………………………………... 6 1.2.4 Extraction and analysis of anthocyanin pigments…………….. 6 1.2.5 Extraction and analysis of RNA……………………………… 7
xi 1.2.6 Plant material…………………………………………………. 7 1.2.7 Microscopy analysis and vacuolar staining…………………... 8 1.2.8 pH measurement……………………………………………… 9 1.3 Results…………………………………………………….…………… 9 1.3.1 BMS cells expressing the transcription factors R and C1 accumulate anthocyanins in the dark…………………………. 9 1.3.2 BMS35S::R+35S::C1 cells darken in the light……………………... 10 1.3.3 The anthocyanin contents or mRNA steady state levels of biosynthetic genes are not altered by white light in BMS35S::R+35S::C1 cells…………………………………………. 13 1.3.4 White light induces alterations in the sub-cellular distribution and vacuolar organization of anthocyanins in BMS35S::R+35S::C1 cells………………………………………………………….... 17 1.3.5 Light-induced vacuolar morphological alterations in anthocyanin-accumulating maize floral organs………………. 25 1.4 Discussion………………………………………………………..…….. 30 1.5 Conclusions ………………………………………………………..….. 37
2 Metabolome and Transciptome Changes in Arabidopsis thaliana Seedlings Induced to Accumulate Anthocyanins……………………………………….. 39
2.1 Introduction……………………………………………………….…… 39 2.2 Materials and Methods………………………………………….……... 52 2.2.1 Plant materials and growth conditions………………………... 52 2.2.2 Naringenin and cycloheximide treatments……………………. 52 2.2.3 Imaging seedlings…………………………………………….. 53 2.2.4 Anthocyanin extraction, and analysis by spectrophotometry, TLC and HPLC……………………………………………….. 54 2.2.5 Extraction of RNA……………………………………………. 55 2.2.6 Northern Blot Analysis……………………………………….. 55 2.2.7 Microarray analysis: RNA extraction and hybridization……... 56 xii 2.2.8 Microarray data analysis……………………………………… 57 2.3 Results and Discussion ………………………………………..………. 58 2.3.1 Developing an anthocyanin inducible system using tt5, wt and naringenin…………………………………………………….. 58 2.3.2 Naringenin induces a new anthocyanin peak – cyanidin-3- glucoside……………………………………………………… 64 2.3.3 There are no significant changes in flavonol accumulation patterns………………………………………………………... 73 2.3.4 280nm HPLC profiles of naringenin treated tt5 and wt seedlings……………………………………………………… 79 2.3.5 Naringenin is an inhibitor of growth…………………………. 83 2.3.6 Components of the tt5 anthocyanin inducible system and rationale for analyzing transcriptome changes using microarrays…………………………………………………… 86 2.3.7 Transcriptome changes in Arabidopsis tt5 and wt seedlings treated with naringenin: Experimental design………………... 90 2.3.8 Naringenin does not feedback regulate its core flavonoid enzymes or transcription factors at the expression level……… 94 2.3.9 Microarray analysis show a total of 48 genes up-regulated and 134 genes down regulated by naringenin in tt5 and wt seedlings………………………………………………………. 100 2.4 Conclusions………………………………………………….………… 123
3 Complementation of Arabidopsis thaliana tt5 with Maize Chalcone Isomerase Mutants: Insights into the Role of CHI in the Flavonoid 126 Pathway…………………………………………………………...
3.1 Introduction………………………………………………………….… 126 3.2 Materials and Methods……………………………………………….... 132 3.2.1 Synthesis of chalcone…………………………………………. 132
xiii 3.2.2 Site directed mutagenesis of ZmCHI1………………………... 133 3.2.3 In vitro transcription/translation……………………………… 134 3.2.4 Expression of recombinant CHI protein in E.coli…………….. 134 3.2.5 Enzyme assays………………………………………………... 135 3.2.6 Plant material and transformation…………………………….. 136 3.2.7 Anthocyanin induction, extraction and analysis by TLC and HPLC…………………………………………………………. 137 3.3 Results……………………………………………………………….… 138 3.3.1 Chalone isomerase dynamics: structure activity and generation of catalytic mutants ZmY104F, ZmT46A and ZmR34A……………………………………………………… 138 3.3.2 ZmCHIY104F mutants show 75% - 80% reduction in activity as compared to ZmCHIYwt and the ZmCHIT46A and ZmCHIR34A mutants are catalytically dead in vitro………… 144 3.3.3 ZmCHIwt, ZmCHIY104F and ZmCHIT46A complement the tt5 mutant phenotype…………………………………………. 147 3.3.4 35S::ZmCHIT46A plants accumulate more anthocyanins and show a different Q:K ratio than 35S::ZmCHIwt plants………. 149 3.3.5 Phenolic profiles of 35S:ZmCHIR34A tt5 plants do not phenocopy tt5…………………………………………………. 150 3.3.6 Inability to detect interactions between the maize flavonoid enzymes……………………………………………………….. 157 3.4 Discussion…………………………………………………………..….. 159
4 Conclusions 163
Appendices:
A Montage of BCECF-AM stained BMS35S::R+35S::C1 Cells…………………….. 170 B List of Primers Used…………………………………………………………. 171
xiv C Epidermal and Subepidermal Accumulation of Anthocyanins in Arabidopsis 173 Cotyledons…………………………………………………………………… D Comparative Spectral Analysis of the Anthocyanin Peaks of Naringenin and 174 Cycloheximide Treated tt5 and wt Arabidopsis Seedlings………………….. E Transient Localization of Maize and Arabidopsis CHS, CHI, DFR and F3H 179 In Nicotiana benthamiana…………………………………………………… F Yeast Two Hybrid Analysis………………………………………………….. 183
Bibliography…………………………………………………………………….. 186
xv LIST OF FIGURES
Figure Page
1.1 Anthocyanins accumulate in maize BMS35S::R+35S::C1 cells in the dark….. 10
1.2 Light induces the darkening of anthocyanin pigmentation……………… 11
1.3 Similar quantities of cyaniding and pelargonidin accumulate in dark and
light-grown BMS35S::R+35S::C1 cells………………………………………. 15
1.4 Northern analysis of flavonoid biosynthetic genes show no alterations in
the steady-state mRNA levels induced by light in BMS35S::R+35S::C1
cells……………………………………………………………………… 16
1.5 BMS35S::R+35S::C1 cells have multiple vacuoles…………………………... 18
1.6 Light induces alterations in the distribution of anthocyanins within
vacuolar compartments………………………………………………….. 19
1.7 Comparison of the size distribution of vacuolar inclusion containing
anthocyanins in BMS35S::R+35S::C1 cells grown in the light or the dark…... 22
1.8 Morphology of vacuoles of dark and light grown BMS and
BMS35S::R+35S::C1 cells loaded with BCECF-AM………………………… 24
1.9 Morphology of anthocyanin-accumulating cells in maize floral organs… 26
1.10 Sub-cellular morphology of BI Pl maize floral cells accumulating 27
xvi anthocyanins……………………………………………………………...
1.11 In situ measurement of vacuolar pH of BMS cells using BCECF-AM…. 34
1.12 HPLC analysis of dark and light grown BMS35S::R+35S::C1 cells show no
significant change in their phenolic profile……………………………… 35
2.1 Schematic representation of the fate of a small molecule within a cell…. 48
2.2 The core flavonoid pathway leading to the major groups of flavonoid…. 49
2.3 3% sucrose and light induce the formation of anthocyanins and
naringenin chemically complements the tt5 mutant…………………….. 61
2.4 Vacuolar localization and quantization of anthocyanins………………... 63
2.5 HPLC profile at 530nm-naringenin induction of cyaniding 3-glucoside.. 68
2.6 Analysis of changes in peak area and peak percent of anthocyanins at
530nm…………………………………………………………………… 70
2.7 Cycloheximide reduces but does not abolish the accumulation of
anthocyanins in naringenin complemented tt5 seedlings………………... 71
2.8 Anthocyanin analysis of naringenin and cycloheximide treated tt5 and
wt seedlings……………………………………………………………… 72
2.9 HPLC analysis of flavonols at 360nm…………………………………... 76
2.10 Analysis of 16.3min peak extracted from 360nm chromatograms of
hydrolyzed tt5 and wt samples…………………………………………... 78
2.11 HPLC analysis of anthocyanins at 280nm………………………………. 81
2.12 Peak eluting at the naringenin standard 27.25min have two alternative
absorption maximas……………………………………………………... 82
xvii 2.13 Northern analysis of AtCHS and AtF3H expression in wt, tt5 and tt3
seedlings treated with naringenin and cycloheximide…………………... 83
2.14 Effect naringenin on tt5 plant growth…………………………………… 84
2.15 Naringenin is an inhibitor of growth…………………………………….. 85
2.16 The three components of the tt5 system…………………………………. 87
2.17 Schematic representation of Arabidopsis tt5 and wt system and
complementation with naringenin……………………………………….. 89
2.18 Microarray experimental variables and experimental break up…………. 91
2.19 Experimental design for the effects of naringenin on tt5 and wt
seedlings…………………………………………………………………. 92
2.20 Venn diagrams depicting number of up and down regulated genes by
naringenin in tt5 and wt seedlings……………………………………….. 106
2.21 Artificial Cluster patterns: based on trends and not expression levels….. 107
2.22 Functional categorization of all the genes affected by naringenin in tt5
and wt seedlings…………………………………………………………. 108
2.23 Functional categorization of genes up-regulated and down-regulated by
naringenin in tt5 and wt together………………………………………… 109
3.1 The core phenylpropanoid and flavonoid biosynthetic pathway in
Arabidopsis thaliana……………………………………………………... 131
3.2 ZmCHI catalyzes the cyclization of 2,4,4’,7’ tetrahydroxychalcone into
2S-naringenin……………………………………………………………. 132
3.3 Spontaneous cyclization of chalcone……………………………………. 140
xviii 3.4 Alignment of ZmCHI1 with representative CHIs from the type I and
type II groups……………………………………………………………. 141
3.5 Cladogram and percentage identities of CHI proteins…………………... 143
3.6 Relative enzyme rate comparisons of TNT synthesized proteins……….. 145
3.7 Comparative enzyme rates for recombinant E. coli expressed GST-
ZmCHI fusion proteins………………………………………………….. 146
3.8 ZmCHIwtt, ZmCHIY104F and ZmCHIT46A complement Arabidopsis tt5
mutants……………………………………………………….………….. 148
3.9 Spectrophotometric quantification of anthocyanins…………………….. 151
3.10 TLC analysis of anthocyanins…………………………………………… 152
3.11 HPLC chromatograms showing anthocyanidin profiles (530nm)…….… 153
3.12 HPLC analysis of flavonol content at 360nm…………………………… 154
3.13 Q:K ratio difference in ZmCHIT46A with no changes in total flavonol
content…………………………………………………………………… 155
3.14 HPLC chromatograms reveal changes in phenolic profiles (280nm)…… 156
xix LIST OF TABLES
Table Page
1.1 Reflectance analysis and L* a* b* values of dark- and light-grown
BMS35S::R+35S::C1 cells in the CIELAB color scale……………………... 12
1.2 Vacuole distribution in dark- and light-grown BMS35S::R+35S::C1 cells… 20
2.1 Proteins characterized to play a role in flavonoid transport to the
vacuole………………………………………………………………… 51
2.2 Microarray relative expression values and fold changes of the enzymes
of the flavonoid pathway…………………………………… 97
2.3 Microarray relative expression values and fold changes of the enzymes
of the phenylpropanoid pathway…………………………… 98
2.4 Microarray relative expression values and fold changes of the known
transcription factors involved in the regulation of the flavonoid
pathway………………………………………………………………... 99
2.5 Naringenin up-regulated genes in tt5 and wt………………………….. 110
2.6 Naringenin down-regulated genes in tt5 and wt……………………….. 113
2.7 Transporters identified in this study that are regulated by naringenin in
wt and tt5 seedlings……………………………………………………. 120
xx 2.8 Transferases identified in this study as being up or down-regulated by
naringenin in tt5 and wt seedlings……………………………………... 121
2.9 The glutathione S transferases identified as being regulated by
naringenin in tt5 and wt seedlings……………………………………... 122
xxi ABBREVIATIONS
Abbreviation Description
~ Approximately
°C Degree centigrade
µg Microgram
µl Micro liter
µM Micro molar
35S Cauliflower Mosaic Virus 35S promoter
4CL 4-coumaroyl:CoA ligase
A Alanine
AS Anthocyanidin synthase
AVIs Anthocyanic vacuolar inclusions
BMS Black Mexican Sweet bp Base pairs
C3G Cyanidin 3-glucoside
C4H Cinnamate 4-hydroxylase
CHI Chalcone isomerase
xxii CoA Coenzyme A d Day
DEPC diethylpyrocarbonate
DFR Dihydroflavonol 4-reductase
DNA Deoxyribonucleic acid
F Phenylalanine
F3’5’H Flavonoid 3’,5’ hydroxylase
F3’H Flavonoid 3’ hydroxylase
F3H Flavanone 3-hydroxylase g Gram h Hours
HPLC High performance liquid chromatography
IPTG Isopropyl beta D-thiogalactopyranoside kDa kilodaltons
LC-MS Liquid chromatography – mass spectrometry
LDOX Leucoanthocyanidin dioxygenase mV.s Milli volt per second min Minutes ml Milliliter
M Molar mg Milligram ng Nano gram
xxiii n moles Nano moles
PAL Phenylalanine ammonia lyase
PCR Polymerase chain reaction
PVC Pre-vacuolar compartment
R Arginine
RNA Ribonucleic acid
RT Room temperature s Seconds
T Threonine
TNT Transcription coupled with translation tt / TT Transparent testa
UFGT UDPglucose: flavonoid 3-O-glucosyl transferase
UGT UDP glycosyl transferase wt Wild type
Y Tyrosine
ε Molar absorption coefficient
λmax Absorption maxima
xxiv CHAPTER 1
LIGHT-INDUCED MORPHOLOGICAL ALTERATION IN ANTHOCYANIN- ACCUMULATING VACUOLES OF MAIZE CELLS
1.1 Introduction
Anthocyanins, the coloured end product of the flavonoid pathway, play an
important role in attracting insects or animals for pollination and seed dispersal. In
addition, they play roles as anti-oxidants and in protecting DNA and the photosynthetic
apparatus from high radiation fluxes (Gould, 2004). Other possible functions of
anthocyanins, such as the protection against cold stress or providing drought resistance,
are likely to be associated with activities restricted to particular classes of plants
(Chalker-Scott, 1999).
The biosynthesis of flavonoids, a large phenylpropanoid-derived group of phenolic compounds, provides one of the best described plant metabolic pathways, with many of the structural and regulatory genes in the pathway identified and cloned (Mol et al., 1998; Winkel-Shirley, 2001). Less is known regarding the mechanisms by which the water-soluble anthocyanins are transported from their site of synthesis, the cytoplasmic surface of the endoplasmic reticulum (Hrazdina and Wagner, 1985; Winkel-Shirley, 1 1999), to the vacuole, where they are usually sequestered (Grotewold, 2004). Plant
vacuoles are highly dynamic, multifunctional organelles that are the primary storage and
turnover sites of macromolecules. These membrane-bound organelles, which can occupy up to 90% of the total cellular volume, are integral part of the endomembrane system,
serving as the terminal products of the secretory pathway (Marty, 1999).
Several plant species store anthocyanins within vacuolar inclusions that have been
loosely termed anthocyanoplasts which start as vesicles in the cytoplasm and appear to be
membrane bound (Pecket and Small, 1980; Nozzolillo and Ishikura, 1988). More
recently, the intravacuolar structures observed in the flower petals of various plants,
including carnation and lisianthus, were termed anthocyanic vacuolar inclusions, or AVIs
(Markham et al., 2000). These inclusions were suggested to be membrane-less,
proteinaceous matrixes that acted as anthocyanin traps, preferentially for anthocyanidin 3,
5-diglycosides (Markham et al., 2000) or acylated anthocyanins (Conn et al., 2003). Once
in the vacuole, many factors influence the in vivo pigmentation provided by
anthocyanins, with important consequences for the eco-physiology of plants (Forkmann,
1991; Markham and Ofman, 1993). Some of these factors include the particular type of
anthocyanidin present (e.g., pelargonidin cyanidin, or myricetin), the shape of the cells in
which the pigments accumulate (Noda et al., 1994), the concentration of the pigment, the
formation of complexes between anthocyanins and co-pigments (Kondo et al., 1992), and
the vacuolar pH (Yoshida et al., 1995; Fukada-Tanaka et al., 2000).
Environmental conditions are known to induce the accumulation of anthocyanin
pigments across the major groups of higher plants, with light being the best studied
(Chalker-Scott, 1999; Winkel-Shirley, 2002; Irani et al., 2003). In Arabidopsis, the
2 anthocyanin pathway is regulated in a circadian fashion, with flavonoid gene expression
peaking at the end of the dark cycle, likely preparing plants for daybreak (Harmer et al.,
2000). In maize, members of the PL1/C1 R2R3 MYB and B/R bHLH families of
regulatory proteins cooperate to regulate synthesis of anthocyanin pigments (Mol et al.,
1998) and are necessary for the expression of the anthocyanin biosynthetic genes, c2
(chalcone synthase), chi1 (chalcone isomerase), f3h (flavanone 3-hydroxylase) a1
(dihydroflavonol 4-reductase), a2 (leucoanthocyanidin dioxygenase/anthocyanin
synthase), bz1 (UDP glucose:flavonoid 3-O-glucosyl transferase) and bz2 (glutathione S-
transferase) (Irani et al., 2003). The light-induced expression of members of these R2R3
MYB and bHLH classes of transcription factors has been proposed to be responsible for
the induction of anthocyanins in maize by light (Taylor and Briggs, 1990; Tonelli et al.,
1991; Procissi et al., 1997; Petroni et al., 2000; Piazza et al., 2002).
Here, we investigated whether light affects the accumulation of maize
anthocyanin pigmentation through pathways independent of the activation of the known
R2R3 MYB and bHLH regulators of the pathway. Towards this goal, we analyzed previously described (Grotewold et al., 1998) transgenic Black Mexican Sweet (BMS) maize cells in culture expressing the Zea mays R and C1 regulators from a constitutive, light-insensitive CaMV 35S promoter (BMS35S::R+35S::C1). We show here that
BMS35S::R+35S::C1 cells are red, even when grown in complete darkness. Upon light
treatment, there is a darkening of the color of the BMS35S::R+35S::C1 cells, without an
appreciable increase in the quantity of anthocyanins or in the type of anthocyanidins present. Consistent with these findings, the steady-state levels of several anthocyanin biosynthetic genes do not increase upon light treatment. Interestingly, at the subcellular
3 level, light induces an alteration in the way the anthocyanins are distributed within vacuolar compartments. A similar alteration in the morphology of anthocyanin- accumulating vacuoles is observed when maize tassel glumes are irradiated with light, suggesting that the phenomenon observed in BMS35S::R+35S::C1 cells in culture also occurs
in planta. Together, our findings suggest a novel mechanism for the action of light on the
packaging of anthocyanins in the vacuole and in sub-vacuolar compartments. This effect
of light could only be uncovered after making the biosynthesis of anthocyanins light
independent.
4 1.2 Materials and Methods
1.2.1 Growth, maintenance and treatment of BMS cells
BMS cells were maintained in conditions previously described (Dias et al., 2003).
In brief, BMS cells were sub-cultured every seven days in liquid MS media supplemented
with 2,4-dichlorophenoxyacetic acid (2,4,D; 0.5 g/L), 3% sucrose (BMS media) on a
rotatory shaker (150 rpm) in the dark at 25 ± 2°C. For dark and light treatments, cells
from suspension cultures were plated on filter paper overlaid on BMS solid media
containing 0.3% phytagel, and were allowed to establish for 20 d in darkness at 25 ± 2°C.
Plates were shifted to total darkness (covered with aluminum foil) or light at 50 ± 5
µmol.m-2.s-1 (Cool white, 215W, F96T12/CW/VHO, Sylvania, Canada).
1.2.2 Transient expression experiments
BMS suspension cells (3 g of cells in 25 ml of BMS media) were treated
overnight with 1.7% (w/v) PEG in BMS media. One ml of cells was overlaid on pre-
soaked filter papers in Petri plates. Ten micrograms of 35S::R+35S::C1 plasmid
[pPHP687 in (Grotewold et al., 1998)] was coated onto gold microprojectiles according
to the manufacturer’s recommendations (Bio-Rad Laboratories, Inc., USA). Coated gold
particles were bombarded into PEG-treated BMS cells using a Biolistic PDS-1000/He
particle gun (Bio-Rad Laboratories, Inc. USA) at 1,100 psi. The plates were kept in the
dark (covered with foil) or exposed to light for a period of 24 h, after which cells were
analyzed microscopically.
5 1.2.3 Reflectance analysis
In vivo reflectance measurements were taken with a Minolta CR-300 reflectometer/colorimeter (Minolta, Japan). The color was represented as CIEL*a*b*
values (for the CIE D65/10° illuminant/observer condition). The L* value represents the
lightness level, ranging from 100 (white) to 0 (black), the a* (+a red; -a green) and b* (+b
yellow; -b blue). The instrument was normalized using a standard white tile provided
with the instrument before performing analysis on cells grown on solid BMS media in
Petri plates.
1.2.4 Extraction and analysis of anthocyanin pigments
BMS35S::R+35S::C1 or control BMS cells after a light or dark treatment were lyophilized for 36 h. Anthocyanins and other phenolics were extracted in 50% methanol
overnight using 1µl of methanol per 50 µg of dry tissue. Methanol extracts were diluted
in 1% (v/v) HCl in 50% (v/v) methanol and absorption spectra were collected between
400 to 700 nm with 5 nm intervals at 0.5 s with a Cary 50 UV-VIS spectrophotometer
(Varian, Inc. USA). Graphs were generated using the Cary WinUV software.
Anthocyanins were measured spectrophotometrically at 530 nm. For the generation of
the anthocyanidins from the corresponding anthocyanins, methanol extracts were
hydrolyzed by the addition of an equal volume of 2 M HCl (37% v/v) and heated in a
boiling water bath for 20 min. Hydrolyzed samples were extracted with isoamyl alcohol.
Chromatographic separation of the anthocyanidins was performed by thin layer
chromatography (TLC) on cellulose plates (5730/7, EM Science, Germany) with
HCl/formic acid/H2O, [3:30:10, v/v] as the mobile phase. Twenty microliters of
6 methanolic extracts of non-hydrolyzed and hydrolyzed samples were injected into a
Waters Alliance® 2695 Separations module (Waters Corporation, Milford, MA) using
separation conditions as described (Dias and Grotewold, 2003). The HPLC profiles were
obtained at 280 nm using the Waters 2996 Photodiode Array Detector and analyzed with
the EmpowerTM software (Waters Corporation, Milford, MA).
1.2.5 Extraction and analysis of RNA
Dark and light grown BMS cells were homogenized in liquid nitrogen and total
RNA was extracted using the TRIzol reagent following the manufacture’s
recommendations (Invitrogen, Life Technologies, USA). For Northern analyses, 25 μg
of total RNA was separated on a formaldehyde-containing 1% agarose gel and blotted onto a nitrocellulose membrane (Bio-Rad Laboratories, Inc., USA). The blot was hybridized with cDNA probes corresponding to c2 (Wienand et al., 1986), f3h (Deboo et al., 1995) and a1 (Schwarz-Sommer et al., 1987). Ubiquitin (Christensen et al., 1992) was used as a normalization control. Comparison of the hybridization signals was performed on a BioRad phosphorimager (BioRad Laboratories, Inc., USA) and ratios of the dark and light grown callus hybridization signal to the ubiquitin normalization control were compared.
1.2.6 Plant material
Maize kernels for C2-Idf the genetic stock 418D C2-Idf1 (Active-1); A1 A2 C1 R1 and BI Pl - 219I B1-I; A1 A2 C1 C2 Pl1-Rhoades r1-r, 219J B1-I; A1 A2 C1 C2 Pl1-
Rhoades r1-g were obtained from the Maize Coop (http://w3.aces.uiuc.edu/maize-coop/).
7 The kernels were planted in the field in the summer and just before anthesis, male tassels
were collected for observation of vacuolar structure of the lemma or the palea.
1.2.7 Microscopy analysis and vacuolar staining
Digital images of the maize floral whorls and callus cells were captured with a
Nikon COOLPIX 5700 camera. Macroscopic images of the transient experiments were
visualized with an Olympus SZH10 Research Stereo microscope (Olympus, Japan) and
images were captured with a Olympus DP10 digital camera. Light and dark grown BMS
cells were examined under a Nikon Eclipse E600 microscope. Differential interference
contrast (DIC) pictures were taken with a SPOT, RT-Slider digital camera and analyzed
using the SPOT imaging software (Diagnostic Instruments, Inc., USA). DIC time lapse
images were taken every 4 s for 2 h and were converted into a movie using the SPOT
imaging software. For vacuolar staining, transformed and control BMS cells were
incubated with 10 µM BCECF-AM (Molecular Probes, USA) in BMS media for 40 min
at room temperature. Cells were spun down, washed twice and re-suspended in BMS
media. Laser scanning confocal microscopy with a PCM 2000/Nikon Eclipse600 system
(Nikon Bioscience Confocal Systems, NY) was used to capture digitized images of the
BCECF stained cells using the Nikon Plan Fluor 40X/0.75 air objective (1 pixel = 0.3
μm) as described (Rose and Meier, 2001). The 488 nm excitation wavelength of the
argon laser was used in conjunction with a 515/30 nm bandpass emission filter
(EM515/30HQ). Images were captured using the SIMPLEPCI software (Compix Imaging
Systems, PA) and assembled using Adobe PHOTOSHOP (Adobe Systems, Mountain View,
CA).
8 1.2.8 pH measurement
BMS cells (6 ml) were grown in 35 mm Petri plates at a concentration of 0.1g/ml
(fresh weight/volume) for 6 days under light (50µmol.m-2.s-1) and dark (foil covered) at
100 rpm. Cells were filtered, weighed and resuspended at a concentration of 0.1 g/ml.
One millilitre of cells were loaded with 10 µM BCECF-AM as described above. One hundred microliters of loaded and washed cells were pipetted into a 96 well microtitre plate. An in situ calibration curve was generated separately for each of the replicates for the dark and light grown BMS cells. One hundred microlitres of 0.1M of various pH buffers from a range of 5.0 to 7.0 with 0.005% digitonin (Moseyko and Feldman, 2001) was added to 100 µl of cells, and incubated for 10 min. Fluorescence emission was measured at 535 nm with excitation at 440 nm and 490 nm using the FLEX stationTM and
data analysis program SOFTmax PRO 4.3 (Molecular Devices, CA). The emission ratio
at 490/440 nm was used for calculation of the pH, where irregularities due to unequal
loading are eliminated. These measurements were carried out in triplicate.
1.3 Results
1.3.1 BMS cells expressing the transcription factors R and C1 accumulate
anthocyanins in the dark
To determine whether the light control of the maize anthocyanin pathway is
mediated by the expression of the B/R and C1/PL regulators and/or the biosynthetic
genes (Procissi et al., 1997), we investigated the pigmentation of BMS cells expressing
the R and C1 genes from the constitutive CaMV 35S promoter (BMS35S::R+35S::C1) 9 (Grotewold et al., 1998). BMS35S::R+35S::C1 cells grown in complete darkness for 30 days were fully pigmented with anthocyanins (Fig. 1.1A). The bombardment of BMS cells with the R and C1 regulators driven from the 35S promoter (p35SR + p35SC1) resulted in the accumulation of red cells within 15 hours, even when cells were kept in complete
darkness after bombardment (compare Fig. 1.1B and 1.1C). These results indicate that
the constitutive expression of the R and C1 regulators is sufficient for the activation of
the pathway, even in the absence of light.
Transient expression 24h 30 day dark
A
Figure 1.1 Anthocyanins accumulate in maize BMS35S::R+35S::C1 cells in the dark: (A) Dark-grown BMS35S::R+35S::C1 cells expressing the R and C1 anthocyanin regulators from the CaMV 35S promoter accumulate anthocyanins. Transient expression of the R and C1 regulators in BMS cells by microprojectile bombardment induce anthocyanins in the dark (B) or the light (C). The magnification bars represent 200 µm. The bar in the inset DIC image is 20 µm.
1.3.2 BMS35S::R+35S::C1 cells darken in the light
To investigate whether light has any additional effect on the pigmentation present
in BMS35S::R+35S::C1 cells, we compared the color of BMS and BMS35S::R+35S::C1 cells grown for six days in complete darkness or under continuous white light (see Materials &
10 Methods). BMS35S::R+35S::C1 cells grown in the light showed a visual darkening, when
compared to cells grown in the dark (Fig. 1.2, BMS35S::R+35S::C1). However, light did not
induce any visible difference in the white-yellow color of the control BMS cells not
expressing the C1 and R regulators (Fig. 1.2, BMS).
BMS BMS35S::R+35S::C1
Dark
Light
Figure 1.2 Light induces the darkening of anthocyanin pigmentation: Images of BMS control and BMS35S::R+35S::C1 cells grown for ten days under total darkness (Dark) or light (Light) conditions.
Visual color differences were quantified in vivo using a reflectometer and the
CIELAB color space value system. L* values, representing the lightness level, were
reproducibly not affected by light (Table 1.1). The a* values for BMS35S::R+35S::C1 cells were positive (+a), consistent with the red color characteristic of these cells. There was
11 however a significant (p < 0.05) reduction in the a-values when the BMS35S::R+35S::C1 cells were exposed to light, which was observed in each of the three times that the experiment was performed (Table 1.1). Although the b* values, contributing to yellow (+b) or blue (- b), were significantly different (p < 0.05) between the dark and light grown
BMS35S::R+35S::C1 cells, they hovered near the zero value, suggesting a low contribution to
the overall observed color shift. Thus, the a* and the b* values observed corresponded to
a quantifiably red color (dull red), with a decreased degree of redness in the light, which
is in agreement with our visual observations (Fig. 1.2). In the absence of a change in the
L* value, the apparent darkening of the cells is likely caused by the spectral shift of the reflected light towards the blue (i.e., less red).
BMS35S::R+35S::C1 L* a* b*
Dark 21.42 ± 1.29 12.06 ± 1.51 0.45 ± 0.48
Light 21.01 ± 0.93 7.02 ± 0.88 -0.45 ± 0.21
Table 1.1 Reflectance analysis and L* a* b* values of dark- and light-grown BMS35S::R+35S::C1 cells in the CIELAB color scale: The a* value contributes to red (a+) or green (a-), the b* value contributes to yellow (+b) or blue (-b) and L* represent the lightness level.
12 1.3.3 The anthocyanin contents and mRNA steady state levels of biosynthetic genes
are not altered by white light in BMS35S::R+35S::C1 cells
To investigate whether the change in color observed between dark- and light
grown BMS35S::R+35S::C1 cells was due to an alteration in the quality or quantity of the pigments, anthocyanins were extracted and quantitated. Methanolic extracts of the cells, normalized for dry weight, were analyzed spectrophotometrically. The absorption spectra of pigments obtained from acidic methanol extracts of dark- and light-treated cells showed identical profiles and very similar absorbance values at 530 nm (Fig. 1.3A,
B). To determine whether light induced an alteration in the type of anthocyanidin present, total anthocyanidins were extracted and separated by thin layer chromatography
(TLC). Similar levels of cyanidin and pelargonidin were present in light- and dark treated BMS35S::R+35S::C1 cells (Fig. 1.3C), consistent with the two major types of
anthocyanidins previously described in BMS35S::R+35S::C1 cells (Dong et al., 2001).
In BMS cells, R and C1 are known to activate several flavonoid biosynthetic
genes (Grotewold et al., 1998). To investigate whether the activation of these genes was
further enhanced by the light treatment, total RNA was extracted from BMS35S::R+35S::C1 cells incubated for six days in the light or the dark and analyzed by Northern blots (Fig.
1.4). No significant alteration in the steady-state mRNA levels for chalcone synthase (c2,
Fig. 1.4), flavanone 3-hydroxylase (f3h, Fig. 1.4) or dihydroflavonol 4-reductase (a1, Fig.
1.4) was observed for the light grown BMS35S::R+35S::C1 cells. The control BMS cells showed no mRNA accumulation for these genes (Fig. 1.4). The expression of the VP24 protein, first identified as being encoded by a light-induced gene in sweet potato
(Ipomoea batata) localize to AVIs in vacuoles, correlate with the accumulation of
13 anthocyanins (Nozue et al., 1997). Thus, VP24 is thought to participate in the transport
or accumulation of anthocyanins to the vacuole (Xu et al., 2000). A similar gene
(GenBank:AI715001) was recently identified to be differentially expressed in endosperm and placenta of water stressed, developing maize kernels (Yu and Setter, 2003). So far,
however, we have not succeeded in detecting transcripts of this putative VP24 ortholog in
BMS or BMS35S::R+35S::C1 cells grown under dark or light conditions (not shown).
