molecular characterization of signaling mechanisms …
TRANSCRIPT
MOLECULAR CHARACTERIZATION OF SIGNALING MECHANISMS IN FLOWERING
TRANSITION IN ARABIDOPSIS AND IDENTIFICATION OF NOVEL FLOWERING QTL
IN SOYBEAN
BY
ERIC JAMES SEDIVY
THESIS
Submitted in partial fulfillment of the requirements
for the degree of Master of Science in Crop Sciences
in the Graduate College of the
University of Illinois at Urbana-Champaign, 2013
Urbana, Illinois
Master’s Committee:
Assistant Professor Yoshie Hanzawa
Professor Lila Vodkin
Professor Steven Huber
ii
Acknowledgements
There are many people who helped me bring this work into existence, but I would like to
extend a special thanks to Dr. Randall Nelson who allowed me to collaborate with him and his
lab group on the soybean results I present here, and to my advisor, Dr. Yoshie Hanzawa, who in
the past 2 years has helped me accomplish more than I thought possible, never letting me settle
for less than my potential.
I would also like to thank all of my friends who were with me up until this point,
simultaneously driving me to accomplish more through their example, and letting me know when
it was time to take a break and enjoy a nice cold beer. I probably would have run myself ragged
in your absence.
Last but not least, I’d like to thank my parents who taught me the value of working until
the job is done, and who never ceased in providing me with the love and support I needed to see
this through. This thesis is as much a testament to their achievements as it is to mine.
iii
Abstract
Flowering response to seasonal photoperiod changes is a critical trait to environmental
adaptation and productivity of plants. To better understand flowering two approaches are
developed. The first is the molecular characterization of signaling mechanisms in flowering
transition in Arabidopsis with the intent to explore basic flowering mechanisms in a model plant
species. To this end TERMINAL FLOWER 1 (TFL1), a flowering repressor that controls the
activity of the shoot apical meristem (SAM), was selected. Through yeast two-hybrid, we
identified nine TFL1 interactors and named them TFL1-IN-LOVE (TIL). Of the nine, TIL3,
showed specific binding to TFL1 at a unique TFL1 residue. For this reason TIL3, which encodes
an inositol 5-phosphatase (5PTase), was selected for further observation. A functional link
between TFL1 and TIL3/5PTase13 was established through root gravitropism experiments, and
supported through flowering time experiments where til3-1/5ptase13-1 mutants were shown to
reduce the late-flowering effect of TFL1 over-expression. Furthermore, in vivo sub-cellular
localization in Nicotiana benthamiana reveals both TFL1 and TIL3/5PTase13 proteins to co-
localize in the nucleus and cytoplasm. Finally, Bimolecular Fluorescent Complementation
(BiFC) demonstrates in vivo protein-protein interaction between TFL1 and TIL3/5PTase13,
supporting our original yeast two-hybrid screen, and reinforcing the hypothesis that
TIL3/5PTase13 is important for proper TFL1 function.
The second approach devised to understanding flowering is the identification of novel
flowering QTLs in Glycine max, the purpose of which is to expand basic knowledge of flowering
gained in model species, and apply that knowledge to a cultivate species. In soybean, we
conducted QTL mapping using a population of 115 BC2F6 recombinant inbred lines (RILs) that
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were created from a cross between cultivated soybean, G. max, and its ancestor, G. soja.
Agriculturally important traits: flowering time (R1), maturity time (R8), height, yield, lodging,
and stem vining were measured for two years in four field locations. QTL mapping analysis
identified many previously unidentified QTLs, including 6 independent flowering QTLs with
candidate genes, 6 independent maturity QTLs with candidate genes, and 14 QTLs that are
observed to affect multiple traits, 3 of which are highly significant.
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Table of Contents
CHAPTER 1 ............................................................................................................................................. 1
Literature review: A brief synopsis of flowering in angiosperms ................................................ 1
1.1 Flowering ........................................................................................................................................................... 1
1.2 Long-day flowering pathway in Arabidopsis thaliana ......................................................................... 1
1.3 Photoperiodic flowering in the short day model species Oryza sativa ......................................... 12
1.4 Flowering in soybean................................................................................................................................... 14
1.5 Tables................................................................................................................................................................ 17
1.6 Figures.............................................................................................................................................................. 19
1.7 Literature cited ............................................................................................................................................... 24
CHAPTER 2 ........................................................................................................................................... 41
Molecular characterization of signaling mechanisms in flowering transition in
Arabidopsis .............................................................................................................................................. 41
2.1 Abstract ............................................................................................................................................................ 41
2.2 Background..................................................................................................................................................... 42
2.3 Materials and methods ................................................................................................................................ 46
2.4 Results .............................................................................................................................................................. 64
2.5 Discussion ....................................................................................................................................................... 68
2.6 Figures.............................................................................................................................................................. 73
2.7 Literature Cited.............................................................................................................................................. 87
CHAPTER 3 ........................................................................................................................................... 93
Identification of novel flowering QTLs in Glycine max ................................................................ 93
3.1 Abstract ............................................................................................................................................................ 93
3.2 Background..................................................................................................................................................... 94
3.3 Materials and methods ................................................................................................................................ 96
3.4 Results ............................................................................................................................................................ 100
3.5 Discussion ..................................................................................................................................................... 105
3.6 Tables.............................................................................................................................................................. 109
3.7 Figures............................................................................................................................................................ 116
3.8 Literature cited ............................................................................................................................................. 122
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CHAPTER 1
Literature review: A brief synopsis of flowering in angiosperms
1.1 Flowering
The initiation of flowering is a major developmental event that occurs in the life cycle of all
angiosperms. It is a finely tuned and highly elaborate set of mechanisms that combines internal
and external stimuli to facilitate the transition from vegetative to reproductive growth. Floral
initiation or repression has a significant impact on the fitness of an individual plant, as well as
determining a plant’s reproductive efficacy. This makes the floral pathway a target for
domestication in cultivated species. For example, varieties of spring wheat and barley have
undergone selection for temperature sensitive alleles (Iqbal, M. et al. 2007; Turner, A. et al.
2005), resulting in the removal of vernalization as a growth requirement. Furthermore, it is
conceivable that alterations in photoperiod would allow for a wider range of viable latitudes, and
corresponding light conditions, for crops to be cultivated. For example, domestication of rice is
marked by a cultivation shift from tropical southern clines to cold northern ones, which indicates
changes in both temperature and photoperiod responses (Izawa, T. 2007; Jung, C. and Miller, AE.
2009).
1.2 Long-day flowering pathway in Arabidopsis thaliana
Arabidopsis thaliana is widely considered the model plant, due to a number of factors such
as a small chromosome size, a short life cycle, prolific seed production, high efficiency
2
transformation using Agrobacterium tumefaciens, and an extensive collection mutant lines and
genomic information. As such, Arabidopsis is a natural choice for inquiry into flowering
initiation and control. It has been revealed that there are four major genetic pathways that
perceive and transmit external and internal inputs that are responsible for proper flowering in
Arabidopsis: the photoperiod, vernalization, autonomous and gibberellin pathways (Figure 1).
Recently, the ambient temperature pathway is emerging as a potent regulator of flowering as
well, usually when photoperiodic conditions are unfavorable for floral initiation (Kumar, SV. et
al. 2012; Lee, H. et al 2010).