Together, these results suggest that the darkening of the BMS35S::R+35S::C1 cells upon light treatment is not due to an alteration in the quantity or quality of the anthocyanin pigments present.
14 A
B 0.8
0.6 A5 30 / 2. 0.4 5 m 0.2 g dr 0.0 Dark Light Dark Ligh C BMS BMS35S::R+35S::C1
Figure 1.3 Similar quantities of cyanidin and pelargonidin accumulate in dark and light-grown BMS35S::R+35S::C1 cells: (A) Spectral profile of methanol-HCl extracts of dark- (black line) or light-grown (green line) BMS35S::R+35S::C1 cells. The red line corresponds to the spectra of control BMS extracts. (B) Quantitation of anthocyanins in dark- and light-grown BMS35S::R+35S::C1 cells. Bars represent the standard deviation of measurements obtained in three independent experiments. (C) Qualitative analysis of the anthocyanins present in dark- and light-grown BMS35S::R+35S::C1 cells by TLC.
15
Figure 1.4: Northern analysis of flavonoid biosynthetic genes show no alterations in the steady-state mRNA levels induced by light in BMS35S::R+35S::C1 cells: Total RNA from dark- (D) and light-grown (L) control BMS and BMS35S::R+35S::C1 cells were analyzed by Northern with probes corresponding to the c2 (chalcone synthase), f3h (flavanone 3-hydroxylase) and a1 (dihyroflavonol 4-reductase) genes. Ubiquitin (ubq) was used as a normalizing control. The numbers indicate the relative hybridization signal (in arbitrary units) obtained with each probe, normalized for the corresponding signal obtained with ubq.
16 1.3.4 White light induces alterations in the sub-cellular distribution and vacuolar
organization of anthocyanins in BMS35S::R+35S::C1 cells
Alterations in rose flower pigmentation were associated previously with the
formation of AVI-like structures (Gonnet, 2003). To investigate if a similar alteration in
the packaging of anthocyanins occurred in BMS35S::R+35S::C1 cells, we investigated the
sub-cellular morphology of dark- and light-treated BMS35S::R+35S::C1 cells. To
unequivocally visualize the vacuole(s), BMS and BMS35S::R+35::C1 were stained with the
cell permeable, acetoxymethyl derivative of the fluorescent vacuolar dye 2’,7’-bis(2-
carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM). In the control BMS cells, there
are typically one to a few large vacuolar compartments (Fig. 1.5A, C). In contrast,
BMS35S::R+35::C1 cells are always multi-vacuolated (Fig. 1.5B, D). Unfortunately,
anthocyanins concentrated in the vacuolar inclusions quenched the fluorescence of the
BCECF-AM dye, as observed in some of the larger vacuoles (Fig. 1.5B).
17
Figure 1.5 BMS35S::R+35S::C1 cells have multiple vacuoles: Laser scanning confocal microscopy (false colored) images of (A) BMS and (B) BMS35S::R+35S::C1 cells loaded with 10 μM of the vacuolar dye BCECF-AM. Laser scanning confocal ‘light transmitted’ images of (C) BMS and (D) BMS35S::R+35S::C1 cells are shown in black and white. The bars represent 20 μm. See Appendix A for montage of B.
18
Figure 1.6 Light induces alterations in the distribution of anthocyanins within vacuolar compartments: Representative DIC images of six day old (A, C, E) dark- grown and (B, D, F) light-grown BMS35S::R+35S::C1 cells. BMS cells grown in the (G) dark or (H) light show no significant alterations. Numbers correspond to the percentage values indicated in Table 2. The bar represents 20µm.
19
% No. of vacuoles Anthocyanin Representation (range) spread 35S::R+C1 Dark (n=239) A 34% 20-30 + C 16% 10-20 - E 11% 20-30 - 35S::R+C1 Light (n=400) B 35% 1-10 ++ D 26% 10-20 + F 12% 1-10 +++
Table 1.2 Vacuole distribution in dark- and light-grown BMS35S::R+35S::C1 cells: The classification is based on the number of vacuoles and the presence or absence of anthocyanins in the vacuolar sap. The A-F letters correspond to the panels in Fig. 1.6, and the presence (+) or absence (-) of anthocyanins in the vacuolar sap (spread) is indicated.
20 Within the vacuole, anthocyanins accumulate in inclusions that, when observed
under polarized light, appear round and regular in shape or aggregated, like an intertwine of fine strings with blebs (Fig. 1.6). The number of vacuoles and inclusions per vacuole
were dramatically affected by light in BMS35S::R+35::C1 cells. In dark-treated
BMS35S::R+35::C1 cells, the major representative cell type (Fig. 1.6A, representing 34% of all the 239 cells observed), had between 20 to 30 observable vacuoles (Table 1.2). In these cells, the anthocyanins were present mainly in the rounded vacuolar inclusions with a characteristic pale pink coloration of the vacuolar sap, a phenomenon called here
“anthocyanin spread”. The next two most abundant cell types present in dark-treated
BMS35S::R+35::C1 cells, corresponding to 16% and 11% of all the cells, had a range of 10 to
20 or 20 to 30 visually observable vacuoles per cell, respectively (Table 1.2). Cells of
these groups were characterized by no observable anthocyanin spread in the vacuolar sap
under the light microscope, yet had red or pale red anthocyanin inclusions in the vacuole
(Fig. 1.6C, E).
In the presence of light, the majority of the BMS35S::R+35::C1 cells (35%, Table 1.2)
showed one to ten pigmented vacuoles with red inclusions that exhibited predominantly
diffuse and “tangled strings” like appearance (Fig, 1.6B). These structures were determined to be 0.1 to 0.3 µm in diameter, appeared in certain cells to be branched with bleb-like structures at their ends. A significant population of cells (26%, Table 1.2) had
10 to 20 vacuoles, that were lightly colored with discrete deeply pigmented, spherical inclusions (Fig. 1.6D). The third most abundant class of BMS35S::R+35::C1 cells present in
the light (12%, Table 1.2) showed deeply pigmented vacuoles and enlarged inclusions
(Fig. 1.6F).
21 Comparison of the size of the vacuolar inclusions in the dark- and light-grown
BMS35S::R+35::C1 cells (Fig. 1.7) reveals that the majority of the inclusions in the dark
grown samples were in the size range of 0.1µm - 1µm, with a noticeable absence of
larger inclusions. The modal range of the vacuolar inclusions in light grown cells was 2
to 3µm in diameter, with some as large as 14 µm. This may reflect the fusion of smaller
vacuolar inclusions to give rise to larger ones (see below). The control BMS cells, grown
in the dark or light, did not display sub-cellular morphological changes (not shown).
120 Dar k
100 Light
80
60
40
Number of AVIs 20
0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 ------1 1 1 1 1 0 1 2 3 4 5 6 7 8 - - - - - 9 0 1 2 3 1 1 1 1 size range (diameter in microns)
Figure 1.7 Comparison of the size distribution of vacuolar inclusion containing anthocyanins in BMS35S::R+35S::C1 cells grown in the light or the dark: Round vacuolar inclusions were measured and classified according to the size range measured in microns (µm) that they fell into. Blue and red bars represent the AVI sizes in dark- and light- grown cells, respectively.
22 To further understand the changes induced by light in anthocyanin-accumulating
cells, we performed laser scanning confocal microscopy of BCECF-AM loaded
BMS35S::R+35::C1 cells. Dark-grown cells showed the presence of multiple vacuoles (Fig.
1.8G), which, upon light treatment, appeared to coalesce to form fewer, much larger
vacuoles (Fig. 1.8H). Light-grown cells display a decrease in fluorescence, likely
because of quenching by the anthocyanins released from the vacuolar inclusions. Dark-
or light-grown BMS cells showed no distinctive differences in visual fluorescence
intensity or vacuolar morphology (Fig. 1.8 C, D), suggesting that the observed
morphological alterations are either a consequence of the (i) expression of the R and C1
regulators, (ii) accumulation of anthocyanins, or (iii) distinctive properties of the
vacuoles in which anthocyanins accumulate.
Together, these results show that light-exposed BMS35S::R+35::C1 cells have a significant reduction in the number of vacuoles with an associated increase in their size, a change in the number, shape and size of the AVIs, and release of anthocyanins from the
AVIs into the vacuole.
23
Figure 1.8 Morphology of vacuoles of dark and light grown BMS and BMS35S::R+35S::C1 cells loaded with BCECF-AM: Laser scanning confocal images of BMS grown cells in the dark and light (C, D respectively) and BMS35S::R+35S::C1 cells cells (G, H) loaded with 10 μM of BCECF-AM. Images were taken with identical gain values.
24 1.3.5 Light-induced vacuolar morphological alterations in anthocyanin-
accumulating maize floral organs
To establish whether the light-induced vacuolar alterations observed in BMS cells
could also be observed also in planta, we looked at the tassels of maize B-I Pl plants that
accumulate large quantities of anthocyanins (Fig. 1.9). The inner, light-protected lemma
and palea (Fig. 1.9B) were the choice of material to observe the light-induced alterations in vacuolar morphology. The epidermal cells of these appendages in a C2-Idf (chalcone synthase-inhibitor diffuse) mutant lacking anthocyanins (Fig. 1.9C) had one to a few
large, observable, colorless, central vacuoles (Fig. 1.9D). In contrast, depending on the
physiological and developmental stage of the florets, the epidermal cells of the lemma or palea of the B-I Pl florets (Fig. 1.9B, E) were either already filled with anthocyanins, or were in the initial stages of accumulation (Fig 1.9F). These cells showed a distinctive multi-vacuolar morphology, and the vacuoles were often heavily loaded with anthocyanins and AVI-like structures (Fig. 1.9F).
25
Figure 1.9 Morphology of anthocyanin-accumulating cells in maize floral organs: The maize male inflorescence, the tassel, is a panicle made of numerous spikes, each formed by numerous, paired spikelets. (A) Single spike with paired spikelets. The spikelet has outer and inner glumes (bracts of the florets) and each floret has a lemma, a palea, a highly reduced lodigule and 3 stamens (B) Spikelet dissection: the two florets with an outer glume and an inner glume; each floret with a lemma, palea, highly reduced lodigule and three stamens. (C) Digital macro images of open florets from a C2-Idf (chalcone synthase) mutant that accumulates no anthocyanins. (D) DIC light micrographs of the lemma from male flowers of C2-Idf plant (E). Digital macro images of open florets from a B-I Pl plant. (F) DIC light micrographs of the lemma from male flowers of a B-I Pl plant. The bar represents 10 μm
26 Figure 1.10 Sub-cellular morphology of BI Pl maize floral cells accumulating anthocyanins: DIC images of a maize lemma from B-I/B-Peru plant over-accumulating anthocyanins. The above are extracted images from a time-lapse series (See Additional file 1: Movie 1). The time points on the images indicate the period from time 0’ i.e. when the sample was mounted onto the stage and exposed to the microscope light. (A) and (B) occur earlier in the series while (D), (C) and (E) are in rapid succession (24 seconds apart). The large central inclusion corresponds to a vacuolar inclusion containing anthocyanins measuring 15 µm in diameter. The green, blue and black arrows indicate, in that order, sequential stages in the conversion of thin tubular anthocyanin-filled structures to thick sheet-like structures. The orange arrows indicate the next step, which is the conversion into round structures. The red arrows indicate large fusion bodies resulting from the fusion of the swollen round structures. The yellow arrows point to clear spherical structures devoid of anthocyanins. The bar represents 10 µm.
27 A
22’ 08” B
50’ 24” C
53’ 28” D
53’ 52” E
54’ 40”
Fig. 1.10
28 Light from the microscope was sufficient to induce dramatic alterations in the
morphology and distribution of the anthocyanin-containing vacuolar structures, as seen in
the time-lapse images taken at four-second intervals (Fig. 1.10, see Additional file 1:
Movie 1 for the original data used to perform this analysis). In these series, the initially
thin, tubular anthocyanin-filled structures (average diameter of 0.6 µm, Fig. 1.10, green
arrows) expanded to a thickness of about 1.4 µm, dynamically filling the entire cell (Fig.
1.10A, blue arrows). These thick, finger-like projections became swollen, sheet-like
structures (~3.3 µm in diameter, Fig 1.10, black arrows), which then become rounded
(Fig 1.10A, B, C; orange arrows) and fuse with each other. These rounded/oval compartments, measuring 1 to 9 µm in diameter, just like the tubular structures, displayed dynamic morphological changes. Swollen “blebs” were observed moving along fine tubules and the ends of the thick tubules swelled up into round structures. Fusion events, once initiated, were very rapid, which resulted in the formation of large fusion bodies
(Fig. 1.10C, D, E; red arrows) containing numerous clear (i.e., no anthocyanin- containing) structures (Fig 1.10C, D, E; yellow arrows). These clear inclusions were also observed initially in the sheet-like structures and were formed as the size of the tubules grew. The fusion bodies progressed rapidly to fill the entire cell, finally coalescing together resulting in the anthocyanin spread throughout the compartment (Fig 1.10E).
The defined margins around the large central AVI-like structure (~15 µm across) become more diffuse with a lighter translucent red halo around the opaque, dark body (Fig.
1.10E).
In contrast to the BMS35S::R+35S::C1 cells, in which anthocyanin production was
uncoupled from the light-induced morphological alterations, the accumulation of
29 anthocyanins in these B-I Pl cells is light induced (Procissi et al., 1997). Thus, the
observed alterations in vacuolar morphology could be a consequence of the light-induced
expression of the transcription factors, of the light-induced accumulation of anthocyanin,
of light-induced alterations in vacuolar morphology observed in BMS35S::R+35S::C1 cells, or a combination of them.
1.4 Discussion
Anthocyanin pigments play many important eco-physiological roles in plants.
While the biosynthesis and regulation of anthocyanins has been extensively described, little is known on how these pigments are sequestered in the vacuole and to what extent their modes of storage affect color. We describe here cytological changes of vacuoles and sub-vacuolar compartments containing anthocyanins in maize cells exposed to light.
Our studies in BMS35S::R+35S::C1 cells show anthocyanins to accumulate even in
total darkness, without any effect of light on the levels of anthocyanins or on the amount
of transcripts of various flavonoid biosynthetic genes. From these results, we conclude that, when the R and C1 regulators are expressed, there is no need for additional light- induced factors to influence the control of the pathway. This conlcusion strongly supports previous inferences that the flavonoid pathway is regulated by light at the level of transcription of the known R2R3 MYB and/or bHLH transcriptional activators and not at the level of the pathway structural genes (Tonelli et al., 1991; Procissi et al., 1997;
Piazza et al., 2002). Thus, the BMS35S::R+35S::C1 cells provide a powerful tool to uncouple
30 the effect of light on anthocyanin accumulation and pigmentation, something not feasible
in most plant systems, where anthocyanins are induced by light.
The similar levels of anthocyanins in the BMS35S::R+35S::C1 cells under dark or light
conditions allowed us to uncover a second effect of light on pigmentation. Light-treated
BMS35S::R+35S::C1 cells were darker in color, when compared to identical cells grown in the
dark (Fig. 1.2). Significant quantifiable reflectance differences were observed between
BMS35S::R+35S::C1 cells grown for six days in continuous light or dark (Table 1.1), changes
that were not associated with a variation in the amount or type of anthocyanins present
(Fig. 1.3). Microscopy studies established that extensive vacuolar morphological
alterations (Fig. 1.6) correlate with the color darkening. BMS cells not expressing the
anthocyanin regulators usually had one or a few vacuolar compartments (Fig. 1.5). In
contrast, constitutive expression of R and C1 in these cells resulted in a remarkable
increase in vacuole number. It remains to be established whether the accumulation of
flavonoids/anthocyanins, the expression of the regulators, or both are necessary and
sufficient to trigger the biogenesis of new vacuolar compartments. Within the vacuoles, anthocyanins accumulate in maize cells in red spherical bodies that resemble vacuolar anthocyanoplasts (Grotewold et al., 1998). Although we have not yet established
whether the anthocyanin inclusions present in BMS35S::R+35S::C1 cells are membrane-
bound, they have very similar characteristics to the recently-described yellow auto-
fluorescent bodies (YFB) present in the vacuoles of BMS cells, induced by the expression
of the P1 regulator of 3-deoxy flavonoid biosynthesis (Lin et al., 2003).
The organization of the anthocyanin inclusions present in BMS35S::R+35S::C1 cells undergo dramatic modifications in the presence of light. These alterations include a
31 reduction in their numbers and an enlargement of their size (Fig. 1.6). In addition, light-
treated vacuoles often showed a spreading of the anthocyanin pigment within the
vacuolar lumen, which may be a result of release from the AVI-like structures. The
morphological changes observed in the vacuoles of BMS35S::R+35S::C1 cells are not a
unique property of cells in culture. We uncovered similar light-induced alterations in the
vacuolar structure of maize floral tissues accumulating high levels of anthocyanins (Fig.
1.8, 1.9). Time-lapse experiments illustrate the formation and fusions of tubular and globular anthocyanin-filled structures that ultimately coalesce to give one or a few large central vacuoles characteristic of pigmented maize cells. These tubular-vesicular structures, some of which are filled with clear vesicles, are reminiscent of observations of the tubular pro-vacuoles found in vacuolating Euphorbia root cells (Marty, 1978, 1999).
The anatomical identity of these structures remains to be established, but similar to what was reported for the Euphorbia root cells, the ontogenesis of larger anthocyanin- containing vacuoles from smaller ones is reminiscent of vacuolar fusion and/or autophagy.
It is possible that these light-induced morphological alterations in the anthocyanin-containing structures are directly associated, if not responsible, for the
observed color changes. However, alterations in vacuolar pH or the association of
anthocyanins with co-pigments can also result in changes in the hue of anthocyanin
pigmentation (Forkmann, 1991; Markham and Ofman, 1993). To investigate whether
light induces a change in the vacuolar pH of BMS cells, we utilized the BCECF-AM
fluorescent dye, for which the ratio of fluorescence emission at the dual excitation
wavelengths of 490nm/440nm can be calibrated to an in vivo generated pH curve
32 (Swanson and Jones, 1996). Using this method, we established that the vacuoles of dark
and light grown BMS cells were acidic at pH 5.8 and showed no significant pH differences (Fig 1.11B). Similarly, pH measurement of crude homogenates of BMS or
BMS35S::R+35S::C1 cells in water did not yield significant pH value differences between
each other or between light- and dark-grown cells (Fig 1.11 E). We were unable to use
the BCECF-AM to measure vacuolar pH in the R+C1 cells as the absorption spectra of
anthocyanins overlaps with the emission spectra of BCECF-AM (Fig 1.3 A; Fig 1.11 A).
Anthocyanins, when ‘spread’ into the vacuolar sap, tend to quench the fluorescence of
BCECF as evident in the light grown BMS35S::R+35S::C1 cells (Fig 1.8H).
33
A C 490nm Calibration of BCECF-AM loaded 6d 2.8 Dark grown BMS cells
2.6
2.4 2.2
440nm
490/440 ratio 490/440 2 Isosbestic point 1.8
1.6
5 6 .2 .4 .6 .8 .2 .4 .6 .8 5 5 5 5 pH 6 6 6 6 www.probes.com B D Calibration of BCECF-AM loaded 6d Light grown BMS cells 2.8 2.00 2.6 1.95 2.4 1.90 2.2 1.85 490/440 ratio 490/440
490/440 ratio 2 1.80 1.8 1.75 Dark Light 1.6 5 6 .2 .4 .6 .8 .2 .4 .6 .8 5 5 5 5 6 6 6 6 pH E BMS pH units ______Dark 5.8 ± 0.1395 Light 5.8 ± 0.0301
Figure 1.11 Measurement of vacuolar pH of BMS cells using BCECF-AM: Equal amounts (0.01g/100 µl fresh wt) of BCECF AM loaded cells were placed in microtiter plates, and the emission measured at 535 nm, 440 nm and 490 nm excitation wavelengths (A). The 490/440 ratio was calculated (B). An in situ calibration curve was generated separately for each of the dark and light samples with various pH buffers with 0.005% digitonin (C, D). Vacuoles of both dark and light grown BMS cells were acidic at ~pH 5.8 and showed no significant pH differences (E).
34 BMS BMS35S::R+35S::C1 0.15 0.15 BMS dark 280nm R+C1 dark 280nm
0.10 0.10 AU AU 0.05 0.05
0.00 0.00 0.15 5.00 10.00 15.00 20.00 25.00 30.00 0.15 5.00 10.00 15.00 20.00 25.00 30.00 BMS light Minutes 280nm R+C1 light Minutes 280nm 0.10 0.10 AU AU 0.05 0.05
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes Minutes 0.030 0.030 BMS dark 360nm R+C1 dark 360nm 0.020 0.020 AU AU 0.010 0.010
0.000 0.000
0.030 5.00 10.00 15.00 20.00 25.00 30.00 0.030 5.00 10.00 15.00 20.00 25.00 30.00 BMS light Minutes 360nm R+C1 light Minutes 360nm 0.020 0.020
AU AU 0.010 0.010 0.000 0.000 5.00 10.00 15.00 20.00 25.00 30.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes Minutes 0.08 0.08 BMS dark 520nm R+C1 dark C P 520nm
0.06 0.06 0.08 0.08 519.5 280.5 519.5 0.06 280.5 0.06
0.04 0.04 0.04 0.04 AU AU AU AU 0.02 0.02 0.02 0.02 0.00 0.00 300.00 400.00 500.00 300.00 400.00 500.00 nm nm 0.00 0.00 0.08 5.00 10.00 15.00 20.00 25.00 30.00 0.08 5.00 10.00 15.00 20.00 25.00 30.00 BMS light Minutes 520nm R+C1 light MinutesC P 520nm 0.06 0.06 0.08 0.08
0.06 0.06 517.1 280.5 518.3 280.5 0.04 0.04 0.04 0.04 AU AU AU AU 0.02 0.02 0.02 0.02 0.00 0.00 300.00 400.00 500.00 300.00 400.00 500.00 nm nm 0.00 0.00
5.00 10.00 15.00 20.00 25.00 30.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes Minutes
Figure 1.12 HPLC analysis of dark and light grown BMS35S::R+35S::C1 cells show no significant change in their phenolic profiles: HPLC analysis of methanolic extracts of dark and light grown BMS and BMS35S::R+35S::C1 cells. Chromatogram profiles were extracted at 280 nm (phenylpropanoids, colourless flavonoids), 360 nm (flavonols) and 520 nm (anthocyanins). Inserts represent the spectra of cyanin (C) with a retention time of ~16.6 min and pelargonin (P) with ~17.8 min.
35 Although unlikely, based on the absence of a shift of the λmax of the pigments
(Fig. 1.3A, B), we also investigated whether light participated in the induction of
phytochemicals that could serve as co-pigments, and hence contribute to the color
change. Reverse phase HPLC analyses exhibited no significant differences in the peak
profiles of phenolic compounds in the dark and light grown BMS and BMS35S::R+35S::C1 cells (Fig 1.12).
While these results do not rule out the possibility of a local and/or transient light- induced pH change or the induction of a minor co-pigment responsible for the color shift, it is possible that the alterations in vacuolar morphology underlie light-induced darkening phenomenon of the anthocyanin containing cells. Similar light-induced effects were previously described for the anthocyanoplasts of red cabbage, an observation that was attributed to an increased accumulation of anthocyanins enhanced by light (Pecket and
Small, 1980). Our results, however, offer the alternative explanation that light itself can affect the packaging and distribution of anthocyanins within the vacuole, independent of variations in the levels of anthocyanins. Similarly, flowers of the “Rhapsody in Blue” rose cultivar show a change in color induced by age, from reddish-purple to bluish- purple, and this variation was associated with a progressive accumulation of anthocyanins into AVI-like structures (Gonnet, 2003). Lisianthus flowers also show a correlation between the packaging of anthocyanins into AVIs, the presence of “blackish-purple” pigmentation at the base of the petal, and the reduction or absence of AVIs in the outer zones, associated with a lighter purple color of this region (Markham et al., 2000). It is thus possible that the observed alteration in the hue of light-grown BMS35S::R+35S::C1 cells reflects a much more general mechanism of light on the packaging of anthocyanins
36 within the vacuole, and hence on pigmentation. If so, the BMS35S::R+35S::C1 cells provide a convenient system for the dissection of the mechanisms of this process because of the molecular and cellular tools available.
1.5 Conclusions and future directions
The results presented here provide evidence that light affects anthocyanin pigmentation by mechanisms beyond the transcriptional regulation of genes encoding pathway enzymes. In maize floral organs and cultured cells, light induces dramatic morphological alterations in the packaging of anthocyanins and distribution of vacuolar and sub-vacuolar compartments. Similar phenomena have been observed before, but the difficulties associated in uncoupling anthocyanin production with morphological alterations in their packaging prevented inferences on cause-consequence relationships.
The dynamic movement of the tubular and vesicular compartments as seen in the maize tassel glume epidermal cells, the presence of AVIs as a sub-vacuolar compartments and the change in vacuole organisation on exposure to light have two important implications. One possibility is that there are two routes of transport of anthocyanins into the central vacuole, one utilizing transporters to directly pump the pigments from the cytoplasm into the central vacuole and a vesicular route where the transporters pump the anthocyanins into the ER or pre-vacuolar compartments which then go on to fuse with the tonoplast delivering its cargo using vesicular transport.
Second, it is possible that autophagy plays a role in the uptake of anthocyanins into the vacuole whereby vesicles can either fuse with the tonoplast and empty their contents in the sap or may be taken up by active autophagy into the vacuole. The presence of AVIs
37 as subvacuolar structures supports the autophagy mechanism. These possibilities are currently being tested in the lab using mutants and inhibitors in a convenient seedling model system developed in Arabidopsis thaliana.
38 CHAPTER 2
METABOLOME AND TRANSCIPTOME CHANGES IN ARABIDOPSIS
THALIANA SEEDLINGS INDUCED TO ACCUMULATE ANTHOCYANINS.
2.1 Introduction:
Plants synthesize more than 200,000 natural products or secondary metabolites,
many of which have immense nutraceutical and pharmaceutical value (Dixon, 2005).
This chemical diversity is a consequence of the sessile characteristic of plants and their
response to the local environment, where their chemical arsenal confers ecological fitness
providing a wide range of functions ranging from mechanical strength to protection
against biotic and abiotic stress (Stafford, 1990; Wink, 1999; Winkel-Shirley, 2001). The
extensive catalogue of natural products is a result of modifications of a relatively limited
number of core chemical structures. For example, the terpenoid and the polyketide
scaffolds are tailored with hydroxylation, glycosylation, acylation, prenylation, and O-
methylation reactions catalyzed by cytochrome P450 monooxygenases and transferases, which are encoded by large gene families (Martinoia et al., 2000b; Dixon et al., 2005).
Furthermore, to exert their effects, secondary metabolites need to be synthesized in large
39 amounts and stored in specific cell types until needed (Martinoia et al., 2000b) and their
proper compartmentalization is essential to prevent plants from poisoning themselves
(Matile, 1987). Thus, understanding the cellular machinery by which plant cells adopt to accumulate and sequester biologically active, highly abundant and inherently toxic secondary metabolites, is paramount to manipulate secondary metabolism in plants
(Martinoia et al., 2000b).
Anthocyanins, being colored pigments, provide a convenient visual marker to address the questions surrounding the transport and uptake of secondary metabolites into the vacuole. Anthocyanins, with a C6-C3-C6 core flavonoid skeleton, are synthesized from the general phenylpropanoid pathway, by the action of at least six flavonoid biosynthetic enzymes (Figure 2.2) (Winkel-Shirley, 2001; Irani et al., 2003).
Anthocyanins are transported to the large central vacuole where pH, co-pigmentation and the formation of anthocyanic vacuolar inclusions (AVIs) influence proper pigmentation, stability, and prevention of oxidation (Mol et al., 1998; Markham et al., 2000). Thus, anthocyanins (or anthocyanin precursors) need to be efficiently trafficked from the cytoplasmic surface of the ER and subsequently be sequestered within the vacuole.
The hydrophobic aglycone forms of flavonoids are toxic and need to be sequestered away from the cytoplasm to either the vacuole as described for anthocyanins, proanthocyanins, flavonol glycosides and phytoalexins or secreted to the extracellular matrix as in the case of polymethylated flavonol glycosides and deoxyanthocyanins
(Hrazdina and Jensen, 1992; Klein et al., 1996; Li et al., 1997; Grotewold et al., 1998).
Many of the mechanisms involved in the storage of flavonoids and other natural products are used by plants to detoxify xenobiotics such as toxins secreted by pathogens and
40 herbicides and pesticides which involve the induction of detoxifying enzymes and transporters (Debeaujon et al., 2001). The process involves distinct phases of activation, conjugation, elimination (compartmentalization) and transformation. In phase I, the compounds are oxidized, reduced or hydrolyzed by cytochrome P450s, hydrolases or peroxidases to expose or introduce a functional group that makes its more reactive. Phase
II involves the deactivation of the compounds by covalent conjugation to hydrophilic molecules such as glycosyl, methyl, sulphonyl, acyl and prenyl groups and glutathione among others, by their respective transferases. In the process of elimation or compartmentalization in phase III, these hydrophilic, conjugated compounds are recognized by specific membrane-associated transporters and pumped either into the endomembrane compartments, for example the vacuole or to the extracellular space.
Once delivered, they may be additionally modified, degraded and/or salvaged, which is sometimes referred to as phase IV. The key aspect of detoxification, especially the conjugation step, is to increase the hydrophobicity and/or net charge of the molecule to reduce non-specific binding to biological membranes, stabilize the molecule and ease the transport across the cytoplasm. These steps may also introduce a functional tag that would determine the transporter and/or transport route it would adopt (Sandermann,
1992, 1994; Coleman et al., 1997; Martinoia et al., 2000a; Debeaujon et al., 2001; Jones and Vogt, 2001; Baerson et al., 2005).
Although the steps in biosynthetic pathways are often known, the cellular events involved in the sequestration to internal compartments or the secretion to the extracellular matrix still remains poorly understood (Grotewold, 2004). Over the past few years, several factors that affect the proper compartmentalization of anthocyanins have been 41 identified (Table 2.1). Phase II enzymes encoding glycosyl-transferases, acyl-tranferases
as well as glutathione S-transferase, have been identified. Anthocyanin accumulation is
suppressed in the bronze 1 (bz1) mutant in maize which encodes a UDP glucose: cyanidin
3-O-glucosyltransferase (Larson and Coe, 1977; Fedoroff et al., 1984). Recently, a functional genomics approach integrating metabolic profiling and transcriptome changes in PAP1 (AtMyb75) over expressing Arabidopsis plants that over-accumulated anthocyanins, identified a flavonoid 3-O-glycosyltransferase (UGT78D2, At5g17050) and anthocyanin 5-O-glucosyltransferase (UGT75C1, At4g14090), which were then functionally characterized, and two anthocyanin 5-aromatic acyltransferases (At1g03940,
At3g29590), which shared homology with their Perilla frutescens and Gentiana triflora counterparts (Tohge et al., 2005).
The bronze 2 (bz2) mutation of maize accumulates cyanidin-3-glycoside (C3G) in the cytoplasm as brown pigments. BZ2 encodes a glutathione S-transferase (GST), which was proposed to conjugate glutathione to C3G. The glutathionylated C3G was proposed to be taken up into the vacuole by a glutathione S-conjugate (GS-X) pump (Marrs et al.,
1995; Alfenito et al., 1998). Similarly, the Petunia AN9 gene encodes a GST and, despite the low identity between AN9 and BZ2, BZ2 complements AN9 mutants (Alfenito et al.,
1998). Interestingly, the GST enzymatic activity of AN9 is not required for the AN9- dependent vacuolar sequestration of anthocyanins, leading to a model in which AN9/BZ2 served as carrier protein for the transport of anthocyanins from the ER to the tonoplast or that the binding of the anthocyanins to the GST prevented its oxidation and accumulation in the cytoplasm as seen in the bz2 mutants of maize (Mueller et al., 2000).
TRANSPARENT TESTA 19 (TT19) mutations affect both anthocyanin accumulation in
42 vegetative tissues and proanthocyanidin (PA) accumulation in seed coats. TT19 encodes
a GST, and interestingly, BZ2 complements the anthocyanins deficiency in the plant
body but not the PA defect of the testa of tt19 mutants (Kitamura et al., 2004). While
TT19 and AN9/BZ2 may have similar functions in the vacuolar transport of
anthocyanins, tt19 mutants have a distinctive phenotype in the seed coat, where PA
precursors accumulate in cytoplasmic, membrane-wrapped structures (Kitamura et al.,
2004).
Transport across the membrane is an active process accomplished by H+ antiporters, electrogenic uniporters and ATP-binding cassette (ABC) transporters. Trans- membrane transporters of the vacuolar type H+-transporting adenosine triphosphatase (V-
ATPase) and the vacuolar type H+-transporting pyrophosphatase (V-PPase) establish a H+ electrochemical gradient across the membrane (Sze et al., 2002) which drives uptake through H+ antiporters and electrogenic uniporters (Martinoia et al., 2000a). ABC
transporters on the other hand are independent of the proton motive force and are
energized by Mg-ATP (Klein et al., 2006). Thus, depending on their conjugation, the
secondary metabolites may be taken up by either the ABC transporter or the H+ antiporters (Martinoia et al., 2002). Extensive in vitro inhibitor studies utilizing isolated vacuoles or microsomal fractions have implicated ABC transporters as well as the antiporters in the uptake of glutathione or glycosyl conjugated compounds (Hopp and
Seitz, 1987; Li et al., 1997). However, very little is known about the molecular identity and characterization of the transporters involved. Recently, a maize tonoplast-localized multidrug resistance-associate protein, ZmMRP3 (Goodman et al., 2004), a member of the ABC transporter superfamily, induced by the C1 and R regulators of anthocyanin
43 biosynthesis (Bruce et al., 2000) was implicated in the transport of anthocyanins.
Antisense lines of ZmMRP3 show reduced levels of anthocyanins and the lack of an
aleurone phenotype suggest the presence of additional redundant transporters (Goodman
et al., 2004). This ABC transporter provides an additional player in a model involving
protein carriers and transporter in the trafficking of anthocyanins from the ER surface to the vacuole. In Arabidopsis, the mutation in the TT12 locus, which encodes a proton coupled multidrug and toxin extrusion transporter (MATE), is involved in loading PAs into the provacuolar compartments of endothelial cells of the seeds (Debeaujon et al.,
2001). The endothelial cells of tt12 seeds accumulate numerous small vacuoles as compared to the large central vacuole observed in wild type (Baxter et al., 2005). Mutants in the plasma membrane H+ pump, auto inhibited H+ ATPase isoform 10 (AHA10), which is primarily expressed in the seeds, resulted in a transparent testa phenotype. There was no change in the accumulation of cyanidin or the flavonols quercetin or kaempferol, but a 100 fold reduction in the accumulation of PAs. As in tt12 mutants, the endothelial cells accumulated numerous small vacuoles. It was hypothesized that AHA10 was recruited from the ER or plasma membrane to acidify endosomes or PVCs, the proton gradient driving cargo uptake through a pump, [e.g, a MATE (TT12)] (Baxter et al.,
2005).
Transport routes may involve the molecule being directly pumped into its final storage site, or alternatively, being transported into intermediate compartments which may involve ER, ER-derived vesicles and/or PVCs which would then deliver the cargo, either to the extracellular matrix or to the vacuole, by vesicle fusion. Evidence for this intermediate vesicle route has come from a number of studies in pathogen induced
44 vesicles containing 3-deoxyanthocyanin phytoalexins (Snyder and Nicholson, 1990), maize cells induced to accumulate anthocyanins (Grotewold et al., 1998; Irani and
Grotewold, 2005), red cabbage (Pecket and Small, 1980), and Arabidopsis mutants in tt12, aha10 and leucoanthocyanidin dioxygenase (LDOX), where endothelial cells of seeds accumulate numerous small compartments instead of a large central vacuole
(Baxter et al., 2005). The ER has also been proposed to be the initial site of PA accumulation, where vesicles would bud off and deliver the cargo to the large central vacuole (Parham and Kaustinen, 1977; Stafford, 1990). Studies in our lab showed some aspects of the vesicle route responsible for the transport of green and yellow fluorescent compounds in maize cells and anthocyanins in Arabidopsis seedlings to be Golgi- independent, and to adopt a direct ER to vacuole, or ER to plasma membrane route (Lin et al., 2003) (unpublished results). Spindle-shaped ER bodies (Matsushima et al., 2003) provide possible vehicles for the transport of proteins, rubber or oil from the ER to the vacuole, by a mechanism resembling autophagy (Herman et al. 2004). Whether ER bodies are involved in the transport of PAs or anthocyanins from the ER to the vacuole remains unclear, but the localization of Arabidopsis flavonoid biosynthetic enzymes to large electron-dense cytoplasmic structures and to the tonoplast (Saslowsky et al., 2005) suggest that this might be indeed the case.
Arabidopsis thaliana, with its genome sequenced, and the availability of mutants, offers an excellent model system to address cellular, biochemical and molecular mechanisms of the sequestration of anthocyanins. The flavonoid pathway is well characterized with single genes coding for the major biosynthetic enzymes (Winkel-
Shirley, 2001). However, steps involved in the conjugation and transport of anthocyanins
45 still remain poorly defined. The availability of a nearly whole genome oligonucleotide
array (Redman et al., 2004) provides the opportunity to probe which of the complex gene
families such as the cytochrome P450s, glutathione S-transferases, glycosyl transferases,
and transporters are involved in the modification and transport of anthocyanins to the vacuole.
In this chapter, I describe the development of a convenient anthocyanins- inducible system in Arabidopsis thaliana by exploiting the transparent testa (tt) mutants.
The series of tt mutants, as the name suggests, do not accumulate proanthocyanidins in their seed coats (Koornneef, 1990). They appear pale yellow (Fig 2.3 A a) as compared to the rich brown seeds of the wild type (Fig 2.3 A b). The chalcone isomerase (At3g55120) tt5 mutant was isolated as a fast-neutron irradiation mutant (Koornneef, 1990) where an inversion within the gene produces a defective transcript that is present at reduced levels as compared to wt (Shirley et al., 1992; Shirley et al., 1995). CHI catalyses the conversion of chalcones to flavanones (Fig 2.2) and the mutation disrupts the flavonoid pathway leading to no accumulation of anthocyanins in the plant body or any proanthocyanins in the seed coat. In a previous study, addition of 100µM of naringenin, the metabolic product of CHI, complements tt5 seedlings (Shirley et al. 1995). A simple
media of liquid 3% sucrose and high light was used to robustly induce synthesis of
anthocyanins in the cotyledons of Arabidopsis seedlings. tt5 seedlings were induced to accumulate anthocyanins by the addition of naringenin. Metabolite profiling shows no change in the type of the accumulated anthocyanidin, but striking changes in anthocyanin profiles. There is de novo synthesis of a new anthocyanin peak on the addition of naringenin which was identified as cyanidin 3-glycoside. Transcriptome profiling was
46 used as a gene discovery tool to identify enzymes involved in the modification and
transport of the flavonoid naringenin or anthocyanins. Transporters, GSTs and UDP
glycosyltransferases were affected on addition of naringenin and may be potential targets
involved in the transport process. There was no effect of naringenin on the expression
levels of the core flavonoid biosynthetic genes and the transcription factors (TF) that control them, indicating no feedback regulation of the pathway genes at the transcriptional level. Many down-regulated genes show root specific expression and appear to be involved in cell elongation and growth of the radicle, consistent with the possibility that naringenin acts as a growth inhibitor at high concentrations. Furthermore, up-regulation of transcription factors and genes involved in signal transduction may provide clues as to which pathway networks are invoked in plant cells in the presence of naringenin or anthocyanins or any of the flavonoid intermediates.
47
Transporters
Transport / Protein carriers Detoxification, Sequestration Vesicles
Combinations of the above
Small Metabolism molecule Feedback regulation of its own pathway at transcription or enzymatic level
Effector of Signal transduction – stimulus as proteins: stress response; biotic or abiotic Activation or repression Transcription factors
Enhance or perturb a cellular process.
Figure 2.1 Schematic representation of the fate of a small molecule within a cell: In addition to being utilized in its metabolic pathway, small molecules may act as modulators of a variety of cellular processes. Transport/detoxification occurs by a number of routes that involves transporters, protein carriers, vesicles and/or combinations of the afore mentioned. The small molecule may affect cellular processes by directly binding to proteins to activate or repress their function. They may act as signaling molecules triggering signal transduction pathway. They may feed-back regulate their own pathway either allosterically or at the expression level.
48 Figure 2.2 The core flavonoid pathway leading to the major groups of flavonoid pigments: The general phenypropanoid pathway converts phenylalanine and tyrosine into the precursor p-coumaryl CoA. The first committed step catalyzed by CHS condenses p-coumaryl-CoA with three molecules of acetyl-CoA derived from malonyl- CoA to form the fifteen carbon skeleton. I show here just the three most common anthocyanins, which upon modifications that include methylations, acylations, hydroxylations, and glycosylations yield a wide variety of derivatives. The abbreviations of the depicted enzymes are as follows: PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumaroyl:CoA-ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3’H, flavonoid 3’hydroxylase; F5’H, flavonoid 5’hydroxylase; F3’,5’H, flavonoid 3’,5’ hydroxylase; F3H, flavanone 3-hydroxylase; DFR, dihydroflavonol 4-reductase; LDOX, leucoanthocyanidin dioxygenase/AS, anthocyanidin synthase; UFGT, UDP glucose:flavonoid 3-O-glucosyl transferase. λmax values correspond to aqueous methanol-HCl solutions. Steriochemistry has been omitted for the sake of simplicity (Adapted from Irani et al. 2003).
49 OH R OH LIGNINS OH O HO SINAPATE ESTERS
H2N STILBENES O PHLOBAPHENES R PAL HO Tyrosine OH HO OH HO OH O
PAL C4H 4CL R HO HO HO SCoA HO H2N OH O O O O Phenylalanine Cinnamic acid p-Coumaric acid p-Coumaroyl CoA OH O
COOH
+ 3 H2C HO HO ISOFLAVONOIDS SCoA CHS Malonyl-CoA
OH R R OH OH OH OH OH O OH O + HO O O n CHALCONE AURONES Tetrahydroxy chalcone HO HO HO 3-DEOXY- CHI FLAVAN-4-OLS ANTHOCYANIDINS [Apiferol (R=H) [Apigeninidin (R=H) Luteoferol (R=OH)] DFR Luteolinidin (R=OH)] OH F3'5'H OH OH OH OH OH O OH O OH OH O F3'5'H F3'H
HO O HO O HO O FLAVANONES Pentahydroxyflavanone Naringenin Eriodictyol
F3H F3H F3H OH OH OH OH OH FLAVONES OH O F3'H OH O F3'5'H OH O OH OH OH OH HO O HO O HO O 5,7-OH-FLAVANONES F3'5'H Dihydrokaempferol Dihydroquercitin (DIHYDROFLAVONOLS) Dihydromyricetin DFR DFR DFR
OH OH OH OH OH FLAVONOLS OH O OH O HO O OH
OH OH OH HO HO HO HO HO HO LEUCOANTHOCYANIDINS Leucodelphinidin Leucopelargonidin Leucocyanidin
LDOX/AS LDOX/AS LDOX/AS OH OH OH OH OH PROANTHOCYANIDINS OH (CONDENSED TANNINS) OH O OH O O + OH + + OH OH OH HO HO HO ANTHOCYANIDINS Delphinidin Pelargonidin Cyanidin
UFGT UFGT UFGT
OH OH OH OH OH
HO O HO O HO O + OH + + O-Glc O-Glc O-Glc HO HO HO ANTHOCYANINS Delphinidin 3-glucoside Pelargonidin 3-glucoside Cyanidin 3-glucoside
OH OH OH OH OH
R'' O R'' O R'' O + OH + + O-R''' O-R''' O-R''' HO HO HO ANTHOCYANIN DERIVITIVES
Fig. 2.2
50
Class Protein Organism Reference UDP glucose: cyanidin 3-O- BZ1 Zea mays (Fedoroff et al., 1984) glucosyltransferase UDP-glucose:flavonoid 3-O- VvUFGT Vitis vinifera (Ford et al., 1998) glucosyltransferase UDP-glucose: flavonoid 3-O- UGT78D2 Arabidopsis thaliana (Tohge et al., 2005) glucosyltransferase (At5g17050) UDP-glucose anthocynin 5-O- UGT75C1 Arabidopsis thaliana (Tohge et al., 2005) glucosyltransferase (At4g14090) Anthocyanin 5-aromatic acyltransferase Gentiana triflora (Fujiwara et al., 1998) Hydroxycinnamoyl-CoA: anthocyanin (Yonekura-Sakakibara Perilla frutescens, 3-O-glucoside-6"-O-acyltransferase et al., 2000) Anthocyanin 5-aromatic acyltransferase Arabidopsis thaliana (Tohge et al., 2005) (At3g29590) Glutathione S-transferase BZ2 Zea mays (Marrs et al., 1995) Glutathione S-transferase AN9 Petunia hybrida (Alfenito et al., 1998) Glutathione S-transferase (At5g17220) TT19 Arabidopsis thaliana (Kitamura et al., 2004) (Debeaujon et al., MATE transporter (At3g59030) TT12 Arabidopsis thaliana 2001) (Bruce et al., 2000; ABC transporter MRP3 Zea mays Goodman et al., 2004) P type H+ ATPase transporter AHA10 Arabidopsis thaliana (Baxter et al., 2005) (At1g17260)
Table 2.1 Proteins that play a role in flavonoid modification and/or transport to the vacuole.
51 2.2 Materials and Methods
2.2.1 Plant materials and growth conditions
Arabidopsis chalcone isomerase (tt5) mutants and wild type (ecotype Landsberg)
seeds were obtained from ABRC (Columbus, Ohio). Approximately 75 µl to 100 µl of
seeds were taken in a 1.5 ml microfuge tube and surface-sterilized. The surface
sterilization regime involved a 1 min 95% alcohol soak, 6 min incubation with a
sterilization mix of bleach:water:Tween20 at 1:1:0.05 (v/v/v) on a rotatory shaker. After
three sterile water washes, a final water suspension was made up to 1.2 ml. Two hundred microliter of an even suspension was plated in 6 ml of liquid 3% sucrose (w/v) or water in 35 mm petri plates. This resulted in a uniform plating density of ~50 to 70 seeds per ml
(1 ml of Arabidopsis seeds = ~25,000 seeds; (ABRC, The Ohio State University,
Columbus, OH). Plates were cold treated at 4°C for two days. Plates were shifted to a culture room at 25°C ± 2°C, with continuous cool white light (GE F30T12-CW-RS) at
~100 ±10 μmol.m-2.s-1, on a rotatory shaker at 100 rpm.
Plants were propagated in soil (PRO-MIX BX, Premier Horticulture Inc., USA).
Seeds were stratified at +4°C for 2 d and then transferred to a growth chamber at
22°C±2°C with a 16 h light, 8 h dark regime and 60 to 80% relative humidity.
2.2.2 Naringenin and cycloheximide treatments
The seedlings were grown for 2.5 d and 100 µM of naringenin (Sigma, USA;
stock 100mM in 95% ethanol) was added to the medium. Twenty four hours after
addition of naringenin, seedlings were harvested. 52 For cycloheximide treatments, 2.5d old seedlings were pre-treated with 100µM
cycloheximide (Sigma, USA; 100mM stock in 95% ethanol) for 30 min after which 100
µM naringenin was added. Ethanol was substituted for the control. Seedlings were
harvested after 24h.
Naringenin inhibition studies were carried out on MS medium (Murashige and
Skoog, 1962) with 3% sucrose, solidified with 0.7% agar. Seeds were plated on MS plates, cold treated for 2 d at 4°C and grown vertically in the growth chamber. 4 to 5 day old seedlings whose roots were ~1.5 cm long were transferred aseptically to new MS plates with concentrations of naringenin ranging from 0 µM to 500 µM. The seedlings were aligned in a straight line 1cm apart. Growth was observed at an end point of 10 d after transfer.
2.2.3 Imaging seedlings
Seeds and seedling were imaged under an Olympus SZH10 Research Stereo microscope (Olympus, Japan), attached to an Olympus DP10 digital camera. Epidermal cells of cotyledons were observed under a Nikon Eclipse 600 compound microscope
(Nikon, Japan) equipped with Nomarski differential interference contrast optics (DIC) using a 100X oil immersion objective. Images were captured and processed with a SPOT
2 Slider charge-coupled device camera and the processed with the SPOT imaging software (Diagnostic Instruments, Sterling Heights, MI). All images were further processed using Adobe Photoshop 7.0 (Adobe Systems, Mountain View, CA).
53 2.2.4 Anthocyanin extraction and analysis by spectrophotometry, TLC and HPLC.
Harvested seedlings were rinsed with water and lyophilized for two days. Dry weight was measured and 50% methanol in water (v/v) was added to a final suspension of 50 µg/µl (dry w/v). Seedlings were allowed to soak overnight at room temperature and stored at -20°C until further use.
For spectrophotometric analysis, 1% HCl (v/v) in 50% MeOH (v/v) (acidic methanol) was added to the methanolic extract in a 2:1 (v/v) ratio and read at 530 nm with a Cary 50 UV-VIS spectrophotometer (Varian, Inc. USA). Forty microliter quartz microcuvettes (1cm path length) were used.
TLC analysis was carried out with hydrolyzed anthocyanins. An equal volume of
2 M HCl was added to the methanolic extract to a final concentration of 1 M HCl and boiled in a boiling water bath for 20 min. Iso-amyl alcohol was added to extract the anthocyanidins at a volume of no more than one-fourth the original volume of the hydrolyzed mix. Equal volumes of the isoamyl alcohol extract were spotted onto cellulose TLC plates (5730/6, Merck, EM Science, Germany) and run in a pre-saturated chamber with water:formic acid:HCl (10:30:3, v/v/v). Plates were dried and photographed in white light using a Nikon Coolpix 5700 digital camera (Nikon, Japan).
HPLC analysis involved injecting 20 µl of methanolic extract of non-hydrolyzed and 40 µl of hydrolyzed samples into a Waters Alliance® 2695 Separations module
(Waters Corporation, Milford, MA) in conditions as described (Dias et al., 2003). The
HPLC profiles were collected from 200 to 600nm using the Waters 2996 Photodiode
Array Detector. Chromatograms and spectra were extracted and analyzed with the
EmpowerTM software (Waters Corporation, Milford, MA).
54 2.2.5 Extraction of RNA
Seedlings were homogenized in liquid nitrogen and total RNA was extracted
using the TRIzol reagent following the manufacturers recommendations (Invitrogen, Life
Technologies, USA). Briefly, homogenized tissue was re-suspended in TRIzol reagent at
a concentration of ~100 mg fresh weight/ml TRIzol and incubated at RT for 5min on a
rotatory shaker. To clear the lysate, the mixture was spun down at 3500 g for 30 min (or
maximum speed for 10min if using microfuge tubes) in a table-top centrifuge (Fisher
Scientific, USA). This additional step was introduced to obtain a cleaner RNA
preparation. The cleared lysate was transferred to a fresh tube and 0.2 volumes of
chloroform per ml of initial ml of TRIzol reagent used was added and incubated at RT for
2 min on a rotatory platform for phase separation. The mixture was spun down at 3500 g
for 30min. The upper aqueous phase was transferred, being careful not to disturb the interphase layer, to a fresh tube and 0.5 volumes of isopropanol, per ml of initial TRIzol reagent used was added, mixed and incubated at RT for 15min. RNA was precipitated by spinning down at 3500 g for 60min. RNA was washed with 75% ethanol made in RNase free water (USB) using 2 ml per ml of TRizol used. The pellets were dislodged and spun down, air dried for 5 min and dissolved in RNase free water by heating at 65°C for
10min. The RNA was spectrophotometrically quantitated at 260 nm.
2.2.6 Northern Blot Analysis
For Northern analyses, 20 μg of total Arabidopsis seedling RNA was separated on a formaldehyde-containing 1% (w/v) agarose gel and blotted onto a nitrocellulose membrane (Bio-Rad Laboratories, Inc., USA). The blot was hybridized with cDNA
55 probes corresponding to AtCHS and AtF3H (Pelletier et al., 1999). Actin-related protein 6
(At3g33520) (Zhao et al., 2006) was used as a normalization control. Comparison of the hybridization signals was performed on a BioRad phosphorimager (BioRad Laboratories,
Inc., USA).
2.2.7 Microarray analysis: RNA extraction and hybridization.
The total RNA prepared by the TRIzol method was further purified using RNA mini spin columns (Qiagen) according to the manufacturer’s protocol. Not more than 100
μg of RNA was loaded onto the column and two subsequent elutions of 50 μl and 30 μl with RNase free water were pooled and quantified spectrophotometrically. The RNA was further processed at the Functional Genomics Core Facilty, Columbus Children’s
Research Institute (Columbus, Ohio; http://www.dnaarrays.org). The RNA integrity and
amount was quantified by capillary electrophoresis using fluorescence with a Bioanalyzer
2100 (Agilent) and data was analyzed using the Degradometer software.
Either biotin labeled cRNA
(http://www.dnaarrays.org/protocols/GeneChip_Protocol.doc) or biotin labeled cDNA
(NuGen Ribo-SPIA RNA amplification,
http://www.nugeninc.com/pdfs/Ovation_Biotin_UserGuide.pdf) was synthesized from 25
ng of total RNA and hybridized to ATH1 whole genome chip (Affymetrix Corporation,
Redman et al., 2004). The hybridized biotin labeled cRNA or cDNA was visualized by
biotin staining with streptavidin R-phycoerythrin. The scanned images contain
quantitative information about fluorescence and the data obtained is further analyzed
using microarray data analysis packages.
56 2.2.8 Microarray data analysis.
The background adjustment, cross-ship normalization was done by Robust
Multichip Analysis (RMA) using a robust linear model to summarize probe level
expression values (Irizarry et al., 2003). This analysis was performed using AffylmGUI
(version 1.5.4) a free shareware program for linear modeling of microarray data,
downloaded from the Walter and Elisa Hall Institute of Medical Research, Australia
(http://bioinf.wehi.edu.au/affylmGUI/index.html). Statistical and additional packages, R-
statistics (http://cran.r-project.org/), Tcl/Tk
(http://www.activestate.com/Products/ActiveTcl/) and Bioconductor packages
(http://www.bioconductor.org/) were downloaded for use with affylmGUI.
The linear values of normalized and scaled relative expression levels were used
for further analysis. Values of the triplicate sets were averaged, and the fold differences or ratios calculated using Microsoft® Office 2003 Excel (Microsoft Corporation, USA).
This spreadsheet was entered into a database manager, Microsoft® Office 2003 Access
(Microsoft Corporation, USA), where the dataset was probed with simple queries to sort and cluster gene lists. A 2 fold ratio cut off was used as an indication of significant changes in gene expression (>=2 : 2-fold up regulated; <=0.5 : 2 fold down regulated).
57 2.3 Results and Discussion
2.3.1 Developing an anthocyanin inducible system using chemical complementation.
As a first step towards robustly inducing anthocyanins in Arabidopsis wt
seedlings, conditions of dark, light, water and 3% sucrose were tested. A previous study
in the lab (Dong et al., 2001) successfully used low nitrogen media to induce
anthocyanins (Hsieh et al., 1998). The low N-containing MS salts was modified to a
simple media of 3% sucrose in water (Xiaoyun Dong, personal communication).
Anthocyanins are known to be induced as a result of elevated sugar levels with sucrose
being the most effective agent (Lloyd and Zakhleniuk, 2004; Teng et al., 2005).
Arabidopsis wt seeds were germinated in the dark or continuous light in water and 3%
(w/v) sucrose for 2 d. The dark grown seedlings were either left in the dark or shifted to
the light. Seedlings grown in 3% sucrose and light (Fig. 2.3 B e, f), showed robust
induction of anthocyanins in the cotyledons and upper hypocotyls. Due to the absence of
any salts in the media and only the presence of sucrose, chlorophyll biosynthesis was
suppressed in the cotyledons of the seedlings grown in the sucrose only media and light,
in contrast to water grown seedlings which showed no accumulation of anthocyanins in
their green cotyledons (Fig. 2.3 B b, c).
Naringenin, the product of CHI, has been previously shown to complement the
pathway in tt5 seedlings (Shirley et al., 1995). Addition of 100 µM naringenin
complemented the tt5 mutant, as clearly seen from the accumulation of anthocyanin in
the cotyledons and upper hypocotyls of 3% sucrose grown seedlings (Fig 2.3 C f). This pathway complementation is also evident in water-grown seedlings where anthocyanins
58 are present at the cotyledon-hypocotyl junction (Fig. 2.3 B b). Addition of naringenin also visibly increased the levels of anthocyanins in wt seedlings, when grown in 3% sucrose (Fig. 2.3 C g; darker purple-red cotyledons) and induced the formation of anthocyanins at the cotyledon hypocotyl zone in water grown seedlings (Fig. 2.3 C d).
Anthocyanins preferentially accumulate in the epidermal to sub-epidermal tissues of plants (Onslow, 1925). Arabidopsis cotyledons show anthocyanins to accumulate in the epidermal cells of the adaxial and abaxial side (Appendix C). Additionally the adaxial side also accumulated anthocyanins in the sub-epidermal layer. There was a developmental delay observed in the accumulation of anthocyanins in epidermal cells of the adaxial side as compared to its sub-epidermal layer and the epidermis of the abaxial side.
At the sub-cellular level, the epidermal cells of the tt5 seedlings showed a colorless large central vacuole (Fig. 2.4 A a). Addition of 100 µM naringenin resulted in anthocyanins to uniformly pigment the central vacuole (Fig 2.4 A b). Depending on the developmental stage and the concentration of the naringenin added to the media, tt5 seedlings (Fig. 2.4 A c) show darkly pigmented sub-vacuolar structures that are reminiscent of the structures described in chapter 1. Epidermal cells of wt seedlings also accumulate these sub-vacuolar structures (not shown).
Naringenin-treated wt seedlings accumulated 40% more anthocyanins than non- treated wt seedlings and this amount was nearly equivalent to the amount of anthocyanins that accumulated in the naringenin complemented tt5 seedlings (Fig 2.4B). A bathochromic shift in the anthocyanins absorption maxima (Fig 2.4C) was observed in the spectral scans of the methanolic extracts of the naringenin treated wt and tt5 seedling.
59 Comparing the absorption maxima of the non-treated wt seedlings (533 nm), the shift of maximum absorption to 526 nm in tt5 seedlings was more dramatic than the shift to 530 nm in the naringenin treated wt seedling. This is reflective of a change in the anthocyanin
profiles.
Thus, these results demonstrate that a simple liquid medium of 3% (w/v) sucrose
in water and high light was sufficient to uniformly induce anthocyanin accumulation in
Arabidopsis cotyledons, without the background influence of chlorophylls. In these
inducing conditions, the pathway genes are “on” and the competent tissues will
synthesize anthocyanins as seen from the accumulation of anthocyanins in the cotyledons
of naringenin treated tt5 seedlings. This convenient seedling system, being in liquid, is
easy to manipulate, especially to evaluate the effect of various drugs/inhibitors of
metabolism and transport on the de novo accumulation of anthocyanins in tt5 or the
additional accumulation in the wt. Furthermore, the shift in absorption maxima of the
total anthocyanins of the naringenin-treated tt5 and wt seedlings as compared to the non-
treated wt is reflective of the possible change in co-pigmentation or in the
anthocyaninsprofile. This was further dissected by HPLC separation of the anthocyanins
as discussed in the next section. The preliminary observations of the formation of sub-
vacuolar structures in the epidermal cells and the changes in the spectral profiles,
indicates that this system is ideal to investigate the modification and transport of
anthocyanins to the vacuole.
60 Figure 2.3 Sucrose and light induce the formation of anthocyanins and naringenin chemically complements the tt5 mutant: (A) tt5 seeds (a) are deficient in tannins and appear pale yellow. In contrast wt seeds accumulate PAs and are a rich brown in color. (B) wt seedlings grown in water (a,b,c) or 3% sucrose (d,e,f) for 2 d were shifted from dark to dark (a,d), dark to light (b,e) and light to light (d,f) for 24 h and then imaged. (C) 3 d old tt5 (a, b, e, f) and wt seedlings (c, d, g, h) grown in water (a, b, c, d) and 3% sucrose (e, f, g, h) in continuous white light. 24 h prior, with 100 µM naringenin (b, f, d, h). Ethanol was used as the control (a, c, e, g).
61 A tt5 wt ab
Seeds
B Dark to Dark to Light to Dark Light Light a b c water
d e f 3% sucrose
C tt5 wt 100µM Naringenin - + - + a b c d
e f g h 3% Sucrose Water
Fig. 2.3
62 A
100µM Naringenin - + + a b c
tt5
B 2.5
2
1.5
1
0.5
Abs 530 / 10mg dry wt / ml 0 tt5-N tt5+N wt-N wt+N
C λmax tt5 -N - tt5 +N 526nm wt -N 533nm wt +N 530nm
Figure 2.4. Vacuolar localization and quantitation of anthocyanins: (A) Epidermal cells of tt5 cotyledons are colorless (a) which on addition of naringenin synthesize anthocyanins that accumulate in the central vacuole. Depending on the developmental stage and concentration of naringenin, seedlings also accumulate anthocyanins in distinct sub-vacuolar bodies (b). (B) Spectrophotometric measurement (530 nm) of anthocyanins from control and naringenin treated tt5 and wt seedlings. (C) Spectral scans of the anthocyanins from the seedlings in (B). The seedlings were 3.5 d old and with a 24 h treatment with either ethanol (control) or 100 µM naringenin.
63 2.3.2 Naringenin induces a new anthocyanin peak – cyanidin-3-glucoside
The metabolic profiles of methanolic extracts of control and naringenin treated
3.5 d old tt5 and wt seedlings were analyzed by HPLC with anthocyanins monitored at
530 nm, flavonols at 360 nm and general phenolics at 280 nm.
The addition of naringenin induces the formation of a new anthocyanin peak in both tt5 and wt seedlings (Fig 2.5 A, ‘a’) with an average HPLC retention time of 16.3 min and λmax that ranges between 512 to 519 nm. This compound was identified as
cyanidin-3-glucoside (C3G) by LC-MS and confirmed by comparing the retention time
with a reference standard. C3G contributes to nearly half (42%) of the total anthocyanins
that are synthesized in tt5+N seedlings, and accounts for 10% of the anthocyanins in
wt+N seedlings (Fig 2.6). Furthermore, naringenin also leads to a 1.5-fold increase in the
amount of a major peak at 19.53 min (Fig 2.5 A, C, peak ‘b’, Fig 2.6, compare wt-N-C to
wt+N-C) and a 3 fold increase in a minor peak at 23.78min (peak ‘d’, Fig. 2.5 A, C).
There was no change in the amount of the major anthocyanin (Fig. 2.5 A, C peak ‘c’,
21.76min) which contributes to the nearly half of the total anthocyanins in untreated wt
seedlings. Structurally, peak ‘c’ is the most decorated (Fig. 2.5C), and the result of no
change in peak height may indicate that the transferases involved in sysnthesis of this
anthocyanin were compartmentalized in a different part of the cell or that the newly
formed C3G is sequesterd away from the cytoplasm.
The different anthocyanins may be characterized by attachment of different co-
pigments (phenypropanoid, flavonoid or flavonol) and/or glycosyl and other moieties like
acyl or prenyl groups (Tohge et al., 2005). The identification of these peaks by mass
spectrometry and NMR and comparison to the published anthocyanins of Arabidopsis
64 pap1-D plants over-accumulating anthocyanins (Tohge et al., 2005) is underway in our
laboratory. The deductions from structures identified so far by LC-MS indicate that the
new anthocyanin molecule induced by naringenin, C3G (Fig. 2.5 a), is a precursor for the
subsequent anthocyanins (Fig. 2.5 b to d), which seem to undergo sequential molecular
transformations with addition of moieties to the core flavonoid structures. Changes in
peak profiles and examination of the anthocyanin structures suggest that overloading the
system with naringenin results in the accumulation of the proposed intermediate
anthocyanin compounds. This may imply that (i) pre-existing glycosyltransferases
function at different rates or efficiencies (ii) naringenin may induce or repress expression
of one or more conjugation enzymes or (iii) the intermediates and/or modification
enzymes are compartmentalized in separate regions of the cell. It would be interesting to
identify and localize the transferases leading to the successive complexity of the anthocyanins.
There are a total of 18 major and minor anthocyanin peaks that are synthesized in the naringenin complemented tt5 and wt seedlings. Not all the peaks are represented in all the samples, especially the minor peaks. For example, apart from the de novo peak induced by naringenin, naringenin-complemented tt5 seedlings do not produce the full complement of wt anthocyanins, and the minor peaks of retention times 18.52, 20.72,
22.63, 23.7 and 25.20 minutes are not detected. Comparative analysis of spectral profiles of peaks present in the treatments resulted in spectral matches for the peaks eluting around the same retention time (Appendix D).