Environmental signals affecting floral induction
The correct timing of floral transition is essential for successful reproduction of plants.
Plants respond to seasonal changes in temperature and light conditions and initiate their
development in tandem with favorable conditions (Simpson, GG. and Dean, C. 2002). While
many plants sense stimuli such as light or temperature to initiate flowering, the strategies
employed to respond to those stimuli can be drastically different for various plant species. For
instance, plants can be divided into three major categories due to their response to daylength:
long-day (LD), short-day (SD), and day-neutral categories. LD plants initiate flowering after
light conditions exceed a certain temporal threshold, whereas SD plants initiate flowering when
night conditions exceed a certain temporal threshold. Day-neutral plants are not dependent on
light conditions for flowering (Garner, WW. et al. 1920).
This ability of plants to detect changes in daylength is known as photoperiodism. Light
detection is carried out in the leaves through oscillating protein and mRNA levels of both
circadian clock and light responsive genes (Suarez-Lopez, P. et al. 2001; Mizoguchi, T. et al
3
2005 ). When light conditions are favorable, it is suggested that a molecular signal known as a
florigen is transported from the leaves to the shoot apical meristem (SAM) where development
of floral organs is initiated for reproduction ( Zeevaart, JAD. 2006).
In conjunction with day length, temperature also plays a vital role in successful floral
initiation. During reproductive growth, plants are highly susceptible to cold temperatures, and in
most cases prolonged exposure to below freezing conditions will result in failure to generate
viable offspring (Olien, CR. 1967). To reduce such damage of reproductive organs by cold
temperature, many plants evolved a mechanism known as “vernalization”, a period of 1-3
months below 7°C in which flowering does not occur until a period of cold has elapsed
(Chouard, P. 1960; Lang, A. 1965). In Arabidopsis, it has been documented that there is a
correlation between the latitude at which a plant is grown, and sensitivity towards vernalization
with more equatorial accessions being more sensitive to vernalization as compared to Northern
accessions (Stinchcombe, A. et al. 2005).
Vernalization requirement of Arabidopsis thaliana is controlled through the key gene
FRIGIDA (FRI). FRI accomplishes this process through up-regulation of the MADS-box
transcription factor FLOWERING LOCUS C (FLC) (Michaels, SD. and Amasino, RM. 1999),
which inhibits floral transition through repression of the floral integrators SUPPRESSOR OF
OVERECPRESSION OF CONSTANS (SOC1) and FLOWERING LOCUS T (FT) (Hepworth,
SR. et al. 2002). Mutations in the FRI gene result in early flowering, and loss of vernalization
requirement (Johanson, U. et al. 2000). Conversely, cold induction leads to the recruitment of
VERNALIZATION INSENSITIVE 3 (VIN3) (Kim, DH. et al. 2010), VERNALIZATION1
(VRN1) (Levy, YY. et al. 2002), and VERNALIZATION2 (VRN2) (Gendall AR et al. 2001).
VIN3 and VRN2 are believed to impart an epigenetic “memory of winter”, preventing continued
4
FLC repression after vernalization has taken place (Gendall, AR. et al. 2001;Kim, DH. et al
.2010). VRN1, a homolog of the meristem identity gene AP1, displays specific binding to the
FLC promoter under cold conditions, resulting in FLC repression (Levy, YY. et al. 2002).
Moreover, the autonomous pathway is tightly interconnected with FLC induction and repression,
which will be addressed in a later section.
In addition to sensitivity to cold environments having an affect on flowering, plants are
also influenced by shifts in ambient temperature (23°C-27°C), sometimes referred to as the
thermosensory (Figure 2). Changes in ambient temperature alter rates of metabolic reactions and
developmental processes (Long, S. and Woodward, F. 1988), as well as flowering (Lee, JH. et al.
2008; Samach A. and Wigge P. 2005). Many genes mediate the thermosensory pathway. SHORT
VEGETATIVE PHASE (SVP) (Hartmann, U. et al. 2000) and FLOWERING LOCUS M (FLM)
(Scortecci, K. et al. 2003), originally characterized as genes in the autonomous pathway, act as
key controllers in ambient temperature induced flowering (Balasubramanian, S. et al. 2006a).
SVP is observed to repress flowering through binding to the promoter region of FT, negatively
regulating FT expression (Lee, JH. et al. 2007). Likewise, FLM is thought to repress FT
expression in Arabidopsis under short day conditions around 23°C; however, it is proposed that
as temperature increases to 27°C, alternative splice variants of FLM are expressed that cannot
repress FT, thus resulting in floral induction despite the short day conditions (Balasubramanian,
S. et al. 2006b). Furthermore, studies have shown that the early flowering phenotype of
phytochromeB (phyB) mutants is temperature-dependent (Halliday, KJ. et al. 2003), which
indicates that the ambient temperature and photosensitivity pathways crossover in their control of
flowering. Similarly, it has been suggested that ELF3 and TFL1 independently regulate
flowering through separate temperature pathways (Strasser, B. et al. 2009; Kim, W. et al. 2013).
5
PHYTOHROME INTEACTING FACTOR4 (PIF4) has been indicated in increasing FT
expression under warm conditions through chromatin remodeling (Kumar, SV. et al. 2012).
Finally, the microRNA miR172 has been indicated in having a role in ambient temperature
control and flowering, possibly mediated upstream by SVP (Lee, H. et al. 2010). These results
illustrate that ambient temperature signaling is intricately connected with other signaling
pathways.
Endogenous floral initiation signals
In addition to external signals having regulatory effects on floral initiation, several
internal signals control flowering. One such mechanism is the autonomous pathway, named
precisely for its lack of reliance on external signals. It is often proposed that the autonomous
pathway maintains pre-reproductive or juvenile development in plants until internal conditions
are ideal for progression into a subsequent developmental state (Poethig, RS. 1990; Poethig, RS.
2010; Amasino, R. M. 2010).
The primary way the autonomous pathway regulates flowering is through control of FLC
(Figure 3). The genes FLOWERING LOCUS D (FLD) and FVE play a critical role in this
regulation by repressing FLC through deacetylation of core histone tails (He, Y. et al. 2003;
Ausin, I. et al. 2004). Moreover, the genes FY and FCA work together to impair proper FLC
mRNA maturation through premature polyadenylation, which results in a truncated, inactive
FLC protein product (Simpson, GG. et al. 2003; Quesada, V. et al. 2003). It is likely that FLC
mRNA is further modified by the RNA binding proteins FPA and FLK, which are thought to
negatively regulation FLC through improper mRNA processing (Schomburg, FM. et al. 2001;
Lim, MH. et al. 2004). While much of the autonomous pathway is focused on repression of
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FLC, members of the RNA polymerase II (Pol II) Associated Factor 1 (PAF1), ELF8 and ELF7
(He, Y. et al. 2004), have been shown to be central to FLC expression. Additionally, other less
characterized proteins such as VERNALIZATION INDEPENDENCE 4 (VIP4) (Zhang, H. and
van Nocker, S. 2002), VIP3 (Zhang, H. et al. 2003), and PHOTOPERIOD INDEPENDENT
EARLY FLOWERING 1 (PIE1) (Noh, YS. and Amasino, RM. 2003) display a similar positive
effect on FLC expression.
Another mechanism that affects floral initiation is endogenous plant hormones.
Gibberellic Acid (GA) in particular has been observed to induce flowering in environmentally
unfavorable conditions (Langridge, J. et al. 1957; Jacobsen, SE. et al. 1993; Chandler, J. et al.
1994). However, the effects of GA on flowering are not universal for all plant species (Zeevaart,
JAD. 1983). So while it is a potent agonist of flowering in Arabidopsis and many other species, it
is not an absolute flowering hormone.
Arabidopsis circadian clock
The circadian clock in plants is a diurnal rhythm of clock gene expression and its actions
is entrained by alternating periods of light and darkness, day and night (Jones, MA. 2009). This
cycle is critical to the proper induction of flowering and involves a complex interaction between
endogenous cues and environmental signals (Bunning, E. 1960 ; Pittendrigh, CS. and Minis, DH.
1964). The circadian clock consists of in three different parts (Dunlap, JC. 1999). The first part is
the central oscillator, the hub of the circadian cycle, which is responsible for modulating 24-hour
rhythms. The second part, the input pathway perceive and transmit external light and dark, or
temperature cycles to the central oscillator. The third part, the output pathway is controlled by the
central oscillator and is responsible for triggering a wide array of developmental and molecular
7
responses. Together, the circadian clock senses external stimuli and induces internal genetic
pathways to trigger floral transition.
The central oscillator in Arabidopsis forms a negative feedback-loop composed of the
genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1), LATE ELONGATED HYPOCOTYL
(LHY), TIMING OF CAB EXPRESSION1 (TOC1), and EARLY FLOWERING4 (ELF4)
(Schaffer, R. et al. 1998; Wang, Z. and Tobin, E. 1998; Strayer, C. et al. 2000; Alabadi, D. et al.
2001; Doyle, M. et al. 2002; Hayama, R. and Coupland, G. 2004) (Figure 4). It is observed that
TOC1 mRNA, expressed at night, acts an activator of CCA1 and LHY so that their transcripts can
accumulate during the initial hours of light exposure. ELF4 also appears to positively regulate
CCA1 and LHY expression. Furthermore, ELF4 protein levels oscillate in phase with TOC1,
suggesting an interaction between the two proteins. Inversely, it appears that increasing the
accumulation of CCA1 and LHY proteins negatively affect TOC1 expression (Mizoguchi, T. et
al. 2002). These competing feedback loops create a daily cycle starting with the increase of
CCA1 and LHY protein levels and decrease of TOC1 protein in the morning. As the day
progresses CCA1 and LHY expression levels decrease, and as evening begins TOC1 levels rise,
only to begin another cycle the next morning (Alabadi, D. et al. 2001). This three-cycle system
can be expanded upon with the addition of the morning loop and the evening loop (Locke, JCW.
2006; Zeilinger, MN. 2006). PSEUDO-RESPONSE REGULATORs (PRRs), PRR5, PRR7, and
PRR9 make up the morning loop, and are observed to repress CCA1 and LHY expression
(Nakamichi, N. et al. 2010). Moreover, PRR5 acts to preserve the TOC1 protein in the nucleus
(Wang, L. et al, 2010), and PRR7 and PRR9 are reciprocally stimulated by CCA1 and LHY
(Nakamichi, N. et al. 2010). The evening cycle is composed of ZEITLUPE (ZTL), FLAVIN-
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BINDING, KELCH REPEAT, F-BOX 1 (FKF1), and LOV KELCH PROTEIN 2 (LKP2), and is
mainly focused on the degradation of TOC1 during the evening hours (Mas, P. et al. 2003).
The input pathway is formed by phytochromes and cryptochromes, which are involved in
red and blue light sensing respectively (Somers, DE. et al. 1998; Devlin, P. and Kay, S. 2000), as
well as EARLY FLOWERING 3 (ELF3) and ZEITLUPE (ZTL). ZTL is proposed to bind to
PHYTOCHROME B (PHYB) and CRYPTOCHROME 1 (CRY1) and repress the light signaling
to the central oscillator. Furthermore, the ZTL protein contains F-box and repeat kelch motifs,
both of which are implicated in proteasome directed degradation (Somers, D. et al. 2000; Jarillo,
J. et al. 2001). The target of this degradation is thought to be TOC1 and PRR5 during the
evening hours for the purpose of generating a more powerful diurnal rhythm (Mas, P. et al.
2003). ELF3 also acts to repress the input of light signals to the central oscillator (McWatters,
HG. et al. 2000) through interaction with PHYB (Liu, JY. et al. 2001). Moreover, ELF3
expression levels are shown to rise under dark conditions, which are thought to prevent the
circadian clock from sensing light. It is proposed this aids in resetting the circadian rhythm for
the next cycle.
Arabidopsis photoperiodism
In the early days of plant biology, it was observed that changes in day length, usually brought
about by the seasonal cycle, greatly affected a plant’s maturity process (Garner, WW. et al.
1920). This led to the initial ideas concerning photoperiod, and how a plant can perceive
changes in photoperiod. Following this insight, it was revealed Arabidopsis is a facultative long-
day (LD) plant, and flowers earlier under 16 hours of light as opposed to short-day (SD)
conditions of 8 or 10 hours of light (Reviewed in Corbesier, L. and Coupland, G. 2005). Several
9
mutants were identified that produced a late flowering phenotype under LD conditions as
compared to wild type, including gigantea (gi), constans (co), flowering locus t (ft), flowering
locus d (fd), and cryptochrome (cry2). It was suggested that these genes make up a single
pathway to promote flowering under LD conditions. Indeed, GI, CO, and FT proteins are
observed to act at the center of the photoperiodic pathway (Figure 4). CO mRNA accumulates
during the night under SD conditions, and CO protein only accumulates late in the day under LD
conditions (Suarez-Lopez, P. et al. 2001). Furthermore, accumulation of CO protein under LD
conditions triggers the production of FT mRNA, and subsequent FT protein. These observations
suggest that CO mRNA regulation is essential to proper floral transition initiation, and that
specific light conditions are required for CO to become post-transcriptionally active (Hayama, R.
Coupland, G. 2004).
Early experiments revealed several key regulators of CO mRNA expression. FLAVIN-
BINDING, KELCH REPEAT, F-BOX PROTEIN 1 (FKF1) and GI, both controlled by the
circadian cycle, are shown to interact in vivo and be essential to CO mRNA accumulation
(Imaizumi, T. et al. 2003; Sawa, M. et al. 2007). It is proposed that this is accomplished through
FKF1 ubiquitination and degradation of a negative regulator of CO transcription. Consistent with
this idea, the transcription factor CYLCLING DOF FACTOR 1 (CDF1) is observed to repress
expression of CO mRNA, and shows direct interaction with the FKF1-GI complex (Imaizumi, T.
et al. 2003; Sawa, M. et al. 2007) that results in degradation of CDF1.