To test if the synthesis and accumulation of total anthocyanins in tt5 seedlings and the new-naringenin induced peak is dependant on the de novo synthesis of protein(s), we
65 utilized cycloheximide, a potent protein synthesis inhibitor. The seedlings were pre-
treated with 100 µM of cycloheximide for 30 min before the addition of 100 µM naringenin. Cycloheximide was unable to completely inhibit the anthocyanin accumulation in tt5 seedlings indicating that the pre-existing machinery is able to synthesize and sequester anthocyanins or that some enzyme is induced by naringenin
(Fig. 2.7). Cycloheximide blocked the de novo synthesis of naringenin-induced C3G
(peak ‘a’, RT 16.34min, Fig 2.5A) in both tt5 and wt seedlings. Naringenin, when added to the cycloheximide treated wt seedlings caused a 2.25-fold increase in accumulation of
anthocyanin peak at 19.53 min (peak ‘b’, Fig 2.5 A; Fig. 2.6 A, B) as compared to non-
treated seedlings. Similar trends were observed with minor peak at 23.78 min in wt
seedlings indicating that when synthesis of C3G is blocked by cycloheximide, the initial
C3G formed by the intact machinery is converted into peak ‘d’ and then‘b’ (Fig 2.5, Fig
2.6). A more comprehensive time course based analysis would be informative of the
changes and potential interconversions of the anthocyanins over time.
There were no new anthocyanidins made as similar profiles of the hydrolyzed aglycones were observed on TLC plates (Fig 2.8 A) which was further confirmed by the
530 nm HPLC chromatogram of the hydrolyzed extracts (not shown).
The HPLC observations of changes in the anthocyanin profiles by naringenin and cycloheximide are reflective in the bathochromic shift of λmax of the total anthocyanins
analyzed spectrophotometry (Fig 2.8 B). The contributions of the individual peaks
influence the spectra of the total anthocyanins. For example, the naringenin treated tt5
seedlings have a λmax of 523 nm, which on cycloheximide treatment, red shifts to 528 nm.
This is in corroboration with the result that the new peak, C3G (λmax in HPLC conditions 66 512 – 519 nm) induced by naringenin, which contributes to nearly half of the anthocyanins in tt5 seedlings, gets inhibited by cycloheximide, thus results in the bathochromic shift from 523 to 528 nm.
Taken together, these results show that naringenin may induce the formation of the C3G by either up-regulating the expression of a gene involved in its modification and/or transport or increases the intermediate metabolite pool (probably an anthocyanidin) such that it now gets modified by a pre-existing enzyme with low affinity for the compound. The fact that cycloheximide is unable to totally inhibit the accumulation of anthocyanins in tt5 demonstrates that in the inductive conditions, the sequestration machinery is expressed in the cells. The perturbation of the anthocyanin profiles by naringenin and cycloheximide illustrates alternative conjugation process and/or transport routes to the vacuole that are operative in the cell. This would be advantageous to the plant where it needs to accumulate high amounts of the phytochemical in a short span of time for e.g. under pathogen attack. Furthermore, the anthocyanin profiles of the tt5 seedlings, where anthocyanin accumulation can be induced, overlaid over the changes in the wt profiles, provide clues as to the possible mechanisms involved in the modifications and the transport routes that the metabolites adopt. This may help us hone in on the enzyme or transporters involved that may either already expressed in the cell or would require de novo transcription/translation. The inhibition of C3G by cycloheximide is further expanded on in section 2.3.4 where cycloheximde induced changes in the endogenous levels of naringenin and transcripts of two main flavonoid enzymes were observed.
67 A Non-hydrolyzed extracts /glycones (530nm)
tt5 – N -C wt – N -C
b c
d
10.00 15.00 20.00 25.00 10.00 15.00 20.00 25.00 tt5 + N -C Minutes wt + N -C Minutes
b c a a b c d d
10.00 15.00 20.00 25.00 10.00 15.00 20.00 25.00 tt5 - N +C Minutes wt - N +C Minutes c b
d
10.00 15.00 20.00 25.00 10.00 15.00 20.00 25.00 tt5 + N +C Minutes wt + N +C Minutes b c
b d c d a
10.00 15.00 20.00 25.00 10.00 15.00 20.00 25.00 Minutes Minutes
3000 B Total 2500 Anthocyanins 2000 1500 1000 Area (mV.s) 500 0
C C C C - -C - N -N + +N + t5 -N - 5 -N +C t t tt5 tt w wt +N wt -N +Ct tt5 +N +C w Fig. 2.5 Continued 68 C a b OH OH
HO O+
O O O HO O O O HO OH OH MeO HO OH OH OMe 16.34 min 19.53 min
c d
OH OH OH OH O HO O+ O + O O HO O HO O O HO O O O O HO O O O O HO OH OH O HO O O O OH O O OH HO OH O O O HO OH OH O OH OH OH
O O OH O OH O HO OH 21.76 min 23.76 min HO OH
Figure 2.5 HPLC profile at 530nm - naringenin induction of a cyanidin 3-glucoside: HPLC chromatograms of anthocyanins at 530nm of non-hydrolyzed methanolic extracts of tt5 and wt seedlings treated with 100 μM naringenin (-/+N) and/or 100 μM cycloheximide (-/+C). ‘a’ marks the new naringenin induced, 16.34 min anthocyanin peak. The peaks ‘b’: 19.53min, ‘c’ : 21.76 and ‘d’ 23.76min indicated also show changes. Inset spectra of the naringenin induced peak ‘a’. (B) The comparative amounts of total anthocyanins as integrated from the chromatograms in (A). (C) Structures of anthocyanin molecules corresponding to the major peaks influenced by naringenin as identified by LC-MS.
69 A 1000 tt5 +N -C 900 tt5 +N +C wt -N -C 800 wt +N -C 700 wt -N +C 600 wt +N +C 500 400
Area (mV.s) Area 300
200 100
0
6 3 8 .5 .55 .98 .99 .76 .5 .72 .01 .5 .76 .63 .78 .89 9.37 2 5 5 6 7 9 0 1 1 1 2 3 4 1 1 1 16.33 1 1 18.52 1 2 2 2 2 2 23.73 2 2 25.20 Retension time (minutes) B 60 tt5 +N -C
50 tt5 +N +C wt -N -C 40 wt +N -C
30 wt -N +C wt +N +C 20
10 % Area of total Anthocyanins total of Area % 0
7 6 5 8 3 9 6 2 3 2 1 8 6 3 3 8 9 0 .3 .5 .5 .9 .3 .9 .7 .5 .5 .7 .0 .5 .7 .6 .7 .7 .8 .2 9 2 5 5 6 6 7 8 9 0 1 1 1 2 3 3 4 5 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 Retention time (minutes)
Figure 2.6 Analysis of changes in peak area and peak percent of anthocyanins at 530nm. Bar graphs of the anthocyanin peaks integrated from chromatograms in Fig 2.5 A (A) Area of anthocyanin peaks in mV.s. (B) percentage representation of total anthocyanins.
70 100µM Naringenin - - + + 100µM Cycloheximide - + - + a b c d
tt5
e f g h
wt
Figure 2.7 Cycloheximide reduces but does not abolish the accumulation of anthocyanins in naringenin complemented tt5 seedlings. 2.5d old tt5 (a,b,c,d) and wt (e,f,g,h) seedlings grown in 3% sucrose and light, were not treated (control, ethanol, a,e), or pre-treated with cycloheximide for 30min (b,f, d,h) and naringenin (d,h) or naringenin alone (c,g) as indicated in the figure. Images were taken 24h after drug treatment
71
A
Cyanidin
- - + + - - + + 100µM Naringenin - + - +- + - + 100µM Cycloheximide
tt5 wt B
tt5-N-C - tt5-N+C - tt5+N-C 523 tt5+N+C 528 wt5-N-C 533 wt5-N+C 533 wt5+N-C 531 wt5+N+C 535
Figure 2.8 Anthocyanin analysis of naringenin and cycloheximide treated tt5 and wt seedlings. (A) Separation of acid-hydrolyzed anthocyanidins by TLC. The major spot represents cyanidin. The minor spots that run near the solvent front are the incomplete hydrolyzed anthocyanins. (B) Spectral scans from 400 to 600nm of the acidified methanolic total anthocyanin extracts.
72 2.3.3 There are no significant changes in flavonol accumulation patterns.
Flavonols in seedlings have been shown to predominantly accumulate in roots,
hypocotyls and the hypocotyl root transition zone (Peer et al., 2001). There is a higher
kaempferol accumulation than quercetin in wt seedlings (Pelletier et al., 1999) and UV irradiation was shown to increase the amount of quercetin (Ryan et al., 2001). However in our seedling system, the quercetin to kaempferol (Q:K) ratio in wt seedlings was 60:40
(Fig 2.9), which is different from the published reports. Accumulation of quercetin has been attributed to the activity of F3’H (Peer et al., 2001; Ryan et al., 2001) and the differences in ratios observed may be due to differences in the growth stage and/or environmental conditions that affect the expression of the entire complement of the pathway genes (Pelletier et al., 1999).
Naringenin, when added to wt seedlings did not significantly increase nor change the flavonol accumulation patterns in the wt. This result was surprising as I had expected increased flux into the flavonol branch of the pathway in the regions of the roots and hypocotyls that do not accumulate anthocyanins. Naringenin can enter the anthocyanin branch of the pathway as well as the flavonol branch, as seen in tt5. The wt seedlings somehow do not utilize the extra precursor for the flavonol pathway, which may be regulated at either the enzymatic level where the pathway is saturated in the conditions used or because naringenin is actively excluded from the root. A recent study in our laboratory has shown that naringenin-treated tt5 seedlings secrete eriodictyol into the media (Lu and Grotewold, unpublished results).
Quercetin and kaempferol were just at the limit of detection in the tt5 mutants
(Fig 2.9). Naringenin-complemented tt5 seedlings accumulated quercetin and kaempferol
73 in a Q:K ratio of ~80:20 as compared to wt that averages at 60:40. This may be
indicative of selective channeling of the intermediates as the enzymes of the flavonoid
pathway have been proposed to be involved in a multienzyme complex based on protein-
protein interaction studies in Arabidopsis (Burbulis and Winkel-Shirley, 1999).
Furthermore, in tt5 mutants, protein levels of enzymes in the pathway were up-regulated
in comparison with wt. Transcript levels were also assessed through microarray analysis
as discussed in Section 2.3.8 and the increase in activities of the enzymes F3’H and FLS may be responsible for the shift in the flavonol ratios when comparing tt5 to wt.
The 360 nm chromatogram of the hydrolyzed extracts revealed a peak at 16.3min
(λmax 343nm) that showed a ~3-fold depletion when naringenin was added to the
seedlings (Fig 2.9, 2.10). The reduction in the relative area of the 16.3 min peak in the
presence of exogenously applied naringenin is intriguing as this reduction trend is in
parallel with the increase of anthocyanins. This is not a flavonol as it is present in the tt5-
N extracts. It is tempting to speculate that maybe this compound is somehow utilized in
the anthocyanin pathway or is modified gene products that are either up- or down- regulated genes by naringenin. The identification of this peak remains to be determined by LC-MS.
The kaempferol 30.7 min peak had a smaller shoulder peak that migrates at 30.4 min and has a λmax similar to that of quercetin at 367nm. The identity of this peak is
unknown, its retention time distinct from the standards and put together with quercetin
and kaempferol represents ~9% of the total flavonols. As noted in a previous study (Ryan
et al 2001), tetrahydroxychalcone, the substrate of CHI (RT 28.9nm) was not detected at
28.9 min in either the tt5 mutants or the wt seedlings in our HPLC run conditions 74 contrary to a report where naringenin chalcone was detected visually in tt5 seedlings using a flavonoid specific stain, diphenylboric acid-2-aminoethyl ester (DPBA, Peer et al., 2001).
Cycloheximide treatment, alone or together with naringenin, also did not show any alterations in the Q:K ratios, where the naringenin-treated tt5 seedlings averaged
80:20 and the wt seedlings averaged 60:40 inhibited the relative abundance of kaempferol and quercetin in tt5+N+C as well as wt-N+C. The levels are compensated for by naringenin in the wt+N-C treatment to the wt control levels (Fig 2.9).
75 Figure 2.9 HPLC analysis of flavonols at 360nm: (A) 360nm chromatograms of hydrolyzed methanolic extracts of tt5 and wt seedlings treated with 100μM naringenin (- /+N) and/or 100μM cycloheximide (-/+C). * marks the 16.3 min peak; Q: quercetin; K: kaempferol (D) Comparison of area (mV.s) of peaks for quercetin and kaempferol in the treated tt5 and wt seedlings (C) Quercetin : Kaempferol ratios of naringenin complemented tt5 and wt seedling extracts. The * indicates either no treatment or treatment.
76
Hydrolyzed extracts/aglycones (360nm) A tt5 – N -C wt – N - C * Q K *
15.00 20.00 25.00 30.00 15.00 20.00 25.00 30.00 tt5Minutes + N -C wtMinutes + N - C Q K Q * * K
15.00 20.00 25.00 30.00 15.00 20.00 25.00 30.00 tt5Minutes – N + C wtMinutes – N + C
* Q * K
15.00 20.00 25.00 30.00 15.00 20.00 25.00 30.00 Minutes Minutes tt5 + N +C wt + N + C Q K Q * * K
15.00 20.00 25.00 30.00 15.00 20.00 25.00 30.00 Minutes Minutes
B C 5000 Quercetin Q : K 4000 Kaempferol 3000 tt5 +N * 80 : 20 2000 w t * * 60 : 40
Area (mV.s) 1000 0
-C -C C C C + +C - + +C N N N + - N -N -C + -N N + t t + tt5 -N t5 t5 wt t t tt5 w w wt
Fig. 2.9
77
A RT 16.3min
343.6 tt5-N-C wt5+N-C 0.20 0.20 343.6 295.9 0.10 529.3 0.10 294.7 554.9 486.7 520.8 0.00 0.00 400.00 600.00 400.00 600.00
B 360nm, 2500 hydrolyzed, 2073 16.3min 2000 1567 1268 1500 1176 960 1000 775
Area (mV.s) 443 387 500
0
C C C - -C -C - + N N -N + -N N 5 + t - tt t5 wt t tt5 -N +Ctt5 +N +C wt w wt +N +C
Figure 2.10 Analysis of 16.3min peak extracted from 360nm chromatograms of hydrolyzed tt5 and wt samples: (A) Representative spectra from seedlings not accumulating anthocyanins (tt5-N-C) and those that are (wt+N-C). (B) Area in mV.s of 16.3min peak comparison across tt5 and wt samples.
78 2.3.4 HPLC phenolic profiles of naringenin treated tt5 and wt seedlings
Endogenous naringenin pools were monitored at 280 nm from HPLC chromatograms of non hydrolyzed and hydrolyzed extracts of the seedlings. Naringenin has an absorption maxima at 290 nm and elutes at 27.2 min in these experimental conditions. The endogenous levels of naringenin are below the levels of detection in the control wt seedlings, indicating that the endogenous naringenin is converted into the pathway end products. Addition of exogenous naringenin to the wt seedlings showed detectable levels of naringenin which was enhanced 60 fold in presence of the protein synthesis inhibitor cycloheximide (Fig 2.11, 2.12). This accumulation of naringenin in the presence of cycloheximide parallels the inhibition of formation of the naringenin induced anthocyanin peak, indicating that naringenin cannot enter the anthocyanin pathway. Northern blots of cycloheximide-treated seedlings as compared to non-treated seedlings showed a significant down-regulation of two of the flavonoid pathway genes
(AtCHS and AtF3H) investigated (Fig 2.13). Thus, the cycloheximide inhibition of the naringenin induced accumulation of C3G and increase of endogenous levels of naringenin provides evidence that C3G is the precursor the the more complex anthocyanins.
Chromatograms of hydrolyzed samples of tt5 seedlings showed 27.2min peaks in the control tt5 and the cycloheximide treated tt5 seedlings. The spectral pattern of these
27.2 min eluting peaks was similar to the naringenin standard, however, they differed in their absorption maxima by 1 nm (Fig 2.12). Tetrahydroxychalcone is known to spontaneously cyclize to naringenin at pH≥7.5 (Moustafa and Wong, 1967) and this peak may represent a small pool of spontaneously cyclized chalcone pool of naringenin.
79 Thus, there is a rapid utilization of naringenin in the naringenin treated seedlings. The endogenous levels of naringenin only start to accumulate in the presence of cycloheximide which may repress the expression of proteins involved in naringenin metabolism.
80 Non-hydrolyzed Hydrolyzed / / glycones 280nm aglycones
0.30 0.30 tt5 – N -C tt5 – N -C 0.20 0.20
0.10 0.10
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 5.00 10.00 15.00 20.00 25.00 30.00 0.30 0.30 tt5 + N -C Minutes Minutes tt5 + N -C 0.20 0.20
0.10 0.10
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 5.00 10.00 15.00 20.00 25.00 30.00 0.30 0.30 Minutes Minutes tt5 – N + C tt5 – N + C 0.20 0.20
0.10 0.10
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 5.00 10.00 15.00 20.00 25.00 30.00 0.30 0.30 Minutes Minutes tt5 + N +C tt5 + N +C 0.20 0.20
0.10 0.10
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes Minutes 0.30 0.30 wt – N - C wt – N - C 0.20 0.20
0.10 0.10
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 5.00 10.00 15.00 20.00 25.00 30.00 0.30 0.30 wt + N - C Minutes Minutes wt + N - C 0.20 0.20
0.10 0.10
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 5.00 10.00 15.00 20.00 25.00 30.00 0.30 0.30 wt – N + C Minutes Minutes wt – N + C 0.20 0.20
0.10 0.10
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 5.00 10.00 15.00 20.00 25.00 30.00 0.30 0.30 Minutes Minutes wt + N + C wt + N + C 0.20 0.20
0.10 0.10
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes Minutes N N
Figure 2.11 HPLC analysis of anthocyanins at 280nm: (A) 280nm chromatograms of hydrolyzed methanolic extracts of tt5 and wt seedlings treated with 100μM naringenin (- /+N) and/or 100μM cycloheximide (-/+C). ‘N’ and the light grey line marks the 27.2 min naringenin peak.
81
288.8 290.0
420.2
440.7 486.7582.9 397.3462.5532.9485.573.15
2500 2353 288.8nm
2000 290nm
1427 1500 987 1000
Area (mV.s) Area 500 347 278 25 0
C C C - - - -N N N +C +N -N +C + tt5 t5 t5 wt - t t t tt5 +N +C wt +N -Cwt -N +Cw
Figure 2.12 Peak eluting at the naringenin standard 27.25min have two alternative absorption maximas. Representative spectra of peaks with absorption spectra at either 288.8 or 290 nm. Comparative analysis of area covered by the peaks eluting at 27.3min with the above mentioned spectra.
82 wt tt5 tt3 - - + + - - + + - - + + 100μM CHX
- + - + - + - + - + - + 100μM N AtCHS
AtF3H
Actin
Figure 2.13 Northern analysis of AtCHS and AtF3H expression in wt, tt5 and tt3 seedlings treated with naringenin and cycloheximide: Total RNA from 3.5 d wt, tt5, and tt3 seedlings were analysed by Northern for expression of CHS (chalcone synthase) and F3H (flavanone 3 hydroxylase). Actin-related protein 6 (At3g22520) was used as the normalizing control.
2.3.5. Naringenin can be a growth inhibitor.
Naringenin, when added to seedlings at higher concentrations inhibits growth. tt5 seedlings grown on MS plates with concentrations of naringenin that range from 0 to
500µM show dramatic arrest of root growth where at concentrations of naringenin
200µM and higher there was total arrest of root tip growth. (Fig 2.14, 2.15 A). Fifty percent inhibition of root growth was observed at 100 µM naringenin as compared to the non treated plants. As observed from the images, there was a promotion of lateral root growth, where lateral roots emerge even after the arrest of root tip growth. The measurement of the area covered by the rosette leaves also revealed arrest of growth where 100 µM of naringenin inhibited growth by 40% (Fig 2.14, 2.15 B).
83
0µM 10µM 50µM 100µM 84
200µM 300µM 400µM 500µM
Figure 2.14 Effect naringenin on tt5 plant growth: Five day old seedlings grown on MS + 3% sucrose was shifted to MS media with concentrations of naringenin ranging from 0 to 500µM. Images and measurements of roots and rosette area coverage were taken after10 days.
84
A 6.00
5.00
4.00
3.00
2.00
Root length (cm) length Root 1.00
0.00
M M µM µ 0 0µM 1 50µM 00µ 100µM 200µM 3 400 500µM Naringenin B 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 Rossette Area (cm2) Rossette Area 0.00
M M M M M µM µ µM 0 0µ 0 1 50 00µ 00µ 00µ 1 2 30 400µM 5 Naringenin
Figure 2.15 Naringenin is an inhibitor of growth: (A) Average root length of 15 day old seedlings after shift to the naringenin containing media represented in Fig 2.13. (B) Average rosette area of the plants. The measurements were made from digital images using NIH Image J software.
85 2.3.6 Components of the tt5 anthocyanin inducible system and rationale for analyzing transcriptome changes using microarrays.
Taking all the results of the experiments done in developing and analyzing the tt5/wt system as a viable model system to analyze molecular players using gene arrays, I took into consideration three components of this system. As represented by the cartoon in
Fig 2.16, the components are the phytochemicals naringenin, anthocyanins and/or intermediates downstream of naringenin and the enzyme CHI. Each of these components can affect the cell in different ways. As depicted in Fig 2.1, the small molecules can be modified, transported directly into the vacuole or pumped out of the cell through transporters, protein carriers or first pumped into vesicles and then pumped into the vacuole or out of the cell. Furthermore, naringenin and anthocyanins may interfere and/or alter cellular processes. The third component, the enzyme CHI which apart from being involved in the biosynthetic pathway may have unknown regulatory function. The localization of CHI, together with other enzymes of the pathway (CHS, DFR and F3H) in the nucleus may point towards regulatory roles (Saslowsky et al., 2005) (Appendix E).
86
Naringenin
Chalcone Anthocyanin Isomerase and / or intermediates downstream of naringenin
Figure 2.16 The three components of the tt5 system: CHI (green circle) is a key flavonoid biosynthetic enzyme. Deficiency of this enzyme in tt5 mutants blocks the production of anthocyanins (pink circle). Naringenin (yellow circle) an intermediate of the pathway and product of CHI can chemically complement the pathway to form anthocyanins.
87
The comparative scheme depicted in Figure 2.17 represents the states of the three components in each of the conditions of tt5 and wt seedling without (tt5-N; wt-N) or with
(tt5+N; wt+N) the addition of naringenin (N). In tt5-N seedlings, the enzyme CHI is absent (white circle), and there is no production of naringenin (white circle) thus no flux to accumulate anthocyanins (light peach circle) in the cotyledons of seedlings which remain non-pigmented. Upon chemical complementation of tt5 by the addition of naringenin, the tt5+N seedlings now have exogenously supplied naringenin (yellow circle) and anthocyanins are made (pink circle), but there is no CHI (white circle). In wt-
N, there is flux through the flavonoid pathway, where the active CHI (green circle) converts endogenous chalcone to form naringenin (yellow circle) and anthocyanins (pink circle). Upon addition of naringenin, the levels of naringenin (dark-yellow circle) increase and there is increased accumulation of anthocyanins (dark-pink circle). The grey circles in the center represent the developmental differences between the tt5 and wt seedlings. As noted in previous studies, tt5 seedlings germinate slower than the wt and temporally are not at the same developmental stage as the wt (Debeaujon et al., 2000).
The microarray experiments would provide insights into many processes, which include feedback regulation, trafficking, functions of flavonoids in development and the processes that plants utilize to deal with large quantities of naringenin. Thus, if we are to compare and contrast the transcriptome of tt5 and wt, with and without the addition of naringenin (Fig 2.17), we may uncover the mechanisms of transport of anthocyanins into the vacuole and/or the effect of naringenin, anthocyanins or pathway intermediates on cellular processes.
88
3% Sucrose, Light
Naringenin Naringenin tt5 -N wt -N
chi Anthocyanin CHI Anthocyanin
+N +N
Naringenin Naringenin tt5 +N wt +N
chi Anthocyanin CHI Anthocyanin
Figure 2.17 Schematic representation of Arabidopsis tt5 and wt system and complementation with naringenin: Representation of the states of the three components in tt5 and wt seedlings treated with and without naringenin (see for explanation in text).
89 2.3.7 Transcriptome changes in Arabidopsis tt5 and wt seedlings treated with naringenin: Experimental design
To uncover the transcriptome changes of tt5 and wt Arabidopsis seedlings on treatment with the flavonoid pathway intermediate naringenin, microarray experiments were performed using the Arabidopsis ‘whole genome’ probe array - Affymetrix ATH1
GeneChip® probe array that consists of approximately 22,750 probe sets representing
~23,750 genes (Redman et al., 2004). Figure 2.18 summaries the experimental variables
and set up. tt5 and wt seedlings were treated with and without naringenin. Each experiment was divided into two sets, which included control (ethanol) and a naringenin
treatment. These experiments were repeated thrice with an experimental set of a control
and a naringenin treatment being performed at the same time under near identical
conditions. The biological triplicates and the number of seedlings (~2000-4000) used per
RNA extraction helped minimize biological variation.
As depicted in the schematic in Figure 2.19 A, an experiment for tt5 consisted of
tt5-N and tt5+N and that for wt consisted of wt-N and wt+N (See materials and methods
for details). Care was taken to ensure equal plating density of the seeds and thus similar
levels of the naringenin treatment. 100μM of naringenin was added to 2.5d old seedlings
and harvested 24 h later. RNA was extracted and quality was assessed by an Agilent
biosystems bioanalyzer (Fig. 2.19 B). Biotin labeled cRNA (or cDNA) was hybridized to
Affymetrix ATH1 Arabidopsis whole genome array (Fig. 2.19 C). The collected raw
fluorescent intensities were normalized and scaled, and the data was analyzed for up- and
down-regulated genes.
90 Experimental variables:
#1 Genotype: mutant : tt5 wildtype: wt
#2 Treatment: Name of the drug: Naringenin (N) No treatment: -N Treatment: +N
#3 Time: 2 time points (age of seedlings at time of harvest) 3.0d 3.5d
Experimental break up:
3 experiments in all.
Each experiment with 2 sets of data i.e. with and without the naringenin treatment.
Each set has biological triplicates, thus 18 chips in all.
Experiment #1: Effects of naringenin on 3.5 d old tt5 mutant seedlings 1. tt5 seedlings –N 3.5d old: biological triplicates 1a, 1b and 1c 2. tt5 seedlings +N 3.5d old: biological triplicates 2a, 2b and 2c
Experiment #2: Effects of naringenin on 3 d old wt seedlings 3. wt seedlings –N 3.0d old: biological triplicates 3a, 3b and 3c 4. wt seedlings +N 3.0d old: biological triplicates 4a, 4b and 4c
Experiment #3: Effects of naringenin on 3.5 d old wt seedlings 5. wt seedlings –N 3.5d old: biological triplicates 5a, 5b and 5c 6. wt seedlings +N 3.5d old: biological triplicates 6a, 6b and 6c
Figure 2.18 Microarray experimental variables and experimental break up: The data for experiment #1 tt5-/+N and #3 wt -/+N 3.5d was further analyzed. The data for experiment #2 i.e. wt -/+ N 3d was not considered for this study due to technical difficulties.
91 Figure 2.19: Experimental design for the effects of naringenin on tt5 and wt seedlings: (A) schematic flow chart of growth conditions of seedlings of tt5 and wt seedlings, treatments with naringenin and times of growth and harvest. (B) RNA quality as assessed by capillary electrophoresis on an Agilent bioanalyzer. (C) A laser scanned fluorescent image of a portion of one of the microarray chips showing the hybridized fluorescent spots.
92 A
tt5 -N
+ethanol
tt5 +N
+100µM Naringenin
wt -N
+ethanol
wt +N
+100µM Naringenin - 2d 0d 2.5d 3.5d 4°C 25°C25°C
Plate surface Culture room, Treatment: Harvest seedlings: sterilized seeds continuous white light +ethanol / •RNA isolation -2 -1 in liquid 3% 100µmole.m .s , +100µM •Anthocyanin sucrose 100rpm Naringenin
B C
wt-N wt+N tt5-N tt5+N M
Fig. 2.19
93 2.3.8 Naringenin does not feedback regulate its core flavonoid enzymes or
transcription factors at the expression level
The flavonoid pathway genes show co-ordinate patterns of expression controlled
by distinct sets of transcription factors in response to development or environmental cues
(Koes et al., 1994; Shirley, 1996). Expression patterns of the enzymes over the course of
seedling development showed CHS, CHI, F3H and FLS encoded by ‘early’ genes and
DFR and LDOX to be encoded by ‘late’ genes. Immunoblot analysis of the enzymes in
the various tt mutants demonstrated higher accumulation of the flavonoid enzymes and
end products as compared to the wt (Pelletier et al., 1999) in contrast to an earlier study where the transcript levels of CHS, CHI and DFR in wt and the tt mutants did not change
(Shirley et al., 1995). tt5 was found to accumulate higher steady-state levels of the enzymes as compared to the wt (Pelletier et al., 1999) and it was suggested that the tetrahydroxychalcone may be involved in the induction of the expression of the enzymes
(Pelletier et al., 1999) while it repressed the synthesis of sinapate esters (Li et al., 1993).
It was further argued that the induction of the flavonoid enzymes by the chalcone could not be explained in the tt3 (encoding DFR) or ttg (encoding a regulatory WD40 repeat protein) mutants, where there were similar elevated expression of the flavonoid enzymes over the wt accumulated elevated levels of flavonols. It was hypothesized that the elevated levels of flavonols in tt3 and ttg mutants and chalcone in tt5 served as signals that post-transcriptionally controlled the accumulation of the flavonoid enzymes
(Pelletier et al., 1999).
Expression data from the microarrays shows that naringenin does not have any significant effect on the expression of the structural enzymes of the flavonoid pathway
94 (Table 2.2), the transcription factors that are known to regulate the pathway (Table 2.4) or the majority of the phenypropanoid pathway genes (Table 2.3) in either tt5 or wt
seedlings. The genes of the flavonoid pathway are robustly expressed indicating that the
conditions used are conducive for anthocyanin induction. This was further demonstrated by northern analysis of AtCHS and AtF3H where there were no changes observed in the naringenin treatment vs. control in tt5 or wt (Fig 2.13). However, comparison of the wt
and tt5 relative expression values (wt-N/tt5-N, Table 2.2) showed that the expression levels of the enzymes CHS, F3H, DFR, F3’H, FLS1, LDOX are expressed >2 fold in tt5
as compared to wt. These results are in agreement with published results where the
protein levels in tt5 and wt were monitored in seedlings (Pelletier et al., 1999) but
contradicts the report where transcript levels of the enzymes show no changes between the mutants and wt as monitored by northern blot in 3 d old seedlings (Shirley et al.,
1995). This difference in the results at the transcript levels between this study and the earlier study (Shirley et al., 1995), may be due to cultural differences between the studies and the microarray results would need to be validated by quantitative RT-PCR or northern analysis.
Examination of the wt vs. tt5 levels of the transcription factors showed no significant changes except for TT8 (Table 2.3), a bHLH transcription factor that coordinately regulates the flavonoid and proanthocyanidin biosynthetic pathway together with the MYB transcription factors PAP1 and TT2, respectively (Baudry et al., 2004).
Thus, the reduction in the expression levels of flavonoid enzymes may be a direct readout of the reduction in the transcript levels of TT8. This down-regulation of TT8 and the enzymes may be attributed to a number of factors. There may be a temporal and
95 developmental regulation, emphasizing the need to take into consideration the developmental differences of wt and tt5 seedlings. The flavonoid intermediate tetrahydroxychalcone, the substrate of CHI may feedback regulate the pathway, however, under our HPLC run conditions, we were unable to detect chalcone in the tt5 seedlings.
Could CHI function as a transcription regulator? CHI together with CHS (Saslowsky et al., 2005), F3H and DFR (Appendix D) are shown to be localized to the nucleus. These enzymes, apart from their biosynthetic function may also ‘moonlight’ as transcriptional regulators as seen in the case of the enzymes of the Krebs cycle, fumarate hydratase and succinate dehydrogenase, which have been show to have tumor suppressing functions
(Jeffery, 2003). More recently Arg5,6 a yeast mitochondrial enzyme involved in arginine metabolism, was shown to bind DNA in vitro and regulate gene expression (Hall et al.,
2004). In plants, a tomato cysteine protease vacuolar processing enzyme (VPE), was shown to bind the promoter and regulate 1-aminocyclopropane-1-carboxylic acid synthase (Moore, 2004; Matarasso et al., 2005).
Thus, the reduction in the levels of flavonoid enzymes may be a direct readout of the reduction in the transcript levels of TT8. This down-regulation of TT8 and the enzymes may be attributed to either a temporal and developmental regulation, emphasizing the need to take into consideration the developmental differences of wt and tt5 seedlings, or that the enzyme CHI, absent in tt5 seedlings and recently shown to be nuclear localized (Saslowsky et al., 2005) (Appendix E) in an unknown manner, is responsible for transcriptional regulation of for example TT8 or of the biosynthetic enzymes.
96 Relative expression values Fold change ATH1 Gene tt5 + wt + wt - AGI No. Gene Annotation Probe ID Symbol tt5 tt5 wt wt N / N / N / - N + N - N + N tt5 - wt - tt5 - N N N
At5g13930 250207_at Chalcone synthase CHS TT4 5212 4736 1665 2240 1.10 0.74 0.47 At3g55120 251827_at Chalcone isomerase CHI TT5 216 213 291 364 1.02 0.80 1.71 At3g51240 252123_at Flavanone 3-hydroxylase F3H TT6 2479 2245 1020 1173 1.10 0.87 0.52 At5g42800 249215_at Dihydroflavonol 4-reductase DFR TT3 2587 2533 823 923 1.02 0.89 0.36 At5g07990 250558_at Flavonoid 3'-hydroxylase F3'H / TT7 760 776 246 284 0.98 0.87 0.37 CYP75B1 At4g12300 254834_at Flavonoid 3', 5'-hydroxylase F3'5'H / 194 197 295 332 0.99 0.89 1.69 CYP706A4 At5g08640 250533_at Flavonol synthase 1 FLS1 1083 862 329 351 1.26 0.94 0.41 At5g63590 247354_at Flavonol synthase 2 FLS2 304 386 305 351 0.79 0.87 0.91 At4g22870 254283_s_at Leucoanthocyanidin dioxygenase / anthocyanidin synthase LDOX / TT18 / 1519 1408 530 576 1.08 0.92 0.41 ANS TDS4 At4g14090 245624_at Anthocyanin 5-Oglucosyltransferase UGT75C1 1281 1509 572 654 0.85 0.87 0.43 At5g17050 246468_at Anthocyanins 3-O-glucosyltransferase UGT78D2 772 862 744 783 0.90 0.95 0.91 97 At3g29590 256924_at Similar to anthocyanin 5-aromatic acyltransferase 347 308 114 133 1.13 0.86 0.43 At1g61720 264401_at Anthocyanidin reductase / dihydroflavonol 4-reductase ANR BAN 18 17 17 18 1.06 0.94 1.06 (dihydrokaempferol 4-reductase) (BAN) At2g38240 267147_at 2OG-Fe(II) oxygenase family protein, similar to flavonol 24 19 25 18 1.23 1.39 0.93 synthase (Citrus unshiu), leucoanthocyanidin dioxygenase (Daucus carota) At5g05270 250794_at Chalcone-flavanone isomerase like family CHI -like 477 430 159 207 1.11 0.77 0.48 At5g48100 248735_at Laccase family protein / diphenol oxidase family protein TT10 58 48 45 50 1.23 0.90 1.05 At5g17220 250083_at glutathione S-transferase AtGSTF12 TT19 2716 3030 1959 2208 0.90 0.89 0.73 At1g02930 262119_s_at glutathione S-transferase, putative AtGST6 3715 3984 3233 2959 0.93 1.09 0.74 At1g02940 262103_at glutathione S-transferase, putative AtGST5 13 13 10 11 0.98 0.88 0.88 At1g17260 262528_at ATPase 10, plasma membrane-type, putative / proton pump 10, AHA10 17 16 14 15 1.09 0.92 0.93 putative / proton-exporting ATPase, putative At3g59030 251504_at multidrug transporter (MATE) TT12 28 24 18 17 1.18 1.05 0.71
Table 2.2 Microarray relative expression values and fold changes of the enzymes of the flavonoid pathway: Relative expression values are the average of the scaled and normalized triplicates. A 2 fold change was considered to be significant. 97 tt5 - tt5 wt - wt tt5 + wt + wt - ATH1 Gene N / N / N / AGI No. Gene Annotation N + N N + N Probe ID Symbol tt5 - wt - tt5 - N N N
At2g37040 263845_at Phenylalanine ammonia-lyase 1 PAL1 877 1211 709 691 0.72 1.03 0.57 At3g53260 251984_at Phenylalanine ammonia-lyase 2 PAL2 399 501 407 445 0.80 0.91 0.89 At5g04230 245690_at Phenylalanine ammonia-lyase 3 PAL3 23 27 43 55 0.85 0.78 2.05 At3g10340 259149_at Phenylalanine ammonia-lyase 164 235 253 274 0.70 0.92 1.17 At2g30490 267470_at Cinnamic acid 4-hydroxylase C4H / 2333 2876 2609 2819 0.81 0.93 0.98 CYP73A5 At1g51680 256186_at 4-coumarate--CoA ligase 1 4CL1 1020 1614 1068 1405 0.63 0.76 0.87 At3g21240 258047_at 4-coumarate--CoA ligase 2 4CL2 430 806 380 463 0.53 0.82 0.57 At1g65060 261907_at 4-coumarate--CoA ligase 3 4CL3 306 314 191 186 0.98 1.03 0.59 At1g20500 259567_at 4-coumarate--CoA ligase family 10 10 10 10 1.04 1.03 1.03 At4g19010 254600_at 4-coumarate--CoA ligase family 56 54 63 56 1.03 1.12 1.04 At1g20480 259569_at 4-coumarate--CoA ligase family 33 33 28 28 0.99 0.99 0.84 At5g63380 247380_at 4-coumarate--CoA ligase family 125 139 139 136 0.90 1.02 0.98 At1g20510 259518_at 4-coumarate--CoA ligase family 405 206 324 402 1.96 0.81 1.95 98 At5g38120 249540_at 4-coumarate--CoA ligase family 16 17 24 17 0.93 1.43 0.97 At1g62940 261096_at 4-coumarate--CoA ligase family 12 12 12 12 0.97 0.95 1.00 At4g05160 255263_at 4-coumarate--CoA ligase, putative 872 996 1008 945 0.88 1.07 0.95 At3g21230 258037_at 4-coumarate--CoA ligase, putative 325 367 415 509 0.89 0.81 1.39 At2g40890 245101_at Coumarate-3-hydroxylase P450 98A3 1339 1940 1157 1300 0.69 0.89 0.67 At5g54160 248200_at Caffeic acid/5-hydroxyferulic acid o- OMT1 1658 2247 1805 2288 0.74 0.79 1.02 methyltransferase / quercetin 3-O-methyltransferase 1 At4g36220 253088_at Ferulate 5-hydroxylase FAH1 / 58 62 38 39 0.94 0.96 0.64 CYP84A1 At3g21560 258167_at Sinapate-1-glucosyltransferase UGT84A2 2810 2762 1436 1789 1.02 0.80 0.65 At5g09640 250501_at Sinapoylglucose:choline-O-sinapoyltransferase 24 19 40 51 1.27 0.79 2.76 At2g22990 267262_at Sinapoylglucose:malate sinapoyltransferase 8 10 8 9 0.79 0.89 0.89
Table 2.3 Microarray relative expression values and fold changes of the enzymes of the phenylpropanoid pathway: A 2 fold change cut off was considered to be significant. Pathway genes downloaded from the TAIR AraCyc resource (http://www.arabidopsis.org:1555/ARA/server.html) 98
tt5 - tt5 wt wt tt5 + wt wt - ATH1 N + N - N + N N / +N / N tt5 - wt - /tt5 -
Gene AGI No. Probe Gene Annotation Symbol N N N ID
At5g24520 249739_at Transparent testa glabra 1, WD40 domain protein TTG1 144 166 190 200 0.87 0.95 1.21 At2g37260 265954_at Transparent testa glabra 2, WRKY family AtWRKY44 TTG2 50 48 32 33 1.03 0.95 0.69 transcription factor At1g56650 245628_at Production of anthocyanin pigment 1 , Myb AtMYB75 PAP1 246 209 220 206 1.18 1.07 0.99 family transcription factor At1g66390 260140_at Production of anthocyanin pigment 2 , Myb AtMYB90 PAP2 285 331 418 449 0.86 0.93 1.36 family transcription factor At1g63650 260242_at Basic helix-loop-helix transcription factor EGL3 87 69 80 84 1.27 0.95 1.23 At4g09820 255056_at Basic helix-loop-helix Transcription factor TT8 96 100 47 55 0.96 0.86 0.55 At5g35550 249704_at Transparent testa 12, Myb family transcription AtMYB123 TT2 13 11 13 12 1.16 1.12 1.08 factor (MYB123)
99 At4g00730 255636_at Anthocyaninless2, homeodomain protein of HD- ANL2 139 146 66 116 0.95 0.57 0.79 GLABRA2 group At5g23260 249851_at Transparent testa 16, MADS-box protein TT16 17 13 12 14 1.27 0.82 1.07 At2g47460 245126_at Myb family transcription factor (MYB12) AtMYB12 159 106 86 88 1.50 0.97 0.84 At3g24650 256898_at Abscisic acid-insensitive protein 3, orthologous ABI3 71 61 65 70 1.17 0.93 1.15 to maize VP1, B3 domain transcription factor
Table 2.4 Microarray relative expression values and fold changes of the known transcription factors involved in the regulation of the flavonoid pathway: Expression cut off was 20 which should be present 50% of the time. A 2 fold change cut off was considered to be significant.
99 2.3.9 Microarray analysis show a total of 48 genes up-regulated and 134 genes down regulated by naringenin in tt5 and wt seedlings.
The addition of naringenin to tt5 and wt seedlings affected a total of 178 genes at the 2-fold cut-off level (Fig. 2.20). Of the 48 up-regulated genes, there were 5 that were induced by naringenin more than two fold in both tt5 and wt seedlings with 28 and 15 genes being unique to tt5+N/ tt5-N and wt+N/wt-N respectively.
There were twice as many genes down-regulated than up-regulated. Of the total of
134 down-regulated genes there were 30 genes that overlapped, 53 unique to the tt5 set and 51 are unique to wt set.
The gene lists were further re-ordered into clusters based on the expression trends using simple queries (Fig. 2.21). This is informative as it broadly classifies the status of the genes in the two parallel systems, where in one system (either tt5 or wt) the genes may be affected whereas the other may show steady state homeostasis levels. For example, cluster 3 represents the genes only up regulated in tt5 upon the addition of naringenin, but did not significantly change in the wt. This is informative as these genes may be implicated in processes involved in the initial responses of the plants to naringenin, anthocyanins or the downstream metabolites leading to anthocyanins (Table
2.5).
Overall, in addition to transcription factors and genes involved in metabolism, and protein modification, there were genes that encoded transporters, transferases, proteins involved in detoxification and a number of genes involved in the metabolism/modification of lipids that were up and down regulated (Fig 2.22). The list of transporters, transferases and glutathione S-transferases are provided in tables 2.7-2.9.
100 Only one UDP-glucoronosyl/UDP-glucosyl transferase family protein (UGT90A,
At2g16890) was up-regulated >2fold in tt5 with a modest increase in wt levels (1.4 fold). It remains to be seen what the substrate for this enzyme would be, either naringenin or an intermediate or cyanidin in lieu of the new anthocyanin peak C3G induced in the presence of naringenin.
Cluster 1 consists of the 5 genes that are up-regulated in both tt5 and wt which may be implicated in the common shared responses to excess naringenin in the process of its detoxification and/or sequestration. These five genes include 2 transporters, an ABC transporter, AtPDR12 (At1g15520) and a cation/hydrogen exchanger AtCHX17
(At4g23700), a peroxidase (At5g05340), a peptide hormone AtPSK3 (At3g49780) and a protease inhibitor/seed storage/lipid transfer protein (At4g12490). Comparison to a recent study using benzoxazolin 2(3H)-one (BOA) as an allelochemical in Arabidopsis identified AtPDR12, AtCHX17 and the lipid transfer protein (At4g12490) together with
DRE-binding protein (DREB2A) and addition clustered LTP genes (At4g12480,
At4g12500) as being up regulated and may represent genes involved in a general detoxification process (Baerson et al 2005). AtPDR12 is one of the 15 members of the pleiotropic drug resistant (PDR) group of the ABC superfamily of transporters (van den
Brule and Smart, 2002) which has been shown to be rapidly induced by a number of compounds including, BOA (Baerson et al., 2005), the antifungal diterpenoid sclaerol, cycloheximide (van den Brule and Smart, 2002), with compatible and incompatible fungi, salicylic acid, ethylene, and methyl jasmonate (Campbell et al., 2003). In a more recent study, AtPDR12 has been implicated in lead detoxification where knockout plants were more susceptible to lead toxicity and the over-expression of AtPDR12 ameliorates the
101 toxic effects of this heavy metal. GFP fusions localize AtPDR12 to the plasma
membrane. Inhibition of glutathione synthesis by buthionine sulfoximine (BSO)
increased toxicity of lead indicating the need for glutathione in the detoxification process
(Lee et al., 2005). A gluthione S-transferase (At2g29450) was up-regulated by naringenin
in both tt5 and wt but was filtered into cluster 3 as wt showed a 1.7 fold up-regulation. It would be interesting to investigate if this GST is involved in modification of naringenin
or a downstream intermediate which would then be pumped out of the cell by AtPDR12.
Recently, results from our laboratory showed eriodictyol to be secreted into the media by
naringenin treated tt5 seedlings, and not the naringenin treated wt seedlings (Lu and
Grotewold unpublished results). It is thought the the excess naringenin in tt5 seedlings is
converted to eriodictyol and then pumped out of the cells. Eriodictyol was not found to be
secreted into the media by naringenin treated Atpdr12 T-DNA insertion mutants, as seen
in the wt. It remains to be tested if the secretion of eriodictyol in Atpdr12 T-DNA
insertion mutants in a tt5 background is affected.
A major portion (13%) of the naringenin affected genes were stress related, which
included peroxidases, heat-shock proteins, genes with unclear functions which have been
reported as being altered in response to pathogen attack or abiotic stress (Fig. 2.23;
Tables 2.5, 2.6). Thus, as seen from the changes in the metabolic profiles these genes
may be potential targets that are actively involved in the modification and transport of
naringenin or anthocyanins or any of the intermediates of the pathway. Furthermore, the
overload of naringenin in the system may trigger stress responses, components of which
are commonly shared between other stress responses. It is interesting that all the nine
102 cytochrome P450’s were down regulated at the 2-fold level, none of which have been functionally characterized.
The majority of the cell-wall associated proteins were down-regulated. As seen in section 2.3.5 (Fig. 2.14, 2.15), naringenin is an inhibitor of growth, especially of root
growth. Inhibition of these cell-wall related genes may arrest cell expansion and growth.
Furthermore, expression profiles in the different organs using the meta-analyzer tool from
Genevestigator (https://www.genevestigator.ethz.ch/at/) show that over 60% of the down-
regulated genes are expressed mainly in the roots.
The stress response viewer from Genevestigator indicated that over 40% of the up
regulated genes overlap (> 2 fold) with Arabidopsis challenged with the biotic pathogens
Botrytis cinerea and Pseudomonas syringae, a phloem feeding aphid Myzus persicae,
chemical treatments of cycloheximide and ozone, and abiotic stress responses of hypoxia,
osmotic and salt (result not shown). This overlap of stress responses shows that there are
common shared pathways that the plant adapts to combat stress, which may act through
jasmonic acid (JA). A lipoxygenase, LOX3, (At1g17420) is up-regulated in tt5 >2 fold
with no change in wt (Cluster 3, Table 2.5). Furthermore, decreasing the cutoff filter to
1.7 fold uncovered sequential enzymes of the JA biosynthetic pathway - lipoxygenase
(At1g72520,) allene oxide cyclase (AOC; At3g25780,), 12-oxophytodienoate reductase
(OPR3; At2g06050,) together with LOX3, to be co-coordinately expressed. Anthocyanins
are known to be induced by methyl-jasmonate (Bate and Rothstein, 1998) and the
induction of genes by methyl-jasmonate alleviates stress (Ellis et al., 2002; Sasaki-
Sekimoto et al., 2005). This is fascinating as it implies that naringenin or intermediates of
the flavonoid pathway function as modulators of cellular processes, as the JA pathway
103 genes are seen to be up-regulated only in tt5 whereas in the wt, they are at steady state levels. Thus, these experiments have uncovered a layer of regulation, which share common genes with the other stress response pathways.
Anthocyanins are classical visual markers for stress, where the common signaling components involved in the plant responses to biotic and abiotic stress have not been identified till date. There are three signal transduction genes, which include a sulfated peptide growth factor, phytosulfokine (AtPSK3, At3g49780), a mitogen activated protein kinase, AtMKK4 (At1g51660) and a calmodulin related protein (At1g76640) were up- regulated by naringenin. Naringenin induced 3 fold AtPSK3 in tt5 and 5 fold in wt, to similar relative expression values. There are five genes in Arabidopsis that encode for
PSK, which were expressed but did not show any change on naringenin treatment. Thus the up-regulation of AtPSK3 is specific to naringenin. PSK is the precursor of a sulfated pentapeptide [Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln] growth factor (PSK-α) which regulates cell proliferation and was first isolated from conditioned media of asparagus mesophyll cell cultures (Matsubayashi et al., 2001).The pentapeptide of PSK-α is cleaved from an approximately 80-amino acid precursor protein which has an N-terminal signal peptide for translocation into the ER and subsequent processing and secretion into the extracellular space (Yang et al., 2001). PSK functions as a plant peptide hormone that stimulates growth and de-differentiation of calli, chlorophyll biosynthesis, formation of adventitious roots and buds and is also involved in differentiation of tracheary elements in cells in culture and in somatic embryogenesis (Yang et al., 2001). Furthermore, the production of PSK-α is co-related with the signal transduction pathway of auxin and cytokinin (Matsubayashi et al., 1999). A leucine rich repeat (LRR) receptor kinase was
104 isolated by ligand-based affinity chromatography from carrot microsomal fractions that
specifically interacted with PSK-α (Matsubayashi et al., 2002). No LRR receptor kinases were filtered through the 2 fold cut off. However, there are two putative LRR transmembrane protein kinase (At1g17750, At1g66830) were up-regulated at ~1.6 fold.
At1g66830 is interesting as it is up-regulated in both tt5 and wt at 1.6 fold while
At1g17750 is only up-regulated in tt5 with no change in wt. It would be interesting to see
if these would be the potential receptors for AtPSK3 in Arabidopsis.
It is also interesting to note that 40% of the genes either up or down regulated
were associated with the endomembrane system according to the GO cellular component
descriptions. There was a down-regulation of genes involved in photosynthesis for
example. porphobilinogen synthase (At1g44318), and ferrochelatase I (At5g26030) This
effect is in addition to the sugar repression of photosynthesis (Sheen, 1990) where
cotyledons of seedlings grown in 3% sucrose do not turn green (Fig 2.3).
105
A tt5+N / tt5-N wt+N / wt-N up up (2 fold) (2 fold) 33 20
28 5 15
B tt5+N / tt5-N wt+N / wt-N down (2 fold) down (2 fold) 83 81
53 30 51
Genes affected by naringenin: Total = 178 Up- regulated = 48 Down- regulated = 134 Shared between up and down sets = 4
Figure 2.20: Venn diagrams depicting number of up and down regulated genes by naringenin in tt5 and wt seedlings
106 1 5 2 30 3 27 4 50
- + - + - + - + - + - + - + - + tt5 wt tt5 wt tt5 wt tt5 wt
5 12 6 50 7 1 8 3
- + - + - + - + - + - + - + - +
107 wt wt wt wt tt5 tt5 tt5 tt5
9 183 10 183
- + - + - + - +
tt5 wt tt5 wt
Figure 2.21. Artificial Cluster patterns: based on trends and not expression levels: Up and down regulated genes in control (-N) and naringenin treated (+) tt5 and wt seedlings were grouped according to their trends of expression into 10 clusters. The cluster number is denoted in red.
107
Cell Wall, 16, 9%
Cell cycle, 1, 1% CYP450, 9, 5% Cytoskeleton, 2, 1% Detoxification, 3, 2% Unknown, 34, 19% Development, 3, 2% DNA transposition, Transporter, 10, 1, 1% 6% Jasmonic acid biosynthesis, 1, 1%
Transferase, 9, 5% Lipid metabolism, 15, 8% Metabolism, 10, 6% Transcription factor, Phenylpropanoid 17, 9% metabolism, 3, 2% Terpenoid Photosynthesis, 5, metabolism, 3, 2% 3% Protein modification, Stress, 23, 13% 9, 5% Signalling, 3, 2% Protein storage, 3, 2%
Figure 2.22 Functional categorization of all the genes affected by naringenin in tt5 and wt seedlings.
108 Detoxification, 2, Up-regulated 4% Development, 1, 2% Cell Wall, 1, 2% DNA transposition, 1, 2% Unknown, 9, 19% Jasmonic acid biosynthesis, 1, 2%
Lipid metabolism, 5, 11% Transporter, 3, 6% Metabolism, 2, 4%
Transferase, 2, 4% Phenylpropanoid metabolism, 1, 2% Photosynthesis, 1, Transcription factor, 2% 4, 9% Protein modification,
3, 6%
Stress, 7, 15% Protein storage, 1, 2% Signalling, 3, 6%
Down-regulated Cell cycle, 1, 1% Cell Wall, 15, 11% Unknown, 25, 19% CYP450, 9, 7%
Cytoskeleton, 2, 2% Detoxification, 1, 1% Transporter, 7, 5% Development, 2, 2%
Lipid metabolism, Transferase, 7, 5% 10, 8%
Metabolism, 8, 6% Transcription factor, 13, 10% Phenylpropanoid metabolism, 2, 2% Terpenoid metabolism, 3, 2% Photosynthesis, 4, 3% Stress, 16, 12% Protein modification, Protein storage, 2, 6, 5% 2%
Figure 2.23: Functional categorization of genes up-regulated and down-regulated by naringenin in tt5 and wt together.
109 ATH1 Gene Relative expression Fold change AGI No. Gene Annotation Function Probe ID Symbol tt5 tt5 wt wt tt5+N wt+N / wt-N/ wt+ N/ -N +N -N +N /tt5-N wt-N tt5 -N tt5+N
tt5 and wt up 5 CLUSTER 1 254832_at At4g12490 Protease inhibitor/seed storage/lipid transfer Lipid metabolism 32 75 71 262 2.35 3.69 2.24 3.52 protein (LTP) family protein /transport 252234_at At3g49780 Phytosulfokines 3 (PSK3) Signaling 126 353 79 362 2.80 4.57 0.63 1.03 250798_at At5g05340 Peroxidase, putative AtPRX52 Stress 106 336 110 289 3.16 2.62 1.04 0.86 261763_at At1g15520 ABC transporter family protein AtPDR12 Transporter 18 62 18 36 3.47 2.02 0.99 0.58 254215_at At4g23700 Cation/hydrogen exchanger, putative AtCHX17 Transporter 76 153 374 815 2.02 2.18 4.93 5.33 (CHX17)
tt5 up 27 wt no change CLUSTER 3 267115_s_ At2g32540 Cellulose synthase family protein --- Cell Wall 66 197 111 97 3.01 0.87 1.69 0.49 at 266299_at At2g29450 Glutathione S-transferase (103-1A) --- Detoxification 148 301 89 150 2.03 1.70 0.60 0.50 261037_at At1g17420 Lipoxygenase LOX3--- Jasmonic acid 50 133 78 77 2.67 0.99 1.57 0.58 biosynthesis
110 259788_at At1g29670 GDSL-motif lipase/hydrolase family protein --- Lipid metabolism 37 81 150 139 2.22 0.93 4.09 1.70 247718_at At5g59310 Lipid transfer protein 4 (LTP4) LTP4 Lipid 175 431 1083 1579 2.47 1.46 6.19 3.66 metabolism/transport 260649_at At1g08080 Carbonic anhydrase family protein --- Metabolism 24 49 27 29 2.03 1.11 1.11 0.60 250149_at At5g14700 Cinnamoyl-coA reductase-related --- Phenylpropanoid 40 100 73 52 2.51 0.71 1.83 0.52 metabolism 247320_at At5g64040 Photosystem I reaction center subunit PSI-N PSI-N Photosynthesis 31 69 88 69 2.24 0.79 2.86 1.00 257130_at At3g20210 Vacuolar processing enzyme, putative / --- Protein modification, 44 91 19 20 2.07 1.05 0.42 0.21 asparaginyl endopeptidase, putative catabolism 261283_s_ At1g35770 Ulp1 protease family protein --- Protein modification, 12 24 9 9 2.04 1.03 0.77 0.39 at catabolism
Continued
Table 2.5 Naringenin up-regulated genes in tt5 and wt : The table consist of genes represented in clusters 1, 3, 5, 7 and 8 (Fig 2.21). A 2 fold cut off was considered to be significant and used as a filter to collate the gene lists. ATHI probe ID are the probe set identification number for the ATH1 GeneChip. AGI no:Arabidopsis genome initiative gene identification number. Gene annotations are from TAIR and functions were allocated manually based on functional description and GO annotations.