Correct light conditions also have a significant impact on CO protein accumulation. This
is illustrated by the observation that CO mRNA accumulation under SD conditions does not
result in the initiation of flowering. It is proposed that this is a result of ubiquitin-mediated
degradation of CO protein, which limits CO accumulation to the end of the day during LD
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(Valverde, F. et al. 2004). Moreover, the photoreceptors PHYTOCHROME A (PhyA) and
CRYPTOCHROME 2 (CRY2) are vital to the proper stabilization of CO proteins, responding to
red and blue light respectively. Conversely, PHYTOCHROME B (PhyB) is observed to promote
CO degradation (Valverde, F. et al. 2004). In addition, SUPPRESSOR OF PHYA-105-1 (SPA1)
together with CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1), an E3 ubiquitin ligase,
likely mediates CO degradation (Laubinger, S. et al. 2006).
Floral integrators
All of the environmental and endogenous inputs that affect flowering transition
eventually converge at a set of genes known as the floral integrators, which include
FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1
(SOC1). SOC1 encodes a MADS box transcription factor, and is promoted and repressed by CO
and FLOWERING LOCUS C (FLC) respectively (Hepworth, SR. et al. 2002; Searle, I. et al.
2006). Moreover, SOC1 expression is also partially dependent on activation of FT, the flowering-
hormone florigen (Moon, J. et al. 2005; Yoo, SK. et al. 2005). SOC1 primarily induces floral
transition by directly binding the promoter of the floral meristem identity (FMI) gene LEAFY
(LFY) (Lee, J. et al. 2008a; Liu, C. et al. 2008) (Figure 4).
FT mRNA expression is induced by CO in the phloem companion cells in leaves (An, H.
et al. 2004). Stem grafting experiments reveal that once FT protein accumulates in the leaves
during LD conditions, it is transported through the vascular tissue to the SAM, were it interacts
with the bZIP transcription factor FLOWERING LOCUS D (FD) (Corbesier, L. et al. 2007; Lin,
MK. et al. 2007; Abe, M. et al. 2005). Together, the FT-FD heterodimer is observed to activate
the FMI genes APETALA 1 (AP1) and FRUITFUL (FUL) (Wigge, PA. et al. 2005; Abe, M. et al.
11
2005). Interestingly, some recent work renews the controversial possibility that FT mRNA is
also transported to the SAM and that the mRNA has an effect on floral promotion despite
previous evidence to the contrary (Lu, HK. et al. 2012).
Characterization of floral repressor TERMINAL FLOWER 1
In plants proper initiation or repression of floral transition requires complex input from
many environmental and internal stimuli and culminate in a single integrated signal. TERMINAL
FLOWER1 (TFL1) is observed to have control over both floral transition and SAM morphology
(Shannon, S. and Meeks-Wagner, DR. 1991; Alvarez, J. et al. 1992). Loss-of-function mutations
in TFL1 result in early floral transition and the appearance of terminal flowers at shoot apices,
marking a shift from indeterminate to determinate growth (Simon, R. et al. 1996; Bradley, D. et
al. 1997). Conversely, overexpression of TFL1 results in an extension in vegetative development
and a delay in transition from inflorescence meristem (IM) to floral meristem (FM), which
produces many secondary flowers and bract-like leaves (Ratcliffe, OJ. et al. 1998; Hanzawa, Y.
et al. 2005). These experiments indicate that TFL1 is a repressor of floral transition, and controls
SAM morphology.
TFL1 is a member of the CETS (CENTRORADIALIS, TFL1, and SELF PRUNING) gene
family and displays homology with phosphatidylethanolamine binding proteins (PEBPs). PEBPs
are a widely conserved protein family plants (Bradley, D. et al. 1997), insects (Pikielny, CW. et
al. 1994), and humans (Tohdoh, N. et al. 1995). In humans, it is indicated that PEBPs bind lipids
and inhibit serine proteases (Hengst, U. et al. 2001), and play an important role in hippocampus
development in the brain (Tohdoh, N. et al. 1995) In plants, PEBPs participate in both repression
12
and induction of floral transition in addition to determining plant architecture (Bradley, D. et al.
1997; Kardailsky, I. et al. 1999; Kobayashi, Y. et al. 1999).
TFL1 demonstrates 55% amino acid identity to FT, another member of the CETS family
(Pnueli, L. et al. 2001). Furthermore, both TFL1 and FT have been reported to act through the
bZIP transcription factor, FD, in order to regulate downstream meristem identity genes (Abe, M.
et al. 2005; Wigge, PA. et al. 2005; Hanano, S. and Goto, K. 2011). Despite the similarity
between the two proteins, TFL1 and FT have opposing effects on floral transition with FT
promoting and TFL1 repressing reproductive phase change. The difference in function for each
protein can be traced back to a single amino acid, histidine-88 in TFL1 and tyrosine-85 in FT.
Exchanging these amino acids will result in TFL1 inducing early flowering, while FT induces
delayed flowering (Hanzawa, Y. et al. 2005). Moreover, TFL1 has been recently indicated in
having a role in protein trafficking to protein storage vacuoles (Sohn, EJ. et al. 2007). It is
proposed that TFL1 possibly shuttles FD protein to storage vacuoles to repress floral transition,
and that FT then extracts FD from the vacuoles to promote floral transition (Hanano, S. and
Goto, K. 2011).
1.3 Photoperiodic flowering in the short day model species Oryza sativa
Recent progress in study of the short-day flowering mechanism in rice clarified similarity
and difference of molecular mechanisms underlying short-day flowering in rice and long-day
flowering in Arabidopsis. It is observed that the GI-CO-FT pathway, key to photoperiod and
flowering in Arabidopsis, is conserved in rice (reviewed by Tsuji, H. et al. 2010). As seen in
Arabidopsis, the GIGANTEA ortholog OsGI is controlled by the circadian clock (Hayama, R. et
al. 2002). OsGI protein accumulates in the evening hours and leads to the increased expression
13
of Heading date 1 (Hd1), the rice CONSTANS ortholog (Hayama, R. et al. 2002). Accumulation
of Hd1 protein then facilitates expression of the FT ortholog, Heading date 3a (Hd3a) (Hayama,
R. et al. 2003; Jang, S. et al. 2008) (Figure 5A). Unlike in Arabidopsis however, Hd1 protein
does not accumulate in the late evening, but rather during the night hours (Hayama, R. et al.
2003). Furthermore, this accumulation does not coincide with an increase in Hd3a expression,
which peaks in the morning (Hayama, R. et al. 2003), marking a notable deviation from the LD
Arabidopsis model.
The exact mechanism by which Hd1 upregulates Hd3a under SD conditions is not well
understood, however, night-break experiments indicate the role of Phytochrome B (Ishikawa, R.
et al. 2005) in Hd3a repression. Additionally, Early heading date1 (Ehd1), a B-typeS response
regulator protein, has been revealed as an important activator of Hd3a expression under SD
conditions (Doi, K. et al. 2004). Ehd1 is of particular interest in that it does not contain a
functional ortholog in Arabidopsis, and creating a distinct gene network (Doi, K. et al. 2004).
Ehd1 expression is upregulated by OsMADS51 (Kim, SL. et al. 2007) and Ehd2 (Matsubara, K.
et al. 2008). Furthermore, OsGI upregulates Ehd1 through both promoting the expression of
OsMADS51 (Kim, SL. et al. 2007), and through control of blue light signaling in the morning
(Itoh, H. et al. 2010).