110 Table 2.5 continued
ATH1 Gene Relative expression Fold change Probe ID AGI No. Gene Annotation Function Symbol tt5 tt5 wt wt tt5+N wt+N / wt-N/ wt+ N/ -N +N -N +N /tt5-N wt-N tt5 -N tt5+N 253894_at At4g27150 2S albumin seed storage protein 2 --- Protein storage 11 22 7 7 2.05 0.95 0.66 0.31 259866_at At1g76640 Calmodulin-related protein, putative --- Signaling 70 163 144 130 2.32 0.91 2.05 0.80 256183_at At1g51660 Mitogen-activated protein kinase kinase AtMKK4 Signaling 111 227 290 252 2.04 0.87 2.60 1.11 (MAPKK), putative (MKK4) 246004_at At5g20630 Germin-like protein (GER3) GER3 Stress, defense 17 51 12 15 3.04 1.24 0.72 0.30 response development, extracellular matrix 249495_at At5g39100 Germin-like protein (GLP6) GLP6 Stress, defense 59 177 105 145 3.00 1.38 1.79 0.82 response, development, extracellular matrix 253259_at At4g34410 AP2 domain-containing transcription factor, --- Transcription factor 58 191 194 167 3.28 0.86 3.33 0.87 putative 259793_at At1g64380 AP2 domain-containing transcription factor --- Transcription factor 38 98 70 77 2.62 1.09 1.87 0.78 248400_at At5g52020 AP2 domain-containing protein --- Transcription factor 57 131 141 115 2.30 0.81 2.49 0.88 266532_at At2g16890 UDP-glucoronosyl/UDP-glucosyl transferase Transferase 46 107 48 69 2.33 1.44 1.05 0.65 111 family protein UGT90A1 257999_at At3g27540 Glycosyl transferase family 17 protein --- Transferase 69 139 165 141 2.00 0.86 2.38 1.02 253832_at At4g27654 Expressed protein --- Unknown 76 242 240 168 3.17 0.70 3.14 0.69 253643_at At4g29780 Expressed protein --- Unknown 311 774 871 687 2.49 0.79 2.80 0.89 266486_at At2g47950 Expressed protein --- Unknown 28 63 37 56 2.25 1.51 1.33 0.89 266901_at At2g34600 Expressed protein --- Unknown 54 113 310 216 2.10 0.70 5.73 1.90 265099_at At1g03990 Alcohol oxidase-related --- Unknown 26 53 13 16 2.03 1.22 0.51 0.30 245982_at At5g13170 Nodulin mtn3 family protein --- Unknown, 443 924 338 349 2.09 1.03 0.76 0.38 development, senescence associated 253632_at At4g30430 Senescence-associated family protein --- Unknown, 65 133 288 299 2.04 1.04 4.41 2.25 senescence associated
tt5 up wt down 1 CLUSTER 7 255937_at At1g12610 DRE-binding protein, putative / CRT/DRE- --- Transcription factor 13 29 64 22 2.20 0.33 4.90 0.75 binding factor, putative
111 Continued Table 2.5 continued
ATH1 Gene Relative expression Fold change Probe ID AGI No. Gene Annotation Function Symbol tt5 tt5 wt wt tt5+N wt+N / wt-N/ wt+ N/ -N +N -N +N /tt5-N wt-N tt5 -N tt5+N wt up tt5 no change 12 CLUSTER 5 266270_at At2g29470 Glutathione S-transferase, putative --- Detoxification 111 124 35 89 1.12 2.56 0.31 0.71 264411_at At1g43240 Mutator-like transposase family --- DNA transposition 12 12 14 39 0.97 2.79 1.18 3.40 254819_at At4g12500 Protease inhibitor/seed storage/lipid transfer --- Lipid Modification 17 20 35 95 1.24 2.69 2.13 4.63 protein (LTP) family protein 254805_at At4g12480 Protease inhibitor/seed storage/lipid transfer --- Lipid Modification/ 404 595 475 1100 1.47 2.31 1.18 1.85 protein (LTP) family protein transport 249101_at At5g43580 Protease inhibitor, putative --- Protein modification 85 62 66 161 0.74 2.44 0.78 2.58 249630_s_ At5g37150 Trna-splicing endonuclease positive effector- --- RNA metabolism 21 22 30 70 1.03 2.35 1.41 3.23 at related 252515_at At3g46230 17.4 kda class I heat shock protein (HSP17.4- Stress 139 185 44 102 1.33 2.32 0.32 0.55 CI) 261838_at At1g16030 Heat Shock protein, HSP70, putative Stress 184 221 117 235 1.20 2.02 0.63 1.06
255807_at At4g10270 Wound-responsive family protein --- Stress 1260 797 237 568 0.63 2.40 0.19 0.71 249492_at At5g39160 Germin-like protein (GLP2a) (GLP5a) /// --- Stress, development, 39 35 188 860 0.89 4.58 4.80 24.55 112 germin-like protein, putative extra cellular matrix 250781_at At5g05410 DRE-binding protein (DREB2A) --- Transcription factor 527 499 191 386 0.95 2.02 0.36 0.77 258487_at At3g02550 LOB domain protein 41 / lateral organ Unknown 1516 1013 479 998 0.67 2.08 0.32 0.99 boundaries domain protein 41 (LBD41)
wt up tt5 down 3 CLUSTER 8 267024_s_ At2g34390 Major intrinsic family protein / MIP family --- Transporter 197 66 23 57 0.34 2.47 0.12 0.86 at protein 258930_at At3g10040 Expressed protein --- Unknown 587 250 114 238 0.43 2.08 0.20 0.95 261567_at At1g33055 Expressed protein --- Unknown 758 318 103 335 0.42 3.24 0.14 1.05
112 Relative expression Fold change ATH1 Gene AGI No. Gene Annotation Function Probe ID Symbol tt5 tt5 wt wt tt5+N / wt+N / wt–N / wt + N/ -N +N -N +N tt5-N wt-N tt5 -N tt5 + N
tt5 and wt down 30 CLUSTER 2 260492_at At2g41850 Endo-polygalacturonase, putative --- carbohydrate 52 141 21 47 0.37 0.45 0.33 0.41 metabolism 246652_at At5g35190 Proline-rich extensin-like family protein --- Cell Wall 28 109 18 45 0.26 0.40 0.41 0.63 248823_s_ At5g46960 Invertase/pectin methylesterase inhibitor --- Cell Wall 28 72 14 54 0.39 0.26 0.74 0.51 at family protein 256352_at At1g54970 Proline-rich family protein --- Cell Wall 54 132 39 116 0.41 0.33 0.88 0.72 251226_at At3g62680 Proline-rich family protein --- Cell wall 305 734 155 402 0.42 0.39 0.55 0.51 250778_at At5g05500 Pollen Ole e 1 allergen and extensin family --- Cell Wall 128 284 59 139 0.45 0.42 0.49 0.46 protein 250801_at At5g04960 Pectinesterase family protein --- Cell Wall 185 390 121 245 0.47 0.49 0.63 0.65 249684_s_ At5g36110 Cytochrome P450 family protein CYP716A1 CYP450 12 58 17 45 0.21 0.38 0.78 1.42 at 248727_at At5g47990 Cytochrome P450 family protein CYP705A5 CYP450 624 1602 428 1434 0.39 0.30 0.90 0.69 249202_at At5g42580 Cytochrome P450 family protein CYP705A12 CYP450 13 29 16 42 0.43 0.39 1.43 1.29
113 245689_at At5g04120 Phosphoglycerate/bisphosphoglycerate --- Glycolysis 81 185 91 207 0.44 0.44 1.12 1.13 mutase family protein 249814_at At5g23840 MD-2-related lipid recognition domain- --- Lipid metabolism 101 468 32 100 0.21 0.32 0.21 0.32 containing protein / ML domain-containing protein 249567_at At5g38020 S-adenosyl-L-methionine:carboxyl --- Lipid metabolism 99 357 68 145 0.28 0.47 0.41 0.68 methyltransferase family protein 248844_s_ At5g46900 Protease inhibitor/seed storage/lipid transfer --- Lipid metabolism 316 974 79 181 0.32 0.44 0.19 0.25 at protein (LTP) family protein /// protease inhibitor/seed storage/lipid transfer protein (LTP) family protein
Continued
Table 2.6 Naringenin down-regulated genes in tt5 and wt: The table consists of genes represented in clusters 2,4,6, 7 and 8 (Fig 2.21). A 2 fold cut off was considered to be significant and used as a filter to collate the gene lists. ATHI probe ID are the probe set identification number for the ATH1 GeneChip. AGI no. is the Arabidopsis Genome Initiative gene identification number. Gene annotations are from TAIR and functions were allocated manually based on functional description and GO annotations.Clusters 7 and 8 are repeated in this list. Peroxidase annotations were obtained from the PeroxiBase: A class III plant peroxidase database (http://peroxidase.isb-sib.ch/index.php)
113 Table 2.6 continued
Relative expression Fold change ATH1 Gene AGI No. Gene Annotation Function Probe ID Symbol tt5 tt5 wt wt tt5+N / wt+N / wt–N / wt + N/ -N +N -N +N tt5-N wt-N tt5 -N tt5 + N 263098_at At2g16005 MD-2-related lipid recognition domain- --- Lipid metabolism 49 143 66 193 0.34 0.34 1.35 1.34 containing protein / ML domain-containing protein 254828_at At4g12550 Protease inhibitor/seed storage/lipid transfer --- Lipid metabolism, 120 606 203 911 0.20 0.22 1.50 1.69 protein (LTP) family protein response to auxin stimulus /// lateral root morphogenesis 256647_at At3g13610 Oxidoreductase, 2OG-Fe(II) oxygenase --- Metabolism 764 1691 666 1337 0.45 0.50 0.79 0.87 family protein 245245_at At1g44318 Porphobilinogen synthase, putative / delta- --- Photosynthesis 97 356 130 268 0.27 0.49 0.75 1.34 aminolevulinic acid dehydratase, putative 245736_at At1g73330 Protease inhibitor, putative (DR4) DR4 Protein modification 166 385 163 364 0.43 0.45 0.95 0.99 260101_at At1g73260 Trypsin and protease inhibitor family protein / --- Protein 211 765 230 583 0.28 0.39 0.76 1.09 Kunitz family protein modification, catabolism 261157_at At1g34510 Peroxidase, putative AtPRX08 Stress 15 35 12 27 0.41 0.45 0.76 0.83 262838_at At1g14960 Major latex protein-related / MLP-related --- Stress, development 174 630 95 386 0.28 0.25 0.61 0.55 114 253353_at At4g33730 Pathogenesis-related protein, putative --- Stress, Pathogen 40 89 26 69 0.45 0.37 0.77 0.64 response 248729_at At5g48010 Pentacyclic triterpene synthase, putative ATPEN1 Terpenoid 496 1323 243 572 0.37 0.42 0.43 0.49 metabolism 254044_at At4g25820 Xyloglucan:xyloglucosyl transferase / XTR9 Transferase 329 886 281 612 0.37 0.46 0.69 0.85 xyloglucan endotransglycosylase / endo- xyloglucan transferase (XTR9) 247871_at At5g57530 Xyloglucan:xyloglucosyl transferase, putative --- Transferase 50 123 25 61 0.41 0.41 0.50 0.50 / xyloglucan endotransglycosylase, putative / endo-xyloglucan transferase, putative 245555_at At4g15390 Transferase family protein --- Transferase, acyl 202 486 188 426 0.42 0.44 0.88 0.93 phenylpropanpid/fla vonoid putative 252605_s_ At3g45070 Sulfotransferase family protein /// --- Transferase 169 544 110 249 0.31 0.44 0.46 0.65 at sulfotransferase family protein 266336_at At2g32270 Zinc transporter (ZIP3) --- Transporter 71 156 97 243 0.45 0.40 1.56 1.37 262301_at At1g70880 Bet v I allergen family protein --- Unknown, stress, 29 59 33 150 0.50 0.22 2.54 1.11 development
114 Continued Table 2.6 continued
Relative expression Fold change ATH1 Gene AGI No. Gene Annotation Function Probe ID Symbol tt5 tt5 wt wt tt5+N / wt+N / wt–N / wt + N/ -N +N -N +N tt5-N wt-N tt5 -N tt5 + N tt5 down wt no change 50 CLUSTER 4
247954_at At5g56870 Beta-galactosidase, putative --- carbohydrate 421 1178 591 868 0.36 0.68 0.74 1.40 metabolism 245148_at At2g45220 Pectinesterase family protein --- Cell wall 69 180 247 314 0.39 0.79 1.75 3.56 251843_x_ At3g54590 Proline-rich extensin-like family protein --- Cell wall 262 526 108 167 0.50 0.65 0.32 0.41 at 249686_at At5g36140 Cytochrome P450 CYP716A2 CYP450 21 150 20 39 0.14 0.51 0.26 0.95 263970_at At2g42850 Cytochrome P450 CYP718 CYP450 35 97 45 75 0.35 0.60 0.77 1.31 249203_at At5g42590 Cytochrome P450 CYP71A16 CYP450 19 40 21 40 0.47 0.53 1.01 1.13 253502_at At4g31940 Cytochrome P450, putative CYP82C4 CYP450 103 208 11 15 0.50 0.71 0.07 0.10 265067_at At1g03850 Glutaredoxin family protein --- Detoxification 135 276 79 108 0.49 0.74 0.39 0.59 259036_at At3g09220 Laccase family protein / diphenol oxidase --- Phenylpropanoid 49 178 226 392 0.27 0.58 2.21 4.65 family protein metabolism; Laccase 257774_at At3g29250 Short-chain dehydrogenase/reductase (SDR) --- Lipid metabolism 321 970 371 738 0.33 0.50 0.76 1.16
115 family protein 258957_at At3g01420 Pathogen-responsive alpha-dioxygenase --- Lipid metabolism 516 1040 784 974 0.50 0.80 0.94 1.52 putative, lipoxygenase activity 259346_at At3g03910 Glutamate dehydrogenase, putative --- Metabolism, amino 36 73 62 62 0.49 0.99 0.85 1.73 acid 254163_s_ At4g24340 Phosphorylase family protein --- Metabolism, nucleic 204 456 201 314 0.45 0.64 0.69 0.98 at acid 254975_at At4g10500 Oxidoreductase, 2OG-Fe(II) oxygenase --- Metabolism, 16 48 27 48 0.33 0.57 1.00 1.71 family protein secondary 256368_at At1g66800 Cinnamyl-alcohol dehydrogenase family --- Phenylpropanoid 139 344 93 149 0.40 0.62 0.43 0.67 metabolism 245264_at At4g17245 Zinc finger (C3HC4-type RING finger) --- Protein modification 69 161 103 100 0.43 1.03 0.62 1.49 family protein 245967_at At5g19800 Hydroxyproline-rich glycoprotein family --- Protein storage 21 51 18 31 0.41 0.60 0.60 0.88 protein 253743_at At4g28940 Nucleosidase-related --- Protein storage 88 186 142 241 0.47 0.59 1.29 1.60 261901_at At1g80920 DNAJ heat shock N-terminal domain- --- Stress 218 500 243 209 0.44 1.16 0.42 1.11 containing protein 261606_at At1g49570 Peroxidase, putative AtPRX10 Stress 36 75 70 138 0.48 0.50 1.84 1.91 247327_at At5g64120 Peroxidase, putative AtPRX71 Stress 525 1078 1196 882 0.49 1.36 0.82 2.28 254539_s_ At4g19750 Chitinase like protein, Glycosyl hydrolase --- Stress, defense 17 44 26 29 0.38 0.89 0.66 1.54 at family 18 protein response 115 Continued Table 2.6 continued
Relative expression Fold change ATH1 Gene AGI No. Gene Annotation Function Probe ID Symbol tt5 tt5 wt wt tt5+N / wt+N / wt–N / wt + N/ -N +N -N +N tt5-N wt-N tt5 -N tt5 + N 265053_at At1g52000 Jacalin lectin family protein --- Stress, defense 20 44 15 16 0.44 0.97 0.35 0.77 response 254537_at At4g19730 Chitinase like protein, glycosyl hydrolase --- Stress, defense 15 33 14 13 0.47 1.01 0.41 0.89 family 18 protein response 258203_at At3g13950 Expressed protein, transmembrane domain --- Stress, defense 50 102 159 250 0.49 0.64 2.45 3.17 response, extracellular 266353_at At2g01520 Major latex protein-related --- Stress, development 207 552 229 429 0.38 0.53 0.78 1.11 266330_at At2g01530 Major latex protein-related / MLP-related --- Stress, development 639 1358 496 949 0.47 0.52 0.70 0.78 245567_at At4g14630 Germin-like protein (GLP9) --- Stress, development, 82 172 778 554 0.48 1.40 3.22 9.52 extracellular matrix 256300_at At1g69490 No apical meristem (NAM) family protein --- Transcription factor 133 528 159 110 0.25 1.44 0.21 1.20 259365_at At1g13300 Golden 2 (G2)-like family --- Transcription factor 117 254 126 121 0.46 1.04 0.48 1.08 259751_at At1g71030 Myb family transcription factor --- Transcription factor 155 335 123 105 0.46 1.17 0.31 0.79 247540_at At5g61590 AP2 domain-containing transcription factor --- Transcription factor 57 121 58 58 0.47 1.00 0.48 1.02 family protein
116 255794_at At2g33480 No apical meristem (NAM) family protein --- Transcription factor 47 99 92 86 0.48 1.07 0.87 1.96 264119_at At1g79180 Myb family transcription factor (MYB63) MYB63 Transcription factor 27 55 31 26 0.48 1.21 0.47 1.17 266669_at At2g29750 UDP-glucoronosyl/UDP-glucosyl transferase UGT71C1 Transferase 105 244 279 451 0.43 0.62 1.85 2.65 family protein 248725_at At5g47980 Acetyl-coA:benzylalcohol acetyltranferase- --- Transferase 799 1715 780 1428 0.47 0.55 0.83 0.98 like protein 252537_at At3g45710 Proton-dependent oligopeptide transport --- Transporter 44 160 70 136 0.28 0.52 0.85 1.59 (POT) family protein 249545_at At5g38030 MATE efflux family protein --- Transporter 65 148 73 112 0.44 0.65 0.76 1.12 246884_at At5g26220 Chac-like family protein, putative cation --- Transporter 82 175 37 54 0.47 0.69 0.31 0.45 transporter 257615_at At3g26510 Octicosapeptide/Phox/Bem1p (PB1) domain- --- Unknown 209 562 99 90 0.37 1.10 0.16 0.47 containing protein 262986_at At1g23390 Kelch repeat-containing F-box family protein --- Unknown 68 170 91 93 0.40 0.98 0.55 1.34 252040_at At3g52060 Expressed protein --- Unknown 171 421 150 144 0.41 1.05 0.34 0.88 255602_at At4g01026 Expressed protein --- Unknown 94 213 68 55 0.44 1.25 0.26 0.73 246001_at At5g20790 Expressed protein --- Unknown 117 265 73 82 0.44 0.89 0.31 0.62 252057_at At3g52480 Expressed protein --- Unknown 23 51 39 34 0.45 1.15 0.66 1.69 248518_at At5g50560 Expressed protein --- Unknown 115 254 218 280 0.45 0.78 1.10 1.90
116 Continued Table 2.6 continued Relative expression Fold change ATH1 Gene AGI No. Gene Annotation Function Probe ID Symbol tt5 tt5 wt wt tt5+N / wt+N / wt–N / wt + N/ -N +N -N +N tt5-N wt-N tt5 -N tt5 + N 265478_at At2g15890 Expressed protein --- Unknown 273 596 152 121 0.46 1.25 0.20 0.56 252730_at At3g43110 Expressed protein, transmembrane domain --- Unknown 35 72 28 32 0.49 0.87 0.45 0.79 258080_at At3g25930 Universal stress protein (USP) family protein --- Unknown, Stress 108 228 95 152 0.48 0.62 0.67 0.88 250464_at At5g10040 Expressed protein, predicted transmembrane --- Unknown 135 303 55 46 0.45 1.19 0.15 0.40 domain
tt5 down wt up 3 CLUSTER 8
267024_s_ At2g34390 Major intrinsic family protein / MIP family --- Transporter 66 197 57 23 0.34 2.47 0.12 0.86 at protein 261567_at At1g33055 Expressed protein --- Unknown 318 758 335 103 0.42 3.24 0.14 1.05 258930_at At3g10040 Expressed protein --- Unknown 250 587 238 114 0.43 2.08 0.20 0.95
wt down tt5 no change 50 CLUSTER 6
117 265633_at At2g25490 F-box family protein (FBL6), putative glucose Unknown 1054 1112 480 1044 0.95 0.46 0.94 0.46 regulated repressor protein 247536_at At5g61650 Cyclin family protein --- Cell cycle 45 66 44 98 0.68 0.45 1.49 0.98 255516_at At4g02270 Pollen Ole e 1 allergen and extensin family --- Cell wall 330 559 144 463 0.59 0.31 0.83 0.44 protein 258765_at At3g10710 Pectinesterase family protein --- Cell wall 79 151 43 111 0.52 0.39 0.73 0.54 261099_at At1g62980 Expansin, putative (EXP18) --- Cell wall 46 91 30 72 0.50 0.42 0.79 0.65 255221_at At4g05150 Octicosapeptide/Phox/Bem1p (PB1) domain- --- Cell wall 876 1018 229 515 0.86 0.45 0.51 0.26 containing protein 266588_at At2g14890 Arabinogalactan-protein (AGP9) AGP9 Cell wall 2836 2648 1114 2303 1.07 0.48 0.87 0.39 262260_at At1g70850 Bet v I allergen family protein --- Cell wall 553 1066 276 566 0.52 0.49 0.53 0.50 249675_at At5g35940 Jacalin lectin family protein --- Cell wall 81 109 33 65 0.75 0.50 0.60 0.40 247765_at At5g58860 Cytochrome P450 86A1 (CYP86) --- CYP450 213 336 146 409 0.63 0.36 1.22 0.69 (CYP86A1) / CYPLXXXVI / P450- dependent fatty acid omega-hydroxylase 254767_s_ At4g13290 Cytochrome P450 71A19, putative CYP71A19 CYP450 14 15 21 44 0.91 0.47 2.96 1.51 at (CYP71A19) /// cytochrome P450 71A20, putative (CYP71A20) 265779_at At2g07370 SH3 domain-containing protein --- Cytoskeleton 101 95 29 67 1.07 0.44 0.71 0.29 255632_at At4g00680 Actin-depolymerizing factor, putative --- Cytoskeleton 53 75 36 75 0.71 0.48 1.00 0.67
117 Continued Table 2.6 continued
Relative expression Fold change ATH1 Gene AGI No. Gene Annotation Function Probe ID Symbol tt5 tt5 wt wt tt5+N / wt+N / wt–N / wt + N/ -N +N -N +N tt5-N wt-N tt5 -N tt5 + N 248178_at At5g54370 Late embryogenesis abundant protein-related, --- Development 84 124 44 98 0.68 0.45 0.79 0.53 root cap protein 2-like protein 252833_at At4g40090 Arabinogalactan-protein (AGP3) AGP3 Development 31 56 14 30 0.55 0.46 0.53 0.45 264147_at At1g02200 CER1 protein --- Lipid metabolism 188 251 36 93 0.75 0.39 0.37 0.19 254326_at At4g22610 Protease inhibitor/seed storage/lipid transfer --- Lipid metabolism 448 809 382 792 0.55 0.48 0.98 0.85 protein (LTP) family protein 262317_at At2g48140 Protease inhibitor/seed storage/lipid transfer --- Lipid metabolism 161 258 120 244 0.62 0.49 0.94 0.75 protein (LTP) family protein 263539_at At2g24850 Aminotransferase, putative --- Metabolism, amino 54 52 14 30 1.05 0.48 0.57 0.26 acid 262659_at At1g14240 Nucleoside phosphatase family protein / --- Nucleic acid 52 65 30 61 0.80 0.49 0.95 0.58 GDA1/CD39 family protein metabolism 244964_at psbE ------Photosynthesis 590 471 147 299 1.25 0.49 0.64 0.25 258055_at At3g16250 Ferredoxin-related --- Photosynthesis 18 16 13 25 1.17 0.50 1.61 0.69 246870_at At5g26030 Ferrochelatase I --- Photosynthesis 902 634 273 793 1.42 0.34 1.25 0.30 252948_at At4g38610 HECT-domain-containing protein / ubiquitin- --- Protein modification 61 58 20 73 1.05 0.27 1.26 0.33
118 transferase family protein 260890_at At1g29090 Peptidase C1A papain family protein --- Protein 426 807 37 196 0.53 0.19 0.24 0.09 modification, catabolism 261215_at At1g32970 Subtilase family protein --- Protein 12 11 17 40 1.03 0.42 3.46 1.41 modification, catabolism 250157_at At5g15180 Peroxidase, putative AtPRX56 Stress 56 67 58 132 0.84 0.44 1.97 1.02 265102_at At1g30870 Cationic peroxidase, putative AtPRX07 Stress 120 196 57 125 0.61 0.45 0.64 0.47 266191_at At2g39040 Peroxidase, putative AtPRX24 Stress 15 20 13 26 0.74 0.49 1.32 0.88 249687_at At5g36150 Pentacyclic triterpene synthase, putative ATPEN1 Terpenoid 23 32 30 63 0.73 0.48 1.95 1.29 metabolism 256736_at At3g29410 Terpene synthase/cyclase family protein --- Terpenoid 86 114 95 193 0.75 0.49 1.70 1.11 metabolism 266821_at At2g44840 Ethylene-responsive element-binding protein, --- Transcription factor 55 34 73 201 1.64 0.36 5.96 1.31 putative 267515_at At2g45680 TCP family transcription factor, putative --- Transcription factor 116 74 50 123 1.56 0.41 1.65 0.43 247508_at At5g62000 Transcriptional factor B3 family protein / --- Transcription factor 348 299 71 171 1.16 0.42 0.57 0.20 auxin-responsive factor, putative (ARF1) 261496_at At1g28360 ERF domain protein 12 (ERF12) --- Transcription factor 98 92 64 148 1.06 0.44 1.60 0.66 266748_at At2g47070 Squamosa promoter-binding protein-like 1 spl1 Transcription factor 65 53 27 60 1.22 0.45 1.14 0.42
118 Continued Table 2.6 continued Relative expression Fold change ATH1 Gene AGI No. Gene Annotation Function Probe ID Symbol tt5 tt5 wt wt tt5+N / wt+N / wt–N / wt + N/ -N +N -N +N tt5-N wt-N tt5 -N tt5 + N (SPL1) 251950_at At3g53600 Zinc finger (C2H2 type) family protein --- Transcription factor 14 17 10 23 0.81 0.45 1.30 0.72 265052_at At1g51990 O-methyltransferase family 2 protein --- Transferase 23 33 15 40 0.70 0.38 1.21 0.66 245399_at At4g17340 Major intrinsic family protein / MIP family --- Transporter 208 230 308 762 0.90 0.41 3.31 1.48 protein 248790_at At5g47450 Major intrinsic family protein / MIP family --- Transporter 18 16 28 58 1.06 0.47 3.54 1.57 protein 250970_at At5g02770 Expressed protein --- Unknown 281 310 93 248 0.91 0.37 0.80 0.33 245209_at At5g12340 Expressed protein --- Unknown 177 163 87 230 1.09 0.38 1.41 0.49 256419_at At1g33470 RNA recognition motif (RRM)-containing --- Unknown 40 39 16 40 1.03 0.41 1.02 0.40 protein 249364_at At5g40590 Expressed protein --- Unknown 11 13 25 57 0.80 0.44 4.26 2.34 249042_at At5g44350 Ethylene-responsive nuclear protein -related --- Unknown 34 39 26 58 0.87 0.44 1.50 0.76 246959_at At5g24700 Expressed protein --- Unknown 185 153 40 87 1.21 0.46 0.57 0.22 257709_at At3g27325 Expressed protein --- Unknown 43 40 16 35 1.09 0.47 0.87 0.37 247215_at At5g64905 Expressed protein --- Unknown 146 191 52 109 0.76 0.47 0.57 0.35 119 258421_at At3g16690 Nodulin mtn3 family protein --- Unknown 37 27 28 59 1.39 0.48 2.19 0.76 262226_at At1g53885 Senescence-associated protein-related --- Unknown, 124 151 104 208 0.82 0.50 1.38 0.84 senescence related
wt down tt5 up 1 CLUSTER 7
255937_at At1g12610 DRE-binding protein, putative / CRT/DRE- --- Transcription factor 29 13 22 64 2.20 0.33 4.90 0.75 binding factor, putative
119
Probe set AGI no. Gene Title Gene symbol Cluster tt5+N wt+N ID /tt5- /wt- N N 261763_at At1g15520 ABC transporter family protein AtPDR12 1 3.47 2.02 254215_at At4g23700 Cation/hydrogen exchanger, AtCHX17 1 2.02 2.18 putative (CHX17)
267024_s_at At2g34390 Major intrinsic family protein / AtNIP2.1/AtNLM3 8 0.34 2.47 NOD26-like intrinsic protein
266336_at At2g32270 Zinc transporter (ZIP3) AtZIP3 2 0.45 0.40 252537_at At3g45710 Proton-dependent oligopeptide 4 0.28 0.52 transport (POT) family protein 249545_at At5g38030 MATE efflux family protein AtDTX30 4 0.44 0.65 246884_at At5g26220 ChaC-like family protein, 4 0.47 0.69
120 putative cation transporter 245399_at At4g17340 Major intrinsic family protein / AtTIP2.2 6 0.90 0.41 tonoplast intrinsic protein 2 delta 248790_at At5g47450 Major intrinsic family protein / AtTIP2.3 6 1.06 0.47 tonoplast intrinsic protein 3 delta
Table 2.7 Transporters identified in this study that are regulated by naringenin in wt and tt5 seedlings: gene symbols are described as from the Aramemmon (http://aramemnon.botanik.uni-koeln.de/index.ep) plant membrane protein database
120
Probe set AGI no. Gene Title Gene Cluster tt5+ wt+ ID symbol N N /tt5- /wt- N N 266532_at At2g16890 UDP-glucoronosyl/UDP- UGT90A1 3 2.33 1.44 glucosyl transferase family protein 257999_at At3g27540 Glycosyl transferase family 17 --- 3 2.00 0.86 protein, N-linked glycosylation
54044_at At4g25820 Xyloglucan:xyloglucosyl XTH14 2 0.37 0.46 transferase / xyloglucan (XTR9) endotransglycosylase / endo- xyloglucan transferase (XTR9) 247871_at At5g57530 Xyloglucan:xyloglucosyl XTH12 2 0.41 0.41 transferase, putative / 121 xyloglucan endotransglycosylase, putative / endo-xyloglucan transferase, 245555_at At4g15390 Transferase family protein --- 2 0.42 0.44 252605_s_a At3g45070 Sulfotransferase family protein --- 2 0.31 0.44 t 266669_at At2g29750 UDP-glucoronosyl/UDP- UGT71C1 4 0.43 0.62 glucosyl transferase family protein 248725_at At5g47980 Acetyl-CoA:benzylalcohol --- 4 0.47 0.55 acetyltranferase-like protein 265052_at At1g51990 O-methyltransferase --- 6 0.70 0.38
Table 2.8 Transferases identified in this study as being up or down-regulated by naringenin in tt5 and wt seedlings: information for the UDP-glycosyl transferases from the Arabidopsis P450, cytochrome b5, P450 Reductase and Glycosyltransferase Family 1 site at PlaCe (http://www.p450.kvl.dk//index.shtml) and xyloglucan transferase obtained from Cell Wall Navigator (http://bioweb.ucr.edu/Cellwall/) 121
Probe set AGI no. Gene Title Gene Cluster tt5+N wt+N ID symbol /tt5-N /wt- N 266299_at At2g29450 glutathione S-transferase - 3 2.03 1.70 (103-1A) 266270_at At2g29470 glutathione S-transferase, - 5 1.12 2.56
Table 2.9 The glutathione S-transferases identified as being regulated by naringenin in tt5 and wt seedlings
122
122 2.4 Conclusions
This study has taken an integrated approach of metabolic and transcriptome
profiling to identify the biochemical changes and molecular players involved not only in the trafficking of anthocyanins to the vacuole but to identify components of a toxic compound defense network. Naringenin, in a simple seedling system, had the ability to complement the flavonoid pathway leading to anthocyanins in tt5 mutants, inducing the accumulation of cyanidin 3-glucoside and changing the anthocyanin profiles of the cotyledons. Recent experiments in our laboratory demonstrated that C3G is the precursor of the different anthocyanins observed. Its accumulation was attributed to several possibilities where its excess accumulation triggered its sequestration, the latter modification enzymes were either low in abundance or exhibited low efficiency or the compounds and the enzymes were spatially separated in different compartments of the cell.
A cellular snapshot of genome wide expression profiles of naringenin treated tt5 and wt seedlings was useful in monitoring not only the changes in expression of the pathway genes but identification of cellular processes induced or repressed by naringenin.
The absence of a change in the expression of the core flavonoid genes in naringenin treated tt5 and wt seedlings rules out the possibility of feedback regulation of the enzymes at the transcriptional level. The confirmation of previously observed results
(Pelletier et al., 1999), that the flavonoid biosynthetic enzymes are up regulated in tt5 as compared to wt, together with the bHLH TT8, puts forth an interesting possibility that
CHI may act as a regulatory protein at the transcription level. Furthermore, the result that naringenin does not change the expression levels of the core flavonoid pathway genes,
123 may indicate that the transferases and transporters may already be robustly induced in the
conditions used for growth. Cluster 1 genes, with induction in both tt5 and wt, implicated in the detoxification process, were in good confirmation as they are shown to be involved in detoxification of xenobiotics. AtPDR12, an ABC transporter superfamily member is a generalist transporter, involved in the elimation of compounds from the cells. A GST
(At2g29450) is proposed to be involved in the conjugation phase of naringenin or its
intermediate metabolite and works together with AtPDR12. Recent results show
eriodictyol to be secreted into the media in the naringenin treated tt5 seedlings (Lu and
Grotewold, unpublished results). Efforts are on validate the expression by quantitative
RT-PCR and characterize the secretion of flavonoids in the Atpdr12 T-DNA insertion
line. A UDP-glucosyl transferase (At2g16890) was induced in the naringenin treated tt5
seedlings with no change in the wt seedlings (cluster 3). This transferase may be involved
in the modification of either naringenin or any of the downstream metabolites.
The induction of a series of JA biosynthetic genes unveils another layer of
regulation by naringenin or its downstream metabolites. Comparison of microarray
results in the Genevestigator response viewer showed forty percent of the induced genes
to be shared between biotic stress responses of pathogenic organisms and abiotic stress
conditions such as hypoxia, salt and osmotic. It is as though the plant sensed a signal in
the naringenin treatment and turned on defense related genes, which may act through JA.
This is true of tt5 where the JA biosynthetic enzymes are up-regulated as compared to wt
where levels are already up. Little is known of the signal transduction pathways leading to the induction of anthocyanins or the pathways that the intermediates induce. It is exciting that phytosulfokines, the sulfonated pentapetides secreted into the extracellular
124 matrix mainly controlling plant cell proliferation is induced in both tt5 and wt. The up regulation of a couple of LRR receptor-like protein kinases leads to interesting possibilities of potential ligand-receptor relationships, which have not been yet characterized in Arabidopsis.
Thus naringenin, or intermediates of the flavonoid pathway may not only be involved in metabolism but also act in modulating cellular processes in an event of stress.
This study provides potential leads into investigation exciting areas of biology.
125 CHAPTER 3
COMPLEMENTATION OF ARABIDOPSIS THALIANA tt5 WITH MAIZE
CHALCONE ISOMERASE MUTANTS: INSIGHTS INTO THE ROLE OF CHI
IN THE FLAVONOID PATHWAY
3.1 Introduction
The metabolic engineering of the flavonoid pathway is an attractive proposition as
it leads to plants with enhanced protection towards biotic and abiotic stress conditions, as
well as increased production of favorable natural products with pharmacological and
agricultural utilities. Manipulation of flavonoid biosynthesis can be achieved by over- expressing or knocking down pathway genes by sense or antisense gene manipulations
(Jorgensen et al., 1996), by targeting the expression levels specifically of regulatory genes in the pathway (Grotewold et al., 1998), or by designing novel enzymatic specificities inferred from protein structure data (Jez et al., 2000). Targeting the catalytic sites of endogenous metabolic enzymes is especially attractive because of the potential of this strategy for the fine manipulation of the internal environment (Dixon and Steele,
1999).
126 Plants use the flavonoid pathway intermediates as well as end products as
attractor of pollinators and seed dispersers, as plant defense molecules, antioxidants and
signaling molecules with many of the enzymes being coordinately regulated in response
to stress (Shirley, 1996). The pathway is highly branched with key enzymes diverting
flux into the different metabolic pathways, (Fig. 3.1). Chalcone isomerase (EC 5.1.1.5)
diverts flux leading either to the general flavonoid pathway forming flavonols, flavones
(like aurones), proanthocyanidins and the anthocyanins, the isoflavonoid pathway unique to the legumes, C-glycosyl flavones or the 3-deoxyanthocyanidins found in the grasses
(Winkel-Shirley, 2001).
CHI catalyzes the stereospecific ring closure of chalcone to form the biologically active 2S isomer of its flavanone (Fig. 3.2), the substrate for the next enzyme in the pathway. Chalcone can also spontaneously cyclize to yield an enantiomeric mixture of 2S and 2R flavanone at a pH greater than 7.5 (Moustafa and Wong, 1967; Mol et al., 1985).
CHI is a monomeric, single substrate catalyzing enzyme, which at ~25 kDa, is physically one of the smallest enzymes of the core flavonoid pathway. Non-legume CHIs, classified as type I, can only catalyze the ring closure of 2’,4,4’,6’ tetrahydroxychalcone
(naringenin chalcone; 6’-hydroxychalcone) to form 5,7,4’ trihydroxyflavanone
(naringenin). CHIs from legumes (Type II) have evolved to utilize both 2’,4,4’,6’ tetrahydroxychalcone as well as 2’,4,4’trihydroxychalcone (isoliquiritigenin;), the latter
forming 7,4’dihydroxyflavanone (liquiritegenin; 5-deoxyflavanone) which feeds into the
isoflavonoid pathway (Shimada et al., 2003).
The crystal structure of the Medicago enzyme (type II CHI), revealed an upside
down bouquet with an open-faced β-sandwich fold. There are a number of conserved
127 amino acids that line the active site and structural and mutational analysis of T48A and
Y106F showed T48A and Y106F to be involved in the catalytic mechanism (Jez et al.,
2000; Jez et al., 2002; Jez and Noel, 2002). The CHI structural fold was initially thought
to be unique to the plant kingdom. However, recent bioinformatics-based studies
identified proteins with the CHI-like fold in mosses, fungi, slime molds and
gammaproteobacteria (Gensheimer and Mushegian, 2004), and more recently in the
human fecal bacterium Eubacterium ramulus (Herles et al., 2004). The physiological
functions of the enzyme in these organisms remain unclear, although a role in metabolism
of flavonoids has been proposed.
The need for this enzyme in the biosynthetic pathway had been questioned due to
the spontaneous ring closure of chalcone at physiological pH (Grotewold et al., 1998).
CHI transcripts and activity was not detected in maize Black Mexican Sweet (BMS) cells
in culture constitutively expressing the transcription factors C1 and R or P1 that regulate
the anthocyanin and the phlobaphene pathways, respectively (Grotewold et al., 1998), nor in tomato fruit pericarp (Muir et al., 2001). However mutants in CHI identified in
Arabidopsis thaliana (tt5) (Shirley et al., 1992), carnation (Itoh et al., 2002), barley
(ant30) (Druka et al., 2003), onion (Kim et al., 2004) and RNAi suppression of CHI in tobacco (Nishihara et al., 2005) accumulate reduced to no anthocyanins, indicating the need for this enzyme activity in these plants.
CHI may be involved in directing flux into the various branches of the pathway as part of the flavonoid metabolome. Protein interaction studies have shown CHI to physically interact with CHS, DFR and F3H (Burbulis and Winkel-Shirley, 1999), and the formation of a multienzyme complex with different partners was proposed to
128 determine which end products would be more abundant in different situations (Winkel-
Shirley, 1999). Over-expression of petunia CHI in tomato produced increased levels of the flavonol rutin in the fruit peel (Muir et al., 2001) and also increased anthocyanins
(Verhoeyen et al., 2002), while the over-expression of CHI in potato singly or together with CHS and DFR, modestly increased the phenolic and anthocyanin content
(Lukaszewicz et al., 2004). Similarly, the over-expression of ZmCHI in Arabidopsis did not increase the amount of anthocyanins (Dong et al., 2001). Maize CHI (ZmCHI1), with less than 60% identity to the Arabidopsis enzyme complemented the tt5 mutant, suggesting that either maize CHI could participate in the macromolecular complex or that the activity of the enzyme was sufficient to catalyze the intermediates for the pathway without the involvement of a complex (Dong et al., 2001). The putative CHS-CHI complex in Medicago sativa was not detected in vitro (Jez et al., 2000).