Under LD conditions, flowering in rice is delayed through repression of Hd3a (Komiya,
R. et al. 2008) (Figure 5B). Interestingly, the source of this LD repression comes from Hd1
(Hayama, R. et al. 2003), which has been shown to act as a promoter of Hd3a expression under
SD conditions. It is demonstrated that this reversal in Hd1 activity is controlled by phytochrome
signaling (Izawa, T. et al 2002). Moreover, while Hd3a expression is reduced under LD
conditions, the gene RICE FLOWERING LOCUS T1 (RFT1), a paralog of Hd3a, is observed to
14
be upregulated under LD conditions (Komiya, R. et al. 2009). RFT1 expression is induced
through the action of OsMADS50, an ortholog of SOC1, and Ehd1 (Lee, SY. et al. 2004). This
illustrates that the core floral components observed in Arabidopsis, GI-CO-FT, are conserved in
both long day and short day plants. However, the molecular action of each component can vary
greatly depending on the plant.
1.4 Flowering in soybean
Soybean is an important oilseed legume that has major uses in human consumption,
animal feed, and industrial processes. Soybean is grown over an extended range of latitudes, but
individual cultivars are limited to specific geographical regions defined by photoperiod
requirements for flowering. This wide array of variance observed in flowering conditions can be
linked to the natural evolution in several major genes and quantitative trait loci (QTL). Currently,
nine major QTLs have been identified that have control over flowering and maturity in soybean,
the eight E-maturity loci (E1-E8) and J (Bernard, R. 1971; Buzzell, RI. 1971; Buzzell, RI. and
Voldeng, H. 1980; McBlain, BA. and Bernard, R. 1987; Bonato, ER. and Vello, NA. 1999;
Cober, E. and Voldeng, H. 2001; Cober, E. et al. 2010; Ray, J. et al. 1995).
In general soybean cultivars have a SD requirement for initiation of floral transition,
which is repressed under LD conditions. With the exception of E6 and J, in which the recessive
alleles delay flowering, the dominant alleles of all the E-loci confer a late flowering phenotype
(Bonato, ER. and Vello, NA. 1999, Ray, J. et al. 1995). Furthermore, as described in a review by
(Watanabe, S. et al, 2012), only E1, E3, E4, and E7 have shown sensitivity to photoperiod. E3
causes a delay in flowering under LD with a variety of light conditions (Cober, E. et al. 1996).
E1, E4, and E7 induce late flowering under LD only with red and far red light (R:FR) (Cober, E.
and Voldeng, H. 2001; Cober, E. et al. 1996), with E1 demonstrating the strongest inhibitory
15
effect (Thakare, D. et al. 2010). E4 and E3 have been identified as homologs to the Arabidopsis
photoreceptor PHYTOCHROME A (PHYA), GmphyA2 and GmphyA3 respectively (Liu, B. et al.
2008; Watanabe, S. et al. 2009). However, only E4 seems to regulate the de-etiolation (greening)
response under constant R:FR conditions as is observed in Arabidopsis PHYA (Liu, B. et al.
2008). GmCRY1a, an ortholog of the Arabidopsis photoreceptor CRYPTOCHROME1 (CRY1), is
observed to promote floral initiation, and exhibits a rhythmic expression pattern connected with
both photoperiodic flowering and latitudinal cline (Zhang, Q. et al. 2008).
Additionally, E2 has been identified as an ortholog to the Arabidopsis GIGANTEA (GI)
gene (Watanabe, S. et al. 2011), and at least ten orthologs to Arabidopsis FLOWERING LOCUS
T (FT) have been isolated (Kong, FJ. et al. 2010). These FT orthologs come in five sets of two,
and only GmFT2a and GmFT5a demonstrate increased expression under SD and decreased
expression under LD (Kong, FJ. et al. 2010, Thakare, D. et al. 2010). When expressed in
Arabidopsis coupled to a CaMv35S overexpression promoter, both GmFT2a and GmFT5a have
produced an early flowering phenotype similar to that of overexpressed Arabidopsis FT. These
results support the hypothesis that GmFT2a and GmFT5a are inducers of floral transition in
soybean (Kong, FJ. et al. 2010, Thakare, D. et al. 2011). Furthermore, both GmFT2a and
GmFT5a expression is affected by the PHYA orthologs E3 and E4, similar to the flowering
pathway in Arabidopsis (Kong, FJ. et al. 2010). However, unlike in Arabidopsis and other model
plants like rice and pea (Takano, M. et al. 2005, Weller, JL. et al. 2001), E3 and E4 PHYA
orthologs repress the expression of the FT orthologs, suggesting that the functions of
photoreceptors can vary widely in plants. E1 has been isolated to a 17.4kb region on
chromosome 6 (Xia, ZJ. et al. 2012). It is observed that mutations in E1 lead to increased
expression of both FT orthologs (Thakare, D. et al. 2011), but mutations in E2 (GmGIa) result in
16
an increase in GmFT2a but not GmFT5a (Kong, FJ. et al. 2010), suggesting the FT orthologs
fulfill different roles in floral initiation.
Soybean possesses two homologs to the Arabidopsis myb transcription factors LHY and/
or CCA1 (Liu, H. et al. 2009) that act in the circadian cycle as core oscillators. In soybean, these
genes exhibit rhythmic expression consistent with Arabidopsis LHY and CCA1 (Liu, H. et al.
2009), but their function in soybean flowering is as of yet uncharacterized. An ortholog of the
Arabidopsis floral repressor, TERMINAL FLOWER 1 (TFL1) is the gene underlying the soybean
determinate stem (Dt1) locus (Liu, B. et al 2010; Tian, ZX. et al. 2010). Eight orthologs of
Arabidopsis CONSTANS (CO) were identified in soybean and their mRNA expression was
characterized (Kim, M. et al. 2012). In Arabidopsis, CO expression is a key determinate of floral
transition. While CO’s role in soybean is presently unknown, the conserved function of CO in
other plant species makes it an attractive choice for functional characterization. There are an
ortholog of CYCLING DOF FACTOR 1 (CDF1) and six orthologs of FLAVIN-BINDING KELCH
REPEAT F-BOX PROTEIN1 (FKF1) that repress and stabilize CO transcription, respectively, in
Arabidopsis (Imaizumi, T. et al. 2005; Fornara, F. et al. 2009).
Further flowering genes have been identified through comparative genomic studies
between soybean, Arabidopsis, and other plant species (Jung, CH. et al. 2012, Hecht, V. et al.
2005; Ehrenreich, IM. et al. 2009). These studies reveal that soybean shares a high number of
orthologs with Arabidopsis flowering genes, and provides future research ample avenues of
inquiry to further characterize flowering in soybean.