CHI may also have secondary functions in other pathways and/or regulation. CHI was proposed to play a role in the sinapate ester pathway, a branch of the phenylpropanoid pathway conferring UV-B protection, since tt5 mutants have significantly lower levels of sinapic acid esters than wt and tt4 (CHS) mutants and are hypersensitive to UV-B radiation (Li et al., 1993). Furthermore, tt5 mutants as compared to wt, accumulate higher levels of the enzymes of the flavonoid pathway (Pelletier et al.,
1999) as well as the bHLH transcription factor TT8 (see Chapter 2). Thus this feedback regulation at the transcriptional level is thought to be either a consequence of the accumulation of pathway intermediates (Pelletier et al., 1999), or as proposed here, by the
CHI protein itself. Nuclear localization of CHI (Saslowsky et al., 2005) (Appendix D)
129 further supports the hypothesis that CHI may also play a secondary role as a regulatory protein.
Thus, the purpose of this study was to characterize and analyze functions of the maize CHI protein that go beyond its catalytic conversion of chalcone to naringenin.
Point mutants in the conserved catalytic site amino acid residues (Y104F, T46A and
R34A) were generated to assay the contribution of these amino acids to catalytic efficiency of a type I CHI. The complementation of Arabidopsis tt5 mutants was used as a convenient measure to assess the function of the maize mutants in terms of catalytic activity in planta and accumulation of the pathway intermediates and end products. I show here that ZmCHIY104F and ZmCHIT46A complemented the tt5 phenotype where as ZmCHIR34A was unable to do so. ZmCHIT46A proved to be the most interesting of the three mutants as it exhibited no activity in vitro but complemented the tt5 mutant and plants expressing this protein exhibited CHI activity in planta. ZmCHIT46A seedlings had increased levels of anthocyanins and no changes in the flavonol content, but a shift in the quercetin to kaempferol (flavonol) ratios of as compared to endogenous wt
Arabidopsis levels. ZmCHIY104F, displaying reduced catalytic efficiency as compared to the wild type maize enzyme synthesized similar levels of anthocyanins showed no changes in phenolic profiles. The R34A mutant that was catalytically inactive was unable to complement tt5, however it still retains some function since the phenolic content of plant expressing this CHI mutant are different form the phenolic contents in tt5. Thus, I have generated an interesting series of maize CHI mutants which can be used further to answer questions of catalytic activity, flux through a biosynthetic pathway and possible regulatory functions.
130
Figure 3.1 The core phenylpropanoid and flavonoid biosynthetic pathway in Arabidopsis thaliana
131
Figure 3.2 ZmCHI catalyzes the cyclization of 2,4,4’,7’ tetrahydroxychalcone into 2S-naringenin
3.2 Materials and Methods
3.2.1 Synthesis of chalcone
2’, 4, 4’, 6’ tetrahydroxychalcone was synthesized according to Moustafa and
Wong 1967. Briefly, 100mg of a racemic mixture of (R/S)-naringenin (Sigma) was dissolved in 1 ml 50% (w/v) KOH and the solution was heated for 10 min at 65°C. The alkaline solution turned from yellow to a bright orange-yellow. HCl was added drop by drop to neutralize and acidify the solution until the orange-yellow chalcone precipitated out. The precipitate was filtered and washed with cold water to dissolve away the KCl.
The filtrate was dried and the chalcone naringenin mix was dissolved in 100% ethanol and separated on a silica gel column using hexane: ethyl acetate (3:1 v/v). The purity of the isolated chalcone was determined on silica TLC plates and by HPLC. Spontaneous cyclization to (R/S)-naringenin was monitored in 50mM HEPES (pH7.5) at 25°C using a
132 Cary 50 Bio UV-VIS spectrophotometer (Varian Inc., USA) with the scanning kinetics
function. The λmax in HEPES, pH 7.5 was 377 nm and in 100% methanol was 365 nm.
The molar absorption coefficient for chalcone in 50 mM HEPES pH 7.5 determined at
-1 -1 -1 -1 λmax was ε377 = 28076 M .cm and at the isosbestic point ε333 = 17519 M .cm . The molar absorption coefficient of naringenin in 50 mM HEPES pH 7.5 at 333nm was calculated and the value was used for ε333 for chalcone.
3.2.2 Site directed mutagenesis of ZmCHI1
The amino acid residues R34, T46 and Y104 were mutated in the ZmCHI1 cDNA
(Grotewold and Peterson, 1994) by site directed mutagenesis using the QuikChange® XL
Site-Directed Mutagenesis Kit (Stratagene, USA). The primer pairs ZmCHIR34A5'and
ZmCHIR34A3’, ZmCHIT46A5' and ZmCHIT46A3'
ZmCHIY104F5’ and ZmCHIY104F3’ were used to generate the mutated clones
ZmCHIR34A, ZmCHIT46A and ZmCHIY104F respectively (Appendix B).
The cDNAs were digested with EcoRI, filled in with the Klenow fragment of
DNA polymerase I, and cloned into the SmaI site of the pBIB121 T-DNA insertion vector. The T-DNA vectors were transfected into Argobacterium tumefaciens strain
GV3101 pMP90 by electroporation and selected on LB supplemented with 50 mg/L kanamycin and 25 mg/L gentamycin.
The open reading frame of the wt and mutant ZmCHIs were amplified using primers CHI N1 and CHI N2 (Appendix B) and cloned into the NcoI and HindIII sites of
GST fusion plasmid pGEX-KG (Guan and Dixon, 1991).
133 3.2.3 In vitro transcription/translation
Transcription coupled with translation for generation of labeled ZmCHI proteins was carried out using the TNT® coupled Wheat Germ and Rabbit Reticulocyte Extract systems (Promega Corporation, USA) as per the manufacturer’s recommendations. The
T7 promoter was used to drive expression of the CHI constructs and L-[35S]-methionine
(Redivue™ Amersham Biosciences, UK) was incorporated to label the proteins. Five microlitres of the reaction was mixed with loading buffer and resolved on a 12% SDS page gel. The gel was stained with Coomassie, dried and exposed to a BioRad phosphorimager (BioRad Laboratories, Inc. USA). Signal intensities of synthesized proteins were calculated using the volume analysis tool of the BioRad Quantity One software (v 4.1.1, BioRad, USA). Relative ratios were calculated with the value of
ZmCHIwt kept as 1. Since equal quantities of initial reaction mix were taken, the total crude protein mix in each reaction was approximately the same, as confirmed by SDS
PAGE, thus automatically normalizing for the proteins synthesized per total protein of the extract
3.2.4 Expression of recombinant CHI protein in E.coli
TOP10F’ E. coli cells (Stratagene, USA ) were transformed with the GST constructs. Cells were grown to an OD600 of 0.8 to 1.0 and induced with 1mM IPTG for
6h. Cells were spun down, re-suspended in 1XPBS, 5mM DTT and sonicated. The solution was cleared by centrifugation and used as the crude lysate.
134 3.2.5 Enzyme assays
In vivo enzyme kinetics were carried out by homogenizing the plant tissue in lysis
buffer [50 mM HEPES, pH7.5; 100 mM NaCl, 20% glycerol, 5 mM DTT and plant
protease inhibitor (Sigma, 0.01:1, v/v) at a ratio of 1:0.5 (fresh wt/v). The homogenate
was cleared by centrifugation and 10 µl of the plant lysate was taken per assay. Assays
were done in 50 mM HEPES, pH7.5 at 25°C using a Cary 50 Bio UV-Vis
spectrophotometer (Varian Inc., USA) using the scanning kinetics function. The buffer
with the plant extract was base-lined before addition of 20 – 35 µM (OD377 should be
≤1.0) of chalcone into the reaction mixture. The substrate concentration in the reaction mixture was monitored at the isosbestic point at 333nm. Velocity (dA377/min) was
measured from the linear part of the graph until the substrate concentration reached
isosbestic point at 333nm and expressed as µmoles of chalcone consumed/min (Fig 3.3).
Measurements were made in triplicate. Km and Vmax were determined for ZmCHI in
planta where substrate concentrations were varied between 5µM and 80µM, initial
velocities were calculated and data were fit to the Lineweaver Burke double reciprocal
plots.
For comparative rate studies in vitro, 10 µl of the rabbit reticulocyte TNT
reactions and 10 µl of an appropriately diluted E. coli crude enzyme extract were used
per assay. Pilots were first run to ensure that rates of reaction were in the limits of
measurement. TNT reaction rates were normalized to the signal intensities imaged by a
phosphorimager. The crude E. coli GST rates were compared after taking equal density
cell cultures for sonication and protein loads were resolved and visualized on Coomassie
stained SDS-PAGE gels.
135 3.2.6 Plant material and transformation
Arabidopsis tt5-1 (tt5) mutants were obtained from the Arabidopsis Biological
Resource Center (The Ohio State University, Columbus). Transformation of plants was carried out by ‘the floral dip method’ (Clough and Bent, 1998). tt5 plants were grown in soil in a growth chamber at 22°C, with a 16h/8h light-dark regime at 50µmole.m-2.s-1 at
50 -80% relative humidity and allowed to bolt. The primary bolts were cut to encourage secondary bolts and were transformed after four to five days. The transfected
Agrobacterium was grown to saturation for 24 h in LB supplemented with 25 mg/L gentamycin and 50 mg/L kanamycin. The cells were spun down and resuspended in half the original volume of 5% sucrose. 0.03% (v/v) Silwet L-77 (Lehle seeds, Round Rock,
TX) was added to the Agrobacterium suspension and the aerial shoots were dipped into the solution for a few seconds. The pots were placed on their sides in flats with water moistened paper towels, covered with plastic domes and left to recover for 24 h. The transformed plants were shifted to the growth chamber for seed set. T1 seeds were surface sterilized (95% ethanol 1min, 50:50:0.05 - bleach:water:Tween 20 v/v/v 6min and three times water wash) and transformants were selected on 0.5X MS media
(Murashige and Skoog, 1962) with 1% sucrose (w/v), solidified with 0.7% agar with 12 mg/L hygromycin for selection of the transgene and 500 mg/L vancomycin to suppress
Agrobacterium growth. Hygromycin resistant plants with first true leaves and well developed roots were transplanted to soil. Three individual transgenic lines of tt5+35S::ZmCHIwt, tt5+35S::ZmCHIY104F, tt5+35S::ZmCHIT46A, tt5+35S::ZmCHIR34 and tt5+35S::empty vector were further selected and bred to homogeneity verifying for simple Mendelian inheritance ratios at each selection step.
136 3.2.7 Anthocyanin induction, extraction and analysis by TLC and HPLC.
Seeds from tt5+35S::ZmCHIwt, tt5+35S::ZmCHIY104F, tt5+35S::ZmCHIT46A and tt5+35S::ZmCHIR34A mutants were surface sterilized and plated in liquid 3% sucrose at a seed density of 75-100 seeds/ml (~25,000seeds/1ml, ABRC). Seeds were stratified in the cold room for 2 days and shifted to the culture room at 25°C,
100µmoles.m-2.s-1 of continuous cool white light (on a rotary shaker at 100rpm. 3d old induced seedlings were harvested and stored at -80C until further analysis.
Anthocyanins were extracted and spectral, TLC and HPLC analyses were performed as previously described (Irani and Grotewold, 2005) and in Chapter 2.
137 3.3 Results
3.3.1 Chalcone isomerase dynamics: structure, activity and generation of catalytic mutants Y104F, T46A and R34A.
2,4,4’,7’ tetrahydroxychalcone (tetrahydroxychalcone), as reported previously
(Moustafa and Wong, 1967), showed the characteristic properties of spontaneous ring
closure to form a racemic mix of 2S2R-naringenin in buffers with pH ≥ 7.5. As observed
from the scanning kinetic curves (Fig 3.3), the rate of the reaction was measured as a loss
of chalcone at 377nm, which shows no interference from the absorption spectra of
naringenin (Bednar and Hadcock, 1988). The isosbestic point at 333nm was monitored to determine initial substrate concentration in the reaction mix.
Maize chalcone isomerase exhibits low sequence conservation across species where it shares ~75% similarity with rice and only ~40-60% similarity with dicot proteins
(Fig 3.4, 3.5 B, Druka et al., 2003). ZmCHI groups together with enzymes from plant
species classified as type I CHI which can catalyze only ring closure of
tetrahydroxychalcone (Fig 3.5 A). These separate from the leguminaceae clade whose
CHIs can catalyze both tetrahydroxy as well as trihydroxychalcones, the latter leading
into the isoflavonoid pathway, which is unique to the group (Shimada et al., 2003). To
confirm if ZmCHI1 is a type I enzyme, a crude E. coli extract expressing recombinant
GST-ZmCHIwt was incubated with trihydroxychalcone. No activity was recorded. A
crude extract of immature soybean pods showed activity converting the
trihydroxychalcone into liquiritegenin (results not shown).
138 Despite the sequence divergence, the residues that line the naringenin binding cleft (Fig 3.3, amino acids marked in green), as inferred from the crystal structure of
Medicago sativa (Jez et al., 2000), are highly conserved. Mutation studies in Medicago have shown Y106 and T48 to facilitate the cyclization of chalcones to flavanones (Jez et al., 2002). Based on the Medicago studies, site directed mutagenesis was used to change
Y104F, T46A and R34A in the cDNA of ZmCHI, (denoted as ZmCHIY104F,
ZmCHIT46A, ZmCHIR34A and the wild type as ZmCHIwt), to address questions if these residues are equally important in maize as in Medicago, and if the catalytically impaired mutants still retained the ability to complement the Arabidopsis tt5 mutant.
139
A Isosbestic point 333nm Chalcone 377nm Naringenin 323nm 50mM HEPES pH 7.5
Region used for Naringenin calculation of 323nm B initial velocity
Isosbestic point 333nm
Chalcone 377nm
Figure 3.3 Spontaneous cyclization of chalcone: (A) Scanning kinetics of spontaneous ring closure of chalcone in HEPES buffer pH 7.5. Rate was calculated as the loss of chalcone at 377nm and the substrate concentration in the reaction was measured at the isosbestic point at 333nm. (B) Kinetics of chalcone loss and naringenin formation. The initial velocity was measured as the slope between the time 0 and the time it took to reach the isosbestic point.
140 Figure 3.4 Alignment of ZmCHI1 with representative CHIs from the type I and type II groups: The multiple alignments were executed with ClustalW (http://www.ebi.ac.uk/clustalw/index.html). The aa residues highlighted in green constitute the naringenin binding cleft. The three residues that were mutated in ZmCHI (i.e. R34A, T46A and Y104F) are boxed in red. The grey highlights represent conserved identical residues and light grey similar residues. Residues marked with pink participate in the hydrogen bonding network of the catalytic site. The secondary structure of the protein is drawn as lavender arrows representing β sheets and pale blue rectangles as α helices. Zm: Zea mays; Os: Oryza sativa; At: Arabidopsis thaliana; Ph: Petunia hybrida; Lj: Lotus japonicus; Ms: Medicago sativa; Ps: Pisum sativum; PV: Phaseolus vulgaris; Gm: Glycine max. Diagram is adapted from information obtained from the crystal structure of Medicago sativa CHI (Jez et al., 2000).
141 β1a β1b β2 β3a β3b ZmCHI ------MAVPEVVVDG--VVFPP-VARPPGSAGSHFLGGAGVRGVEIGGNFIK 44 OsCHI ------MAAVSEVEVDG--VVFPP-VARPPGSGHAHFLAGAGVRGVEIAGNFIK 45 AtCHI MSSSNACASPSPFPAVTKLHVDS--VTFVP-SVKSPASSNPLFLGGAGVRGLDIQGKFVI 57 PhCHIA ------MSPPVSVTKMQVEN--YAFAP-TVNPAGSTNTLFLAGAGHRGLEIEGKFVK 48 LjCHI2 ------MALPSVTALQVEN--VAFPPTLIKPPASANTLFLGGAGERGLHIQDKFVK 48 LjCHI1 ------MAPAKGSSLTPIQVEN--LQFPA-SVTSPATAKSYFLGGAGERGLTIEGKFIK 50 MsCHI ------MAASITAITVEN--LEYPA-VVTSPVTGKSYFLGGAGERGLTIEGNFIK 46 PsCHI ------MCCSILHHRNPRREHEFPA-VVTSPVTENHIFLGGAGERGLTINGTFIK 48 PvCHI ------MATAPTITDVQVEF--LHFPA-VVTSPATAKTYFLGGAGERGLTIEGKFIK 48 GmCHI ------MATISAVQVEF--LEFPA-VVTSPASGKTYFLGGAGERGLTIEGKFIK 45 :: : . .. : **.*** **: * ..*:
β3b α1 α2 α3 β3c α4 ZmCHI FTAIGVYLED-AAVPALAKKWGGKTADELASDAAFFRDVVTGDFEKFTRVTMILPLTGEQ 103 OsCHI FTAIGVYLEEGAAVPALAKKWAGKSADELAADAAFFRDVVTGDFEKFTRVTMILPLTGEQ 105 AtCHI FTVIGVYLEG-NAVPSLSVKWKGKTTEELTESIPFFREIVTGAFEKFIKVTMKLPLTGQQ 116 PhCHIA FTAIGVYLEE-SAIPFLAEKWKGKTPQELTDSVEFFRDVVTGPFEKFTRVTMILPLTGKQ 107 LjCHI2 FTAIGIYLQD-TAVPSLAVKWKGKPVDELTESVQFFRDIVTGPFEKFMQVTMILPLTGQQ 107 LjCHI1 FTGIGVYLED-TAVDSLATKWKGKSSQELQDSLDFFRDIISSPSEKLIRGSKLRPLSGVE 109 MsCHI FTAIGVYLED-IAVASLAAKWKGKSSEELLETLDFYRDIISGPFEKLIRGSKIRELSGPE 105 PsCHI FTCIGVYLED-KADKSLATKWEGK-LEELLETLDFYRDIISGPFEKLIRRSKIKELSGPE 106 PvCHI FTAIGVYLED-KAVASLATKWKGKPSEELINTLDFYRDIISGPFEKLIRGSKILQLSGTE 107 GmCHI FTGIGVYLED-KAVPSLAAKWKGKTSEELVHTLHFYRDIISGPFEKLIRGSKILPLAGAE 104 ** **:**: * *: ** ** .** *:*::::. **: : : *:* :
α4 α5 β3d β3e ZmCHI YAEKVTENCVAFWKAAGLYTDAEGVAVEKFREVFKPETFAPGRSILFTHSPAGVLTVAFS 163 OsCHI YSDKVTENCVAAWKAAGVYTDAEGAAADKFKEAFKPHSFPPGASILFTHSPPGVLTVAFS 165 AtCHI YSEKVTENCVAIWKQLGLYTDCEAKAVEKFLEIFKEETFPPGSSILFALSPTGSLTVAFS 176 PhCHIA YSEKVAENCVAHWKGIGTYTDDEGRAIEKFLDVFRSETFPPGASIMFTQSPLGLLTISFA 167 LjCHI2 YSEKVSENCVAIWKHLGIYTDEEGKAIDKFVSVFKDQTFPPGSSILFTVLPKGSLAISFS 167 LjCHI1 YSRKVMENCVAHMKSAGTYGEAEATAIEKFAEAFRKVDFPPGSSVFYRQSTDGKLGLSFS 169 MsCHI YSRKVMENCVAHLKSVGTYGDAEAEAMQKFAEAFKPVNFPPGASVFYRQSPDGILGLSFS 165 PsCHI YSRKVMENCVAHLKSVGTYGDAEVEAIQNLQKLSRMLIFHLVLLKKNRQSPDGILGLSSS 166 PvCHI YSRKVMENCVAHLKSVGTYGDAEAKGIEEFAEAFKKVNFPPGASVFYRQSPDGILGLSFS 167 GmCHI YSKKVMENCVAHMKSVGTYGDAEAAAIEKFAEAFKNVNFAPGASVFYRQSPDGILGLSFS 164 *: ** ***** * * * * . ::: : * . * * :: : β3f α6 α7
ZmCHI KDSSVP--AAGGVAIENKRLCEAVLESIIGERGVSPAAKLSLAARVSELLAKETAAAADA 221 OsCHI KDSSVPEGAVAAAAIENRALCEAVLDSIIGEHGVSPAAKRSIAARVSQLLKAESTG--DV 223 AtCHI KDDSIP--ETGIAVIENKLLAEAVLESIIGKNGVSPGTRLSVAERLSQLMMKNKDEKEVS 234 PhCHIA KDDSVT--GTANAVIENKQLSEAVLESIIGKHGVSPAAKCSVAERVAELLKKSYAEEASV 225 LjCHI2 KDGSIP--EVESAVIDNKLLSEAVLESMIGAHGVSPAAKQSLASRLSELFKHHAEV---- 221 LjCHI1 LDDTIP--EEEAVVIENKALSEAVLETMIGEHAVSPDLKRCLAERLPIVMNQGLLLTGN- 226 MsCHI PDTSIP--EKEAALIENKAVSSAVLETMIGEHAVSPDLKRCLAARLPALLNEGAFKIGN- 222 PsCHI KDISIP--EKEDAIIENKAASSAVLETMIGEHAVSPDLKRCLAARLPALLNEGTFKIGN- 223 PvCHI EDATIP--GEEAVVIENKAVSAAVLETMIGEHAVSPDLKRSLASRLPAVLNGGIIV---- 221 GmCHI EDATIP--EKEAAVIENKAVSAAVLETMIGEHAVSPDLKRSLASRLPAVLSHGIIV---- 218 * ::. . *.*: . ***:::** ..*** : .:* *:. ::
ZmCHI PQAEPVSITA------231 OsCHI AAAEPAPVSA------233 AtCHI DHSVEEKLAKEN---- 246 PhCHIA FGKPETEKSTIPVIGV 241 LjCHI2 ------LjCHI1 ------MsCHI ------PsCHI ------PvCHI ------GmCHI ------Fig.3.4 142 A
Zea mays
Oryza sativa
A. thaliana (TT5) Type I
L. japonicus CHI2
P. hybrida CHIA
L. japonicus CHI1
Phaseolus vulgaris Type II Glycine max
Medicago sativa
Pisum sativum
B
Percentage Identity 1 2 3 4 5 6 7 8 9 10 1 74.9 57.6 58.9 44.2 57.0 47.3 47.1 49.5 40.4 1 Zea mays 2 51.5 54.9 41.6 53.4 47.7 44.3 49.5 37.7 2 Oryza sativa 3 57.3 48.2 63.3 46.4 47.5 50.0 39.5 3 Arabidopsis thaliana 4 49.1 63.3 47.7 49.8 51.4 41.3 4 Petunia hybrida CHIA 5 50.2 75.2 76.9 74.8 60.1 5 Lotus japonicus CHI1 6 49.8 51.6 53.2 43.0 6 Lotus japonicus CHI2 7 80.1 80.7 73.0 7 Medicago sativa 8 86.7 65.2 8 Phaseolus vulgaris 9 64.7 9 Glycine max 10 10 Pisum sativum
Figure 3.5 Cladogram and percentage identities of CHI proteins: (A) The cladogram was created using ClustalW as in figure 3.4. The leguminous members with type II CHIs are labeled in blue. (B) Percent identities were calculated using MegAlign sequence analysis package (DNASTAR, Madison, WI, USA).
143 3.3.2 ZmCHIY104F mutants show 75% - 80% reduction in activity as compared to
ZmCHIYwt and the ZmCHIT46A and ZmCHIR34A mutants are catalytically
dead in vitro.
To test the activity of the CHI mutant clones, activities of in vitro transcribed and
translated (TNT, rabbit reticulocyte extracts) ZmCHIwt, ZmCHIY104F and ZmCHIT46A
proteins were measured. ZmCHIY104F had about 20% activity of ZmCHIwt (Fig 3.6A).
The ZmCHIT46A and the double mutant ZmCHIT46A/Y104A showed no detectable
activity and rates were comparable to the spontaneous reaction. The enzyme rates were
normalized to the intensity of the L- [35S] -methionine labeled CHI band on an
autoradiogram (Fig 3.6 B) where equal amounts of total protein from the TNT reaction
were used. Activity for ZmCHIR34A was not determined in this system.
In vitro enzyme activity was further validated with E. coli expressed, GST-tagged,
recombinant proteins. There was a 75% reduction of wt activity observed for
GST:ZmCHIY104F (Fig. 3.7 A,B). GST:ZmCHIT46A and GST:ZmCHIR34A showed no activity and rates were comparable to the spontaneous reaction. The GST tagged recombinant proteins for the double and triple mutants were also catalytically inactive
(not shown).
Initial kinetic measurements for GST:ZmCHIwt from crude E. coli extracts measured apparent Vmax at 9 nmoles chalcone catalyzed/min and apparent Km as
11.2µM. These values were not corrected for the spontaneous reaction.
The cell extracts were initially normalized to the OD600 value of the cultures and
subsequent visualized on a SDS page gel (Fig 3.7 C). Equal volumes of the diluted
extracts were taken for measurement at a fixed substrate concentration.
144
A 120
100
80
60 100 40
% activity of ZmCHIwt 20 18.4 00nd 0
t F 4 6A HIw 46A IT4 T ZmC CHIY10 CH Zm Zm ZmCHIR34A
B ZmCHI Y104F
T46A ZmCHI wt Y104F T46A Y104F 24kDa
Figure 3.6 Relative enzyme rate comparisons of TNT synthesized proteins: ZmCHIwt and site directed mutagenesis generated CHI constructs ZmCHI Y104F, ZmCHI T46A and ZmCHI T46A Y104F were transcribed and translated using the rabbit reticulocyte TNT system using 35S-labeled methionine. (A) Relative rates of the enzymes as compared as a percentage of ZmCHIwt. Values are the averages of % activity measured from three independent experiments. (B) Autoradiogram of the labeled proteins, the relative intensities of which were determined using a BioRad Phoshorimager. These values were used to correct the percentage activity in (A). The TNT enzyme rate values for ZmCHI R34A were not determined (nd). Activity measurements were done in triplicate. Error bars represent standard deviation.
145 20 A
15
10
5 nmoles chalcone / min / chalcone nmoles 0
F A 4 6 s GST 0 4 34A IT IR H HIY1 CH m mC mC :Z :Z GST:ZmCHIwt:Z T T Spontaneou ST G GS GS
B 120 100 100 80 60
40 23.4 20
% activity of ZmCHIwt 0
ST 4F G 10 HIwt IT46A eous C Y n HI :Zm C
GST T:ZmCH Sponta ST:Zm S G G GST:ZmCHIR34A
C kD M 1 2 3 4 5 M: Protein Marker 1: GST 2: GST:ZmCHI wt 75 3: GST:ZmCHI Y104F 4: GST-ZmCHI T46A 50 GST:CHI 5: GST-ZmCHI R34A 37
25 GST
Figure 3.7 Comparative enzyme rates for recombinant E. coli expressed GST- ZmCHI fusion proteins: (A) Initial velocities as measured with 35µM of chalcone (B) Activity as expressed as a percentage of the average ZmCHIwt activity. Percentages were calculated after subtraction of spontaneous rate. (C) Coomassie stained gel showing the relative amounts of proteins in the samples evaluated for enzyme activity.
146 3.3.3 ZmCHIwt, ZmCHIY104F and ZmCHIT46A complement the tt5 mutant phenotype.
ZmCHI has been shown previously in our laboratory to functionally complement the tt5 mutant (Dong et al., 2001). To test if the catalytically impaired mutants of ZmCHI had the ability to complement the tt5 phenotype of Arabidopsis thaliana, independent transgenic lines of tt5 plants expressing 35S::ZmCHIwt, 35S::ZmCHIY104F,
35S::ZmCHIT46A, and 35S::ZmCHIR34A were generated. The tt5 plants, as described in
Chapter 2, have yellow seed coats and do not accumulate anthocyanins in their vegetative tissue (Fig 3.8, tt5). ZmCHIY104F, with only 20% of the activity of ZmCHIwt as measured in vitro, complemented the anthocyanin and the proanthocyanidin phenotype of the seedlings and the seed coats of the tt5 mutant respectively (Fig 3.8). Even more exciting was the result of in vitro catalytically inactive ZmCHIT46A complementing the tt5 mutant (Fig 3.8).
The 35S::ZmCHIR34A mutant plant seeds remained yellow and did not accumulate anthocyanins in the cotyledons of the seedlings (Fig 3.8 ZmCHIR34A). The tt5 mutants transformed with double and the triple mutant showed no complementation of the phenotype.
147
wt (Ler) tt5 tt5
empty vector
tt5 tt5 tt5 tt5
ZmCHI ZmCHI Y104F ZmCHI T46A ZmCHI R34A
tt5 tt5 tt5 tt5
ZmCHI Y104F T46A ZmCHI Y104F T46A ZmCHI Y104F R34A ZmCHI T46A R34A R34A
Figure 3.8 ZmCHIwtt, ZmCHIY104F and ZmCHIT46A complement Arabidopsis tt5 mutants: Complementation of seedling anthocyanins and seed coat proanthocyanindins in tt5 plants transformed with the maize CHI wt and catalytic mutants, 35S::ZmCHIwt, 35S::ZmCHIY104F and 35S::ZmCHIT46A. 35S::CHI R34A and the rest of the double and the triple mutant were unable to complement tt5 and show yellow cotyledons and seeds.
148 3.3.4 35S::ZmCHIT46A plants accumulate more anthocyanins and show a different
Q:K ratio than 35S::ZmCHIwt plants
To test if the catalytically altered ZmCHI mutants have an effect on the metabolic profile, we measured the changes in phenolic profiles in 3d old seedlings grown in anthocyanin inductive conditions. The spectrophotometric measurements of acidic methanol extracts at 530 nm showed no significant changes in the total anthocyanin content for the endogenous wt Arabidopsis levels, 35S::ZmCHIwt and
35S::ZmCHIY104F plants (Fig 3.9). There was a 25% increase in anthocyanin levels in the 35S::ZmCHIT46A mutant plants.
TLC profiles show no appearance of any new anthocyanidins in the Arabidopsis plants expressing ZmCHIwt, ZmCHIY104F and ZmCHIT46A (Fig 3.9A, B). This is supported by HPLC chromatograms at 530 nm where the total anthocyanidin profiles remain the same across the lines (Figure 3.10). An increase in peaks with retention time
16.5 min and 19.6 min reflected an increase in the total anthocyanins in
35S::ZmCHIT46A seedlings
There was no significant change in the total flavonol content (Q+K) (Fig 3.12 B).
However there was a striking change in the Q:K ratios in the ZmCHIT46A mutant expressing plants where the 60:40 ratios observed in the others changed to a 50:50 ratio.
This is attributed to a decrease in the synthesis of quercetin which correlates with the increase in the amount of anthocyanins in these plants (Fig 3.12 A). There are two alternative possible explanations for the change in the Q:K ratio. First, there could be selective channeling as the flavonoid enzymes in Arabidopsis have been shown to interact and may form multienzyme complexes. Second, change in the kinetic properties
149 of the maize enzyme could result in change of the intermediate metabolite pools which
may be shuttled into different pathways due to the different affinities of the downstream enzymes.
3.3.5 Phenolic profiles of 35S:ZmCHIR34A tt5 plants do not phenocopy tt5
The catalytically inactive ZmCHIR34A protein was unable to complement the
flavonoid pathway leading to accumulation of anthocyanins in the seedling cotyledons
and proanthocyanidins in the seed coat. However, the phenolic profiles are not identical
to the tt5 plants as seen by TLC and HPLC analysis. The phenolic profiles, when separated by TLC and visualized under UV, show an increase in intensity of a faint blue fluorescent spot ~1 cm from the origin in the ZmCHIR34A mutant extracts (Fig 3.9 B, lane 6). This is also present in the tt5 plants transformed with the empty plasmid (Fig3.10
B, lane 2) although at a lower intensity. The presence of this spot cannot be ruled out in
the other samples as it may be quenched by the quercetin and kaempferol spots that
migrate at the same position (Fig. 3.10 B). Furthermore, the 360 nm chromatogram from
the hydrolyzed samples, the intensity of peak with retention time of 15.5 min was higher
as compared to tt5 plants (Fig 3.11). This is also represented in the 280 nm HPLC
profiles (Fig 3.13, peak marked with a red star) with a major decrease in peak at 23.7 min
(Fig 3.13, peak marked with a green star) which was also observed in the other CHI
mutants as well as Arabidopsis wt. The identity of the peaks needs to be determined.