17
1.5 Tables
Primer name Sequence 5`-3` Tm (°C)
TIL3 (AT1G05630)-S ATGGATTCGCTAATTATCGA 58.6 TIL3 (AT1G05630)-A CCGGCTTTTACCTCGTCTAA 62.9
TIL3end (813bp)-S TGGTGTGACCGAGTTATATAT 56 TIL3end (813bp)-A TCACCGGCTTTTACCTCGTCT 62.9
TIL3L1 (AT2G31830)-S ATGGATTCGGTAATTATTGAAC 58.3 TIL3L1 (AT2G31830)-A CTGTACCCTGAAGAACAGTCTCA 62.2
TIL3L2 ( AT2G43900)-S ATGGATATCATCAACAACAACCAC 63.4 TIL3L2 ( AT2G43900)-A ACGTTTGTTGTGGTTATTCCG 63.4
TFL1 (AT5G03840)-S ATGGAGAATATGGGAACTAGAGT 58.5 TFL1 (AT5G03840)-A GCGTTTGCGTGCAGCGGTTTC 75.6
TIL3 BiFC N-ter-S GGAGTCGACGGATGGATTCGCTAATT 72.8 TIL3 BiFC N-ter-A AAACCCGGGTCACCGGCTTTTACCTCG 78.8
TIL3 BiFC C-ter-S GGGGTCGACATGGATTCGCTA ATT 71.2 TIL3 BiFC C-ter-A AATCCCGGGCCCCGGCTTTTACCTCG 81.4
TFL1 BiFC N-ter-S GTCA AGCTTCATGGAGAATATGGGA 67.9 TFL1 BiFC N-ter-A AAACCCGGGGCGTTTGCGTGCAGC 8.6
TFL1 BiFC C-ter-S GGCAAGCTTCGTT GAGAATATGGGA 73.6 TFL1 BiFC C-ter-A AATCCCGGGCCCTAGCGTTTGCGTGC 82.1
TIL3end BiFC Ntag_S GGAGTCGACGGATGTGGTGTGACCGAGTT 79.5
TIL3end BiFC Ntag_A AAACCCGGGCCACCGGCTTTTACCTCG 81.2
TIL3end BiFC Ctag_S GGGGTCGACTGGTGTGACCGAGTT 75 TIL3end BiFC Ctag_A AATCCCGGGCCTCACCGGCTTTTACC 79
Table 1. List of primers showing primer name (and TAIR IDs for gene specific primers), 5`-3`
sequence, and melting temperatures (Tm (°C)). (S) indicates a sense primer and a (A) indicates an anti-sense primers. Green, blue and red sequences represent a SalI, XmaI, and HindIII restriction digest sites respectively.
18
Vector Promoter Tags/ Fusions Bacterial Selection Plant Selection Type Supplier
pCR 2.1/TOPO T7 Kan/ Amp Cloning Invitrogen
pCR8/GW/TOPO T7 Spec Entry Vector Invitrogen
pH7RWG2 35S: C tagged-RFP Spec Hyg Destination UCSD
pH7WGR2 35S: N tagged-RFP Spec Hyg Destination UCSD
pMDC43 2x 35S: N tagged-GFP6 Kan/ Chlor Hyg Destination ABRC
pSAT1-nVenus-C 2x 35S: C tagged-nVenus Amp Multi-color BiFC ABRC
pSAT1-nVenus-N 2x 35S: N tagged-nVenus Amp Multi-color BiFC ABRC
pSAT1-nCerulean-N 2x 35S: N tagged -nCerulean Amp Multi-color BiFC ABRC
pSAT1-nCerulean-C 2x 35S: C tagged -nCerulean Amp Multi-color BiFC ABRC
pSAT1-cCFP-N 2x 35S: N tagged-cCFP Amp Multi-color BiFC ABRC
pSAT1-cCFP-C 2x 35S: C tagged-cCFP Amp Multi-color BiFC ABRC
pSAT5-DEST-c(175-end)EYFP-C1 2x 35S: C tagged-cEYFP Amp/ Chlor Gateway BiFC ABRC
pSAT5A-DEST-c(175-end)EYFP-N1 2x 35S: N tagged-cEYFP Amp/ Chlor Gateway BiFC ABRC
pSAT4-DEST-n(1-174)EYFP-C1 2x 35S: C tagged-nEYFP Amp/ Chlor Gateway BiFC ABRC
pSAT4A-DEST-n(1-174)EYFP-N1 2x 35S: N tagged-nEYFP Amp/ Chlor Gateway BiFC ABRC
Table 2. List of vectors show vector name, type of promoter the vector contains, the position of any protein fusion tags, the bacterial
and plant selection markers, type of vector, and construct supplier. (Marker abbreviations: Kan= kanamycin, Amp=ampicillin,
Chlor= chloramphenicol, Spec= spectinomycin, Hyg= hygromycin). (Supplier abbreviations: UCSD= University of California San
Diego-Tsien lab, ABRC= Arabidopsis Biological Resource Center).
19
1.6 Figures
Figure 1. Floral induction pathways in the LD model plant, Arabidopsis. Pathways with colored
boxes denote environmental input pathway, pathways with black boxes denote endogenous input
pathways.
20
Figure 2. Vernalization and ambient temperature pathways in Arabidopsis.
21
Figure 3. Autonomous Pathway and PAF1 histone remodeling complex in Arabidopsis.
22
Figure 4. Circadian clock and photoperiod inputs facilitating or inhibiting floral initiation.
23
Figure 5. Simplified diagram of the floral initiation pathway in rice under (A) short day, and (B) long day conditions
The red, orange and yellow boxes indicate the conserved GI-CO-FT flowering motif characterized in Arabidopsis.
24
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CHAPTER 2
Molecular characterization of signaling mechanisms in flowering transition in
Arabidopsis
2.1 Abstract
TERMINAL FLOWER 1 (TFL1) is a flowering repressor that controls the activity of the
shoot apical meristem (SAM), however, the molecular action of TFL1 is unknown. To better
understand the nature of TFL1 molecular action, we identified possible TFL1 interactors via
yeast two hybrid screening. Nine such interactors were identified, and were named TFL1-IN-
LOVE (TIL).
TIL3 showed specific binding to TFL1 at a unique TFL1 residue. TIL3 is a member of a
small gene family that encodes inositol polyphosphate 5-phosphatase (5PTase) and contains WD-
40 repeats. Of the members in this family, TIL3-Like1 (TIL3L1) and TIL3-Like2 (TIL3L2) share
the highest homology to TIL3. To clarify the role of TIL3, TIL3L1 and TIL3L2 in the TFL1
pathway, T-DNA insertion mutants were identified and crossed with transgenic plants carrying
35S:TFL1. til3-1, til3l1-1 and til3l2-1 mutants were seen to suppress the late flowering
phenotype generated by 35S::TFL1. Moreover, both overexpressed 35S:TFL1 and mutant tfl1-1
display increased sensitivity to gravity in root gravitropism similar to til3-1 mutants.
We created transgenic Arabidopsis plants carrying 35S:GFP:TFL1 and observed both
nuclear and cytoplasmic localization in root tissue using fluorescent microscopy. Supporting our
observation in the TFL1 localization and interaction with TIL3, transient expression of
35S:GFP:TFL1 and 35S:GFP:TIL3 in Nicotiana benthamiana leaf tissue exhibited co-
localization for TFL1 and TIL3 proteins in the nucleus and cytoplasm. Furthermore, transient
42
bimolecular fluorescent complementation (BiFC) experiments in N. benthamiana confirmed in
vivo protein-protein interaction between TFL1 and TIL3 in both the nucleus and cytoplasm,
supporting the hypothesis that TIL3 affects TFL1 floral repression.
2.2 Background
It is known that floral transition in Arabidopsis is controlled by multiple genetic
pathways that perceive and transmit the input of environmental and endogenous signals.
Environmental signals include photoperiod and light quality that are processed by the
photoperiod pathway (Suarez-Lopez, P. et al. 2001; Mizoguchi, T. et al. 2005), and temperature
(Wellensiek, SJ. 1964; Sheldon, CC. et al. 1999; Searle, I. et al. 2006) that is processed by the
vernalization pathway and the ambient temperature pathway. Endogenous flowering cues include
Gibberellic Acid acting as a floral inducer (Langridge, J. et al. 1957; Chandler, D. and Dean, C.
1994; Domagalska, MA. et al. 2010), and are processed in the autonomous pathway, which
maintains vegetative growth until internal conditions are ideal (Poethig, RS. 1990; Poethig, RS.
2010; Amasino, R. M. 2010). These pathways converge at the floral integrators FLOWERING
LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), which
induce meristem identity genes that are responsible for the transition from vegetative to
reproductive organ development (Abe, M. et al. 2005; Liu, J. et al. 2008a).
A key inhibitor of floral transition is TERMINAL FLOWER 1 (TFL1), which suppresses
the expression of meristem identity genes (Liljegren, S. et al 1999; Abe, M. et al. 2005) in
Arabidopsis. The function of TFL1 is conserved in many flowering plant species such as
Determinate stem1 (Dt1) in soybean (Tian, ZX. et al. 2010), ZEA CENTRORADIALIS1 (ZCN1)
in maize (Danilevskaya, ON. et al. 2010), and RCN1 and RCN2 in rice (Nakagawa, M. et al.
43
2002). It is then interesting that TFL1 reveals itself as a homolog of the florigen FT (Pnueli, L. et
al. 2001), but displays the opposite phenotypic effect (Hanano, S. and Goto, K. 2011).
It is proposed both TFL1 and FT act through the DNA binding protein FD (Abe, M. et al.
2005; Wigge, PA. et al. 2005; Hanano, S. and Goto, K. 2011), though the exact mechanism of
these interactions and signaling is unknown. It is possible that FT and TFL1 interact with FD at
the promoter regions of meristem identity to induce or repress floral initiation respectively
(Wigge, PA. et al. 2005; Ahn, JH. et al. 2006). Another hypothesis is that TFL1 represses
flowering through sequestering FD to the vacuoles, and FT induces flowering through retrieving
FD from the vacuoles and inducing floral meristem identity genes (Hanano, S. and Goto, K.
2011). Additional experimentation is necessary to determine the molecular action of FT and
TFL1.
Characterization of the amino acid sequence of FT and TFL1 reveals that the difference
in function for each protein can be traced back to a single amino acid, His-88 for TFL1 and Tyr-
85 for FT (Hanzawa, Y. et al. 2005). Exchanging these amino acids will result in TFL1 inducing
early flowering and FT delaying flowering (Hanzawa, Y. et al. 2005; Ahn, JH. et al. 2006). To
clarify the action of this key amino acid in TFL1, yeast-two hybrid experiments were conducted
to identify proteins that interact with TFL1 in a His-88 specific manner (Hanzawa, Y.
unpublished). Several classes of interactors were isolated and named TFL1-IN-LOVEs (TILs).
Of these TILs, only TIL3 was shown to interact with TFL1 specifically at His-88 (Hanzawa, Y.
unpublished). Further analysis reveals that TIL3 encodes inositol polyphosphate 5-phosphotase
13 (5PTase13). TIL3/5PTase13 is a member of the lipid signaling pathway (Astle, MV. et al.
2006) that affects a wide variety of downstream responses such as auxin levels (Lin, WH. et al.
2005), sucrose metabolism (Ananieva, S. et al. 2009), blue light signaling (Chen, X. et al. 2008),
44
and vesicle trafficking (Wang, Y. et al. 2009). The function of TIL3/5PTase13 in flowering is
currently uncharacterized.
In plants, the inositol polyphosphate 5-phosphatase (5PTase) family composes a large
group of enzymes that cleave 5-phophatates from a wide array of polyphosphate inositol (PPI)
substrates (as reviewed by Munnik, T. et al. 2010; Astle, MV. et al. 2006). In Arabidopsis, there
are sixteen 5PTases that can be divided into Group A and Group B according to function and
similarity. Group A contains At5PTases 1 - 11 and Group B contains At5PTases 12 - 14 and
FRAGILE FIBER3 (FRA3) (Berdy, SE. et al. 2001). Both Group A and B contain 5-phosphatase
regions, but only Group B 5PTases contain WD-40 repeats, which are often considered protein
interaction sites (Zhong, R. and Ye, ZH. 2004). Group A At5PTases 1 and 2 were characterized
first as being able to hydrolyze 5-phosphates from inositol 1,4,5-trisphosphate (InsP3) which
leads to altered levels of abscisic acid (ABA) (Burnette, R. et al. 2003; Sanchez, J. and Chua, N.
2001) and is shown to regulate seed growth. At5PTase 5 and 6 are involved in root hair initiation
(Jones, MA. et al. 2006) and cotyledon vasculature (Carland, FM. and Nelson, T. 2004),
respectively, through InsP3 5-phophate hydrolysis (unpublished data Gillaspy, G. referenced in
Munnik, T. et al. 2010).
Of the Group B At5PTases, only FRA3 and 5Ptase13 are currently characterized in their
functions in various aspects of plant development and signaling. FRA3 is expressed in stem
tissue (Zhong, R. et al. 2004), and is suggested to regulate both InsP3 and phosphatidylinositol
4,5 bisphosphate (PtdInsP2) in a tissue specific manner (Zhong, R. et al. 2004). Mutations in
FRA3 include modified actin organization, a decrease in secondary cell wall thickness and
reduced stem vigor (Zhong, R. et al. 2004). At5PTase13 has been indicated in using both InsP3
(Zhong, R. and Ye, ZH. 2004) and InsP4 (Chen, X. et al. 2008) as substrates, and demonstrates
45
roles in multiple different signaling pathways, making it arguably the most versatile of the
5PTases. It regulates auxin levels (Lin, WH. et al. 2005), modulates sucrose in response to stress
through the SNF1-like Kinase (SnRK) (Ananieva, S. et al. 2009), controls blue light signaling
through calcium fluctuation (Chen, X. et al. 2008), monitors hypocotyl growth (Chen, X. et al.
2008), and is involved in root gravitropism through vesicle trafficking (Wang, Y. et al. 2009).
The substrates of 5PTases, membrane bound phospholipids and soluble inositol
phosphates, are found commonly in all eukaryotic cells, controlling many cellular functions
(Martin, TFJ. 1998; Cockcroft, S. and De Matteis, MA. 2001; Di Paolo, G. and De Camilli, P.