Thus, these subtle changes in the metabolic profile of the 35S::ZmCHIR34A seedlings
shows that it does not phenocopy the tt5 background and even through catalytically
inactive, influences the metabolic pathway.
150 A
2.0
1.5
1.0
0.5 AU 530nm/AU 10mg dry wt 0.0
5 1 r) t5 -2 - a -5 - e t 4 5 3 1-3 2 L 3-7-3 3- _ 1-4-1 _ _ 1 t ( 9 9 3-14 - w -3-1-4 a wt 6A 3-4-1A 5 1 I G G F F 46 4A IB H 4 4 T4 T 3 B F G9_3-5-1 a R R34A 5-2_ p CHI wt mC1-10-2 10 4 10 I m Z ZmCHI wtY 3-5-3 Y CHI Z I I m CHI H HI H H Z m CHI R34A 5-3_ C Z m mC m Z ZmC Z ZmC Z ZmCHI T46A 3-15- B ZmCHI Y10
2.0
1.5
1.0
0.5
0.0
5 r F A o wt A er) tt 04 6 34 L ect R t ( v Y1 w I HI H C m mpty Z Absorbance 530nm / 10mg dry / wt ml ZmC +e tt5 + ZmCHI+ t5 t t5 tt5 + ZmCHItt5 T4 + t
Figure 3.9 Spectrophotometric quantification of anthocyanins: Acidic methanolic extracts from lyophilized, 3d old ‘induced’ Arabidopsis seedlings were spectrophotometrically quantified at 530nm. (A) Anthocyanin content of three independent lines of the 35::ZmCHI wt and mutants Y104F, T46A and R34A transformed into tt5. (B) Averages of the transgenic lines representative of the maize wt and mutant CHI expressed in Arabidopsis tt5 mutants. AU: absorbance units.
151 A
Sinapic Acid
Pelargonidin
Cyanidin
Kaempferol Quercitin
1 2 3 4 5 6 7 8 9 10 B
Sinapic Acid
Pelargonidin
Cyanidin
Kaempferol Quercetin
1 2 3 4 5 6 7 8 9 10
1 wt (Ler) 6 tt5 + ZmCHI R34A 2 tt5 + empty vector 7 Maize B-I/B-Peru 3 tt5 + ZmCHIwt 8 Kaempferol 4 tt5 + ZmCHI Y104F 9 Quercetin 5 tt5 + ZmCHI T46A 10 Sinapic acid
Figure 3.10 TLC analysis of anthocyanins: Isoamyl alcohol extracts of acid hydrolyzed 3d lyophilized seedlings separated on cellulose TLC places with water: formic acid: HCl (3:30:1). (A) Separation profile as visualized under white light and (B) is the UV excited version of the same plate. The extracts were normalized to dry weight.
152 530nm non-hydrolyzed
tt5 + ZmCHI wt
wt (Ler)
15.00 20.00 25.00
tt5 + ZmCHI Y104F
15.00 20.00 25.00
tt5 + empty vector 15.00 20.00 25.00
tt5 + ZmCHI T46A
15.00 20.00 25.00 Minutes
15.00 20.00 25.00
tt5 + ZmCHI R34A
15.00 20.00 25.00 Minutes
Figure 3.11 HPLC chromatograms showing anthocyanidin profiles (530nm)
153 360nm (hydrolyzed)
wt (Ler) Q K
10.00 15.00 20.00 25.00 30.00 tt5 + empty vector
10.00 15.00 20.00 25.00 30.00 tt5 + ZmCHI wt Q K
10.00 15.00 20.00 25.00 30.00 tt5 + ZmCHI Y104F Q K
10.00 15.00 20.00 25.00 30.00 tt5 + ZmCHI T46A Q K
10.00 15.00 20.00 25.00 30.00 tt5 + ZmCHI R34A
10.00 15.00 20.00 25.00 30.00
minutes
Figure 3.12 HPLC analysis of flavonol content at 360nm: 360nm chromatograms of hydrolyzed samples are represented here. Q: quercetin and K: kaempferol.
154
A 6000 Quercetin
Kaempferol 5000
4000
3000 2000 Area (mV.s)
1000
0 5 t tt Iw 4F H 10 R34A C I wt (Ler) Zm mCH ZmCHI Y ZmCHI T46AZ B 10000 9000 Q+K 8000 7000 6000 5000 4000 (mV.s)Area 3000 2000 1000 0
t5 F t Iwt 4 H 10 R34A t (Ler) Y w HI ZmC HI mC mC Z ZmCHI T46AZ C 100 90 Quercetin 80 Kaempferol 70 60 50 40 30
Percentage area 20 10 5941 57 43 59 41 51 49 0 5 tt 6A 4 I T wt (Ler) ZmCHIwt mCH ZmCHI Y104FZ ZmCHI R34A
Figure 3.13 Q:K ratio difference in ZmCHIT46A with no changes in total flavonol content: (A) Comparative analysis of the area (mV.s) for quercetin (Q) and kaempferol (K) peaks integrated from the HPLC chromatograms at 360nm. (B) Total flavonol content as expressed as the sum of Q and K. (C) Ratios of the Q:K content.
155 280nm (hydrolyzed) wt (Ler) * *
5.00 10.00 15.00 20.00 25.00 30.00 tt5 + empty vector * *
5.00 10.00 15.00 20.00 25.00 30.00 tt5 + ZmCHI wt * *
5.00 10.00 15.00 20.00 25.00 30.00 tt5 + ZmCHI Y104F * *
5.00 10.00 15.00 20.00 25.00 30.00 tt5 + ZmCHI T46A * *
5.00 10.00 15.00 20.00 * 25.00 30.00 tt5 + ZmCHI R34A *
5.00 10.00 15.00 20.00 25.00 30.00
Figure 3.14 HPLC chromatograms reveal changes in phenolic profiles (280nm)
156 3.3.6 Inability to detect interactions between the maize flavonoid enzymes.
Changes in the metabolic profiles with the various mutants may indicate selective
channeling of intermediates where the metabolites would be kept concentrated in a local environment and would not need to equilibrate with the free cytoplasmic pools (Achnine
et al., 2004). Yeast two hybrid investigations in Arabidopsis show the Arabidopsis CHS,
CHI and DFR to interact as CHS-CHI, CHI-DFR and no interaction between CHS and
DFR). Affinity chromatography and immunoprecipitations from seedling extracts demonstrated that CHI interacts with CHS and F3H (Burbulis and Winkel-Shirley, 1999).
Using the yeast two-hybrid, I intended to demonstrate interactions between the maize flavonoid enzymes, and whether the maize enzymes interact with the Arabidopsis
enzymes. I had difficulty in detecting interactions. CHS, CHI, DFR and F3H from maize
and Arabidopsis were cloned into pAD-GAL4 and pBD-GAL4 vectors and transformed into yeast. Interactions were not detected using the stringent adenine marker. Out of the four independent transformation sets, three yielded negative results and one transformation set yielded growth scoring for the histidine (HIS3) reporter, but not for the adenine (ADE2) reporter present in the pJ69.4a yeast strain (James et al., 1996). Clonings were repeated once again into the Gateway based pAD-GAL4 and pBD-GAL4 vectors and there was failure to reproducibly detect interactions (Appendix F).
Immunoprecipitations from maize plants using α-ZmCHS antibodies or α-ZmF3H and pull downs using GST:ZmCHI did not detect F3H or CHS in their respective experiments. A far western using GST:ZmCHI as a probe against E.coli expressed his-
tagged flavonoid enzymes showed no signal either (results not shown).
157 These experiments, although failed to yield results confirming the interactions, indicated that interactions if present, may be transient, and may be stabilized when present in association with a complex or in planta may be organized on a scaffold. It is conceivable that the yeast two hybrid does not capture all the possible interactions. In in vitro studies in Medicago, protein-protein interaction between CHS and CHI were not detected using gel filtration and analytical ultracentrifugation (Jez et al., 2000). Thus, in planta based experiments are needed to substantiate the existence of the maize flavonoid metabolome or to explain the mechanisms by which ZmCHI may complement tt5 mutants.
158 3.4 Discussion
The Arabidopsis transparent testa mutants of the enzymes of the flavonoid pathway provide a powerful and convenient system to characterize in planta functionality of catalytically impaired mutants (Dong et al., 2001). tt5 was transformed with the
ZmCHI Y104F, T46A and R34A mutants to test the contribution of these residues in the catalytic mechanism of the ring closure of chalcone and their functionality in vivo.
ZmCHIT46A and ZmCHIY104F complemented the tt5 phenotype restoring the production of anthocyanins to that seen with ZmCHIwt and endogenous Arabidopsis levels and with ZmCHIR34A unable to do so.
The complementation of tt5 by ZmCHIT46A is extremely appealing as it does not show any activity in vitro but has the ability to complement the tt5 mutant. Possible explanations for this finding are as follows:
− The CHI enzyme could be modified in vivo, making in vitro catalytically
inactive mutants functional
− CHI may function as a dirigent protein or a structural protein, which in
vivo may participate in an enzyme complex and direct flux.
− CHI controls at some level (transcriptionally or post-translationally)
another enzyme with CHI activity.
In the first option, the enzyme may either be modified in vivo by a plant specific modification or interaction. It has been reported that the Arabidopsis CHI undergoes post translational modification through a thioester linkage to the cysteine residues present in the protein (Burbulis and Winkel-Shirley, 1999). Of the cysteines present in both the
Arabidopsis and maize CHI, only one is conserved and homology modeling does not
159 show it to be solvent exposed (Dong et al., 2001). Furthermore, initial CHI activity from
Phaseolus vulgaris induced by elicitors was proposed to be from activation of pre-
existing enzyme followed by de novo synthesis of the protein (Dixon et al., 1983).
However, if there was a post translational modification affecting the activity of the maize
ZmCHIT46A mutant, the same should be true for the ZmCHIwt and ZmCHIY104F
which are still active in vitro either as E. coli expressed proteins or as synthesized
proteins in the rabbit reticulocyte cell extracts. Western blots would need to be performed
to investigate the migration patterns of the ZmCHI band from the transgenic Arabidopsis
plants.
The second possibility is that ZmCHIT46A may act as a dirigent protein. Dirigent
proteins are a unique class of proteins that do not have catalytic activity but ‘guide’ the
biochemical reaction in a regio-specific manner (Davin and Lewis, 2000). Dirigent
proteins were first described in the synthesis of lignans and lignins where these small,
extracellular, glycoproteins bind to the monolignols- p-coumaryl, conferyl or sinapyl
alcohols and together with laccases and peroxidases lead to the linkage specific coupling
of the phenoxy radical radicals (Davin and Lewis, 2000; Davin and Lewis, 2005). Thus,
if ZmCHIT46A behaves like a dirigent protein, it would preferentially convert the
chalcone into the biologically active 2S-naringenin. The participation in a multienzyme complex may explain the activity observed in vivo where increased local concentration would be directed into the preferential formation of 2S naringenin by CHI. To test this hypothesis, the ratios of the two isomers of naringenin would need to be monitored by chiral HPLC.
160 The third possibility suggests that CHI may be able to transcriptionally or post- transcriptionally control the activity of a protein with CHI like activity. However if this were to be the case, similar kinetic parameters should have been detected in the
ZmCHIY104F mutant. To assess this option, 35S::ZmCHIT46A plant extracts would need to be separated and CHI activity tracked along with the ZmCHIT46A protein.
Furthermore, tt5 seedlings expressing T46A mutant of ZmCHI, accumulated 25% more anthocyanin than the ZmCHIwt, ZmCHIY104F expressing tt5 seedlings and the
Arabidopsis wt seedlings. Although there was no significant change in the total amount of flavonols, the 35S::ZmCHIT46A seedlings showed a change in quercetin to kaempferol ratio to 50:50 from the average 60:40 observed in the ZmCHIwt. There was a drop in the levels of quercetin, which is co-related with the increase in the total amount of anthocyanins, where flux now either gets shuttled into the anthocyanin branch of the flavonoid pathway or the flavonol pathway.
These changes in metabolic profiles may be explained by the amount of protein being expressed or catalytic characteristics of the enzyme. The protein expression levels need to be determined in the plants. However, the consistently increased levels of anthocyanins exhibited by the three independent transgenic lines suggest that the T46A mutant may be involved in channeling the intermediates towards anthocyanins or that the change in the dynamics of the catalytic site may make it insensitive to inhibitors.
Kaempferol and quercetin were shown to inhibit Phaseolus vulgaris CHI together with the isoflavonoids kievitone and coumestrol (Dixon et al., 1982). The effect of the flavonols on ZmCHI activity remains to be determined.
161 Thus, this series of ZmCHI mutants demonstrate that the role for CHI in the flavonoid pathway is not only catalytic may also perform a structural role diverting flux into the pathways. Future exciting directions would be to use these catalytic mutants to address the moonlighting role of CHI as a regulatory protein.
162 CHAPTER 4
CONCLUSIONS
Not much is know about the transport mechanisms of small molecules in cells. Do they randomly diffuse in the cytoplasm until they encounter a transporter that specifies their correct intracellular destination or are there transport processes in the cells that keep high localized concentrations, guiding the synthesized molecules to their destination without interfering with cellular processes? Here, we used anthocyanins, colored pigments from the well studied flavonoid biosynthetic pathway as a model system to understand their transport from the site of synthesis on the cytoplasmic face of the ER
(Hrazdina and Wagner, 1985; Winkel-Shirley, 1999), to thier sequestration site in the central vacuole (Grotewold, 2004).
Light is known to be one of the best studied environemental cues to induce anthocyanins (Chalker-Scott, 1999). In transgenic maize BMS35S::R+35S::C1 cells in culture,
over-expressing the MYB (C1) and bHLH (R) transcription factors and which
accumulate pigments constitutively, light induced the darkening of the calli. Biochemical
and molecular investigations showed light not to induce additional pigment synthesis or
influence the expression of core flavonoid biosynthetic genes, but to dramatically alter
163 the vacuolar morphology and anthocyanin accumulation of the cells. BMS35S::R+35S::C1
cells grown in the dark were multi-vacuolated with irregularly-shaped AVIs and little
coloration of the vacuolar sap. Light induced the fusion of vacuoles and anthocyanins
“spread” from the AVIs into the vacuolar sap. Similar light induced fusion of membrane
compartments were observed in planta. Epidermal cells of maize tassel glumes exhibited
light induced formation of anthocyanin filled dynamic tubular and vesicular compartments which fused and spread into the entire cell. Although AVIs have been described as a membraneless, proteinaceous matrix, trapping anthocyanins (Markham et al., 2000), observations of the AVIs in the BMS35S::R+35S::C1 cells as rounded structures, appeared membrane bound. Isolation of AVIs and characterization of their biochemical and molecular contents would provide clues to thier biogenesis and potential uptake into the vacuole by autophagy. A novel effect of light on vacuole fusion was uncovered using the BMS35S::R+35S::C1 cells, which may help us understand the role of light in
vesicle/vacuole fusion in plants. Investigating which wavelength of light is responsible
for bringing about the fusion may implicate which of the photoreceptor pathways is
involved. The organelle identity of the dynamic compartments in the maize glume cells needs to be determined, for example using immuno-cytochemistry. Furthermore, proteomic studies of the constituents of the endomembrane system of cells accumulating anthocyanins versus. those that do not may be used as a direct means of identification of the kegs and wheels of the transport machinery.
Cellular observations in maize of a vesicle based transport of anthocyanins lead us
to investigate anthocyanin transport in the model plant Arabidopsis thaliana. A
convenient seedling system was developed utilizing the anthocyanin-less tt5 mutant,
164 deficient in chalcone isomerase, and chemically complementing the mutant seedlings
with naringenin, the product of CHI, to accumulate anthocyanins. Metabolic analysis of
the tt5 and wt seedlings compared with their naringenin treated counterparts showed that
naringenin induced the accumulation of a new anthocyanin peak identified as C3G.
Recent experiments from our laboratory have identified more than 15 anthocyanins in
Arabidopsis and demonstrated C3G to be the precursor of more complex anthocyanins. It
would be interesting to track the sub-cellular localization of C3G, and identify and
localize the transferases to address questions if C3G gets into the vesicular route to the
vacuole, where does its further modification occur – the cytoplasm or the vacuole and
which transporters are involved in the sequestration of the various anthocyanins into the
vesicles or vacuoles.
Transcriptome profiling of naringenin-treated and non treated tt5 and wt seedlings
revealed the flavonoid biosynthetic enzymes to be robustly expressed in our conditions of
3% sucrose and continuous white light. Naringenin did not feedback regulate the
flavonoid enzymes at the transcription level. However, comparing relative expression
values in tt5 to wt, a significant 2 fold up-regulation of the core flavonoid pathway
enzymes CHS, F3H, F3’H, LDOX and FLS1 together with TT8, a bHLH transcription
factor was observed which is in good confirmation with a previous study comparing the
enzyme levels on a protein level (Pelletier et al., 1999). The up-regulation of the enzymes
in tt5 could be attributed to the feedback regulation by the accumulated flavonoid
intermediate, chalcone or more intriguingly, transcription regulation by the metabolic enzyme CHI which is shown to be localized to the nucleus (Saslowsky et al., 2005)
(Appendix E). Further studies involving validation of the observed results by northern
165 analysis and investigating the potential moonlighting role of CHI as a nuclear regulator
using chromatin immunoprecipition (ChIP) coupled with gene expression arrays (ChIP-
CHIP), would further help us understand what a metabolic enzyme might be doing in the
nucleus.
Naringenin affected a total of 178 genes in the tt5 and wt seedlings. Genes were clustered based on their expression trends. The common up-regulated genes in both tt5 and wt were primarily implicated in a detoxification process. Interestingly naringenin also induced the sulfated peptide growth factor, phytosulfokine (AtPSK3), previously shown to stimulate growth and differentiation (Matsubayashi et al., 2001; Yang et al., 2001). In carrot, an LRR receptor kinase was recently identified to interact with the five amino acid phytosufokine (Matsubayashi et al., 2002). It would be interesting to see if any of the two putative LRR transmembrane protein kinases (At1g17750, At1g66830), up-regulated at
~1.6 fold by naringenin are the potential receptors for AtPSK3. The stress response viewer from Genevestigator showed that naringenin induced a number of genes that overlapped with the genes upregulated in Arabidopsis challenged with Pseudomonas syringae, Botrytis cinerea and abiotic hypoxia, salt and osmotic stress. This common stress defense response may be mediated through jasmonic acid where sequential JA biosynthetic enzymes, lipoxygenases (LOX3; At1g17420 and At1g72520), allene oxide cyclase (AOC; At3g25780) and 12-oxophytodienoate reductase (OPR3; At2g06050) were up-regulated by naringenin in tt5 at the >1.7 fold level (cluster 3). It would be interesting to see which of the cluster 3 transcription factors regulates the co-ordinate expression of these JA bisynsthetic genes. Apart from the two transporters being commonly up-regulated in tt5 and wt, naringenin did not induce any other transporters,
166 which may indicate that the transporters, together with the flavonoid biosynthetic enzymes, were robustly and coordinately expressed in the conditions that were used for seedling growth. Furthermore, naringenin is an inhibitor of growth as seen from root
growth assays on plates and reflected in the number of down regulated genes which were
cell wall related and expressed in the roots. The genes identified from this experiment
would need to be first validated by quantitative RT-PCR. Genetic approaches using
mutants and biochemical approaches using protein-protein, protein-ligand interaction
studies would help in understanding how the pieces identified here fit into the cellular
network puzzle.
The role of CHI in the flavonoid pathway and its participation in the flavonoid
metabolome was investigated. The reaction catalyzed by CHI can occur spontaneously at
pH ≥7.5. Maize cells constitutively expressing either transcription factors P or C1 and R
(Grotewold et al., 1998) and tomato fruit pericarps (Muir et al., 2001) do not show
detectable transcripts or enzyme activity of CHI. However, mutations in this enzyme in
Arabidopsis and several other species leads to no accumulation of anthocyanins. The
need for the enzyme in Arabidopsis and no need in maize led to generation of a series of
mutated maize CHI enzymes asking if a catalytically inactive enzyme had the ability to
complement the Arabidopsis tt5 mutant. Based on studies in Medicago sativa (Jez et al.,
2000), point mutants in the aa residues Y104, T46 and R34 in ZmCHI were generated. In vitro activity determined ZmCHIY104F to have 20% of the ZmCHIwt activity while the
ZmCHIT46A and the ZmCHIR34A were catalytically inactive. ZmCHIwt,
ZmCHIY104F and ZmCHIT46A complemented the tt5 mutant, with ZmCHIR34A unable to do so. It is most interesting that the in vitro catalytically inactive ZmCHIT46A
167 complements the tt5 mutant. CHI activity in these plants was detected above the spontaneous levels. This may be due to a number of different possibilities including post
translational modifications, activation of another protein with CHI like activity or that the
protein may act as a dirigent protein (Davin and Lewis, 2000) preferentially biasing the
spontaneous reaction towards the formation of the biologically active 2S form of
naringenin. Furthermore, the ZmCHIT46A complemented tt5 seedlings accumulated
about 25% more anthocyanins than the wt Arabidopsis, the ZmCHIwt and the
ZmCHIY104 complemented seedlings. This increase in accumulation of anthocyanins
could be attributed to the expression levels of the transgenes in the plants, or to changes
in sensitivity to the feedback inhibition by flavonols, as previously reported in Phaseolus
vulgaris (Dixon et al., 1983). Thus, future studies using the ZmCHIT46A allele of CHI,
would lead to insights of the role of CHI in the flavonoid pathway. Lastly, the in vitro
protein-protein interaction of CHI with the flavonoid enzymes CHS, DFR and F3H from
both maize and Arabidopis using the yeast two-hybrid or GST pull downs were unable to detect the interactions. The interactions, if present, may be transitory and further in vivo interaction investigations using TAP tagging or gel filtration would be useful to identify the presence, and members of the complex. The formation of a complex is attractive as this would prevent free diffusion of the intermediates into the cytoplasm and direct the compounds to their correct sub-cellular destination.
Thus, understanding the processes involved in the vesicular route of sequestration anthocyanins to the vacuole would also address questions in vacuole biogenesis and autophagy. The seedling system developed in Arabidopsis can be conveniently used to assay numerous mutants and evaluate the effects of various biochemical inhibitors on the
168 accumulation of anthocyanins. Genes identified in the microarray have opened up
exciting leads into the field of signaling and defense mechanisms. And finally the
exciting potential role of a metabolic enzyme, CHI, as a regulatory protein and the
generation of an in vitro catalytically inactive CHI mutant that complements the
flavonoid pathway in tt5 mutants may redefine functions of enzymes in regulation and biochemistry.
169 APPENDIX A
MONTAGE OF BCECF-AM STAINED BMS35S::R+35S::C1 CELLS
Montage of a stack of ten individual LSCM optical sections through a dark grown BCECF-AM loaded BMS35S::R+35S::C1 revealing the multi-vacuolate nature of the anthocyanin accumulating cells
170 APPENDIX B
LIST OF PRIMERS USED
Primer name Sequence
ZmCHIR34A5' 5’GGCGGCGCAGGCGTGgcAGGCGTCGAGATCGGAGG 3’
ZmCHIR34A3’ 5’CCTCCGATCTCGACGCCTgcCACGCCTGCGCCGCC3’
ZmCHIT46A5' 5'AACTTCATCAAGTTCgCGGCCATCGGCGTGTAC3'
ZmCHIT46A3' 5'GTACACGCCGATGGCCGcGAACTTGATGAAGTT3'
ZmCHIY104F5’ 5’CGGGCGAGCAGTtCGCGGAGAAAGT3’
ZmCHIY104F3’ 5’ACTTTCTCCGCGAACTGCTCGCCCG3’
CHI N1 5’AATAAGGATCCGAATTCACCATGGCCGTGCCGGAG3’
CHI N2 5’AATATAAGCTTCTC GAGTCAGGCGGTGATGGAGAC3’
ZmCHI pENTR-5’ 5’ CACCATGGCCGTGCCGGAGGTGGTG 3’
ZmCHI pENTR-3’ 5’ TGCGGCGGTGATGGAGACGGGCTCC 3’
ZmCHS-pENTR-5’ 5’ CACCATGGCCGGCGCGACCGTGACC 3’
ZmCHS-pENTR-3’ 5’ TGCGGCGGTGGCCGCTCCGGTGGTG 3’
ZmA1-pENTR-5’ 5’ CACCATGGAGAGAGGTGCCGGTGCG 3’
ZmA1-pENTR-3’ 5’ TGCAGCGCCAATCGTCGCCTCCGTC 3’
ZmF3H pENTR-5’ 5’ CACCATGGCTCCCGTGAGCATCAG 3’
171 ZmF3H pENTR-3’ 5’ GGCAAAAATGGCGTCGAGAGGCTTG 3’
AtCHS-pENTR-5’ 5’ CACCATGGTGATGGCTGGTGCTTCT 3’
AtCHS-pENTR-3’.2 5’ GAGAGGAACGCTGTGCAAGACGAC 3’
AtCHI pENTR-5’ 5’ CACCATGTCTTCATCCAACGCCTG 3’
AtCHI pENTR-3’ 5’ GTTCTCTTTGGCTAGTTTTTCCTC 3’
AtDFR pENTR-5’ 5’ CACCATGGTTAGTCAGAAAGAGACC 3’
AtDFR pENTR-3’ 5’ GGCACACATCTGTTGTGCTAGCATG 3’
AtF3H pENTR-5’ 5’ CACCATGGCTCCAGGAACTTTGAC 3’
AtF3H pENTR-3’ 5’ AGCGAAGATTTGGTCGACAGGCTTG 3’
The small letters in the nucleotide sequence indicate the corresponding mutations introduced into the wt sequence. The pENTR 3’ primers do not include a stop codon.
172 APPENDIX C
EPIDERMAL AND SUBEPIDERMAL ACCUMULATION OF ANTHOCYANINS
IN ARABIDOPSIS COTYLEDONS.
Addition of naringenin to tt5 seedlings causes accumulation of anthocyanins in epidermal and subepidermal tissues: Sub-epidermal tissue of the adaxial surface of the cotyledons of tt5 seedlings treated with 100µM naringenin for 24h, seen as ‘unfocused’ cells in the center (a), accumulate anthocyanins before the epidermal cells. On the abaxial surface, only epidermal cells accumulate anthocyanins (b)
a b
Adaxial Abaxial
173 APPENDIX D
COMPARTIVE SPECTRAL ANALYSIS OF THE ANTHOCYANIN PEAKS OF NARINGENIN AND
CYCLOHEXIMIDE TREATED tt5 AND wt ARABIDOPSIS SEEDLINGS
Spectra extracted from the HPLC chromatogram at 530nm. Blanks indicate that the peaks were absent in the sample. RT= retention time in minutes.
174 RT tt5 +N -C tt5 +N +C wt -N -C wt +N -C wt -N +C wt +N +C 9.37
12.56
174 RT tt5 +N -C tt5 +N +C wt -N -C wt +N -C wt -N +C wt +N +C 15.55
15.98
16.33
175 16.99
17.76
175 RT tt5 +N -C tt5 +N +C wt -N -C wt +N -C wt -N +C wt +N +C 18.52
19.53
20.72 176
21.01
21.58
176 RT tt5 +N -C tt5 +N +C wt -N -C wt +N -C wt -N +C wt +N +C 21.76
22.63
23.73 177
23.78
24.89
177 RT tt5 +N -C tt5 +N +C wt -N -C wt +N -C wt -N +C wt +N +C 25.20
178
178 APPENDIX E
TRANSIENT LOCALIZATION OF MAIZE AND ARABIDOPSIS CHS, CHI, DFR
AND F3H IN NICOTIANA BENTHAMIANA
The enzymes of the flavonoid pathway of maize and Arabidopsis CHS, CHI, A1/DFR and F3H were cloned into the Gateway pENTR-SD-TOPO vector and mobilized into a binary T-DNA plasmid harboring GFP as a C-terminal fusion (pGW5). Agrobacterium tumefacience strain GV3101 pMP90 harboring the enzyme-GFP constructs were infiltrated into the abaxial side of the leaves of two to three week old Nicotiana bethamiana plants. Two days after infiltration, images of epidermal cells were obtained using laser scanning confocal microscopy. The enzymes were localized in the nucleus as well as the cytoplasm, similar to free GFP expression pattern but not as robustly expressed as the free GFP (D.1 a.).
179 E.1 ZmCHIwt-GFP is localized to the nucleus and cytoplasm:LSCM images of the epidermal cells of the leaves show ZmCHIwt-GFP (c) localized to the cytoplasm as well as the nucleus. The gain levels for free GFP (a) and ZmCHI-GFP (c) were the same and fluorescence intensities of ZmCHIwt-GFP was not as robust as free GFP. Size bar represents 10µm.
180 E.2 Transient localization of the maize CHS, CHI, A1 (DFR) and F3H GFP fusions in N. benthamiana: The enzymes show nuclear and cytoplasmic localization. The fluorescence intensities of the maize enzymes are not as robustly as compared to the Arabidopsis enzymes in N. bethamiana. Gain levels are not the same. Size bar represents 10µm.
a b
35S::ZmCHS-GFP
c d
35S::ZmCHI-GFP
e f
35S::ZmA1-GFP
g h
35S::ZmF3H-GFP
181 E.3 Transient localization of the Arabidopsis CHS, CHI, DFR and F3H GFP fusions in N. benthamiana: The enzymes are localized to the nucleus and cytoplasm. AtCHS- GFP shows lower fluorescent signal in the nucleus as compared to the cytoplasm. Dynamic sub-vacuolar structures were also observed in all the Arabidopsis enzyme constructs (not shown). Size bar represents 10µm.
a b
35S::AtCHS-GFP
c d
35S::AtCHI-GFP
e f
35S::AtDFR-GFP
g h
35S::AtF3H-GFP
182 APPENDIX F
YEAST TWO HYBRID ANALYSIS
Directed yeast two hybrid analysis was carried out using the enzymes of the flavonoid pathway from maize and Arabidopsis : ZmCHS, ZmA1, ZmF3H and AtCHS, AtDFR, AtF3H. The interaction studies using the yeast two hybrid were carried out in four independent transformations. The enzymes were cloned into the pAD-GAL4 and the pBD-GAL4 vectors (Stratagene). The first three independent transformations were carried out with these constructs. The yeast strain pJ694a was used and transformants were selected on leucine and tryptophan deficient plates (-L-T). Six colonies from each of the enzyme combinations were screened on the histidine (-L-T-H) and adenine (-L-T-H- A) selective plates. Two of the three independent transformations yielded negative results where there was no growth recorded on the -L-T-H or the -L-T-H-A plates. One transformation event yielded growth on the –L-T-H plates for the possible recombinations but exhibited no growth when selected for the adenine reporter. The experiment below represents the fourth set of independent transformations done.The enzymes were re-cloned into the pAD-GAL4 and pBD-GAL4 vectors modified with a Gateway recombination cassette – pAD-GAL4 GW C and pBD-GAL4 GW C. ZmCHI and AtCHI in pAD and pBD were utilized in a combination grid with the other enzymes. As seen in the figures, the six colonies from each combination grew on plasmid selective –L-T plates but not on the reporter plates –L-T-H or –L-T-H-A. Lac Z assays for the colonies did not yield positive results. GS99 and GS88 were the positive controls, combinations with the pAD and pBD vectors were the negative controls. Taken together, these experiments suggest that the interactions, if present, are transitory and the yeast two hybrid system could not reproducibly and robustly detect the interactions.
183
pAD-ZmCHI
-L-T -L-T-H -L-T-H-A Lac Z pBD-ZmCHI pBD-ZmCHS pBD-ZmA1 pBD-AtCHS pBD-AtCHI pBD-AtDFR pBD-AtF3H pBD-ZmF3H pBD
pBD-ZmCHI -L-T -L-T-H -L-T-H-A Lac Z pAD-ZmCHS pAD-ZmA1 pAD-AtCHS pAD-AtCHI pAD-AtDFR pAD-AtF3H pAD-ZmF3H pAD
184
pAD-AtCHI -L-T -L-T-H -L-T-H-A Lac Z pBD-ZmCHS pBD-ZmA1 pBD-AtCHS pBD-AtCHI pBD-AtDFR pBD-AtF3H pBD-ZmF3H pBD
pBD-AtCHI -L-T -L-T-H -L-T-H-A Lac Z pAD-ZmCHS pAD-ZmA1 pAD-AtCHS pAD-AtDFR pAD-AtF3H pAD-ZmF3H pAD
Controls -L-T -L-T-H -L-T-H-A Lac Z pAD - pBD GS88 – GS99 pAD – GS99 GS88 - pBD
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