2006). In the phosphatidylinositol pathway, the membrane-associated phospholipid,
phosphatidylinositol (PtdIns), is phosphorylated by lipid kinases to produce phosphatidylinositol
4-phosphate (PtdInsP) and PtdInsP2. Under specific stress conditions, PtdInsP2 becomes cleaved
by PHOSPHOLIPASE C (PLC), and the cellular secondary messengers, inositol 1,4,5-
trisphosphate (InsP3) and diacylglycerol are released (Berridge, MJ. 1993). Once a sufficient
cellular response has been attained, 5PTases then hydrolyze the 5-phophate of these secondary
messengers as described above, effectively eliminating the signal. This system creates an ideal
connection between membrane sensing and signal propagation to induce specific regulatory
programs. For example, IP3 has been characterized as a key intermediate to plasmodesmata
opening and closing, facilitating cell-to-cell communication (Tucker, E. and Boss, W. 1996),
therefore 5PTase13 degradation of IP3 directly affects cell-to-cell communication (Perera, IY. et
al. 2006). Furthermore, 5PTase13 has been shown to maintain diurnal calcium levels (Tang, RH.
et al. 2007).
Here I report characterization of the role of TIL3/5PTase13 in signaling mechanisms in
Arabidopsis flowering transition. Both overexpressed TFL1 and mutant tfl1 are revealed to have
46
increased sensitivity to root gravitropism similar to TIL3/5PTase13. Furthermore, til3-1/5ptase-1
tfl1-1 double mutants do not have an additive affect in root gravitropism, suggesting that they act
in the same pathway. Mutations in til3-1/5ptase-1 significantly reduce the late flowering
phenotype of overexpressed TFL1, thus establishing a functional link between TFL1 and
TIL3/5PTase13 in floral regulation. Finally, transient bimolecular complementation experiments
in Nicotiana benthamiana demonstrate that TFL1 and TIL3/5PTase13 proteins physically
interact in both the nucleus and cytoplasm of live cells.
2.3 Materials and methods
Polymerase chain reaction
The polymerase chain reaction (PCR) solution used in most experiments was compose of
1 l of DNA template (1g), 2 l 10x PCR buffer (New England Bio Labs), 1 l of dNTPs (2.5
mM), 0.5 l of both forward and reverse primers (Table 1) (10 M), 0.1 l Taq polymerase (New
England Bio Labs), and 14.9 l of water. This mixture was the placed in the Eppendorf
Mastercycler® pro for automated thermal cycling.
The following conditions were used for PCR reactions. The initialization step heated
samples to 94° C for 2 minutes, after which point the cycles began. The first step was DNA
denaturing at 94° C for 20 seconds. The primer annealing step followed with temperatures
ranging from 55-71° C depending on the primer (Table 1) for 45 seconds. Finally, the elongation
phase proceeded at 68° C with variable times, all of which adhered to the rule of 1 minute of
elongation per 1 kb of amplified DNA. These three steps were repeated for a total of 32 cycles. A
final elongation occurred after this time for 5 minutes at 68° C, with an indefinite holding step of
47
4° C at the end of the PCR reaction. The PCR product was then immediately used for gel
electrophoresis or stored at -20° C.
Gel electrophoresis
DNA products from PCR or restriction digest were visualized using gel electrophoresis to
confirm both the presence and length of the desired DNA fragment. Gel electrophoresis
accomplishes this through generating an electric field through an agarose matrix, which results in
a DNA gradient based on size. The first step was to create an agarose gel matrix. A 1% gel is
created through mixing 1 g of agarose per 100 mL of 1x Tris-acetate-EDTA (TAE) solution. This
solution is then heated to melt (MP= 85° C) and homogenize the agarose with the TAE. When
the mixture becomes clear, the agarose is completely melted. 3 l of ethidium bromide (EtBr)
(10 mg/mL), which aids in DNA visualization by intercalating with DNA and fluorescing under
UV light, was then added to the molten mixture. The result was then pored into a gel mold with a
well comb. The gel is allowed to cool for 30 minutes until it completely hardens.
Once this occurs, a Thermo EC 105 Classic™ electrophoresis tank is filled with 1x TAE
with trace amounts of EtBr (5 l per use). The gel is placed in the tank, and 2 l of bromophenol
DNA marker is added to each DNA sample with subsequent agitation. The DNA products along
with a Quick-Load® 2-Log DNA ladder (New England Bio Labs) are added to their own
individual wells in the gel. The gel then proceeds at 130 V until the DNA bands are separated.
After completion the gel is imaged with UV light in a Gel Doc™ XR+ (BIO-RAD) using the
program Image Lab™.
48
Cloning
PCR products were first cloned into the Original TA cloning® Kit pCR2.1® vector
(Invitrogen) for both amplification (see E-coli transformation) and sequence fidelity checks (see
DNA sequencing). pCR2.1® is a linearized 3.9 kb vector carrying both ampicillin and kanamycin
antibiotic resistance. The insert site contains 3’ poly-T overhangs and is flanked by M13 forward
and reverse primers, embedded within a LacZα coding region. The DNA fragment to be cloned
must have a poly-A over hang at its 5’ ends for successful ligation with pCR2.1®. To produce a
poly-A over hang, a 20 l PCR sample is mixed with a solution of 3 l 10x PCR buffer-mg2+, 0.5
l dNTPs (2.5 mM), 1 l MgCl (50 mM) and 0.2 l Native Taq polymerase (New England Bio
Labs) and run on an Eppendorf Mastercycler® pro at 72° C for 10 minutes. Once the DNA
fragment has the necessary 5’ poly-A ends, 6 l are mixed in a ligation solution containing 1 l
ligation buffer, 2 l pCR2.1® vector, and 1 l T4 ligase. This mixture is incubated at 4°C
overnight, after which it is transformed into competent bacterial cells (see E-coli transformation).
Bacterial transformation
For bacterial transformation the heat shock method with either Subcloning Efficiency™
DH5α™ (Invitrogen) competent cells or One Shot®TOP10 chemically competent E-coli
(Invitrogen) cells were used. 25 l aliquots of competent cells (109 cells/mL) were removed from
-80° C storage and added to 10 l of concentrated DNA plasmid. The mixture was briefly
agitated and left on ice for 30 minutes. After this interval, the mixture is heat shocked for 30
seconds at 42° C on an aluminum heating block. The sample is then returned to the ice for 60
seconds. 250 l of Super Optimal broth with Catabolite repression (SOC) (Invitrogen) is added
to the culture, followed by a 1 hour incubation at 37° C. Once the incubation is complete the
49
resulting culture is spread in 50 l and 250 l allotments onto two LB-ager plates (1.5% agar)
containing the appropriate antibiotics. The plates are left overnight in a dark 37° C growth
chamber. Once colonies appeared, single colonies are picked and incubated in 100 l of LB
media with the appropriate antibiotics for 2 hours at 37° C with constant agitation (200 rpm).
The resulting culture was analyzed for the presence of the plasmid-insert by polymerase chain
reaction (PCR) (see section “Polymerase chain reaction” for cycle details). Upon confirmation of
plasmid insert, an additional 2 mL of LB-broth plus antibiotic is added to the single colony
culture and incubated overnight for at 37° C with constant agitation (200 rpm). The culture is
now at a sufficient bacterial concentration to extract large amounts of plasmid DNA (see plasmid
purification).
Plasmid purification
Plasmid purification is accomplished using the QIAprep® Spin Miniprep kit (QIAGEN)
which includes all the reagents necessary for the process. Purification began by centrifuging 2
mL of bacterial culture containing a plasmid of interest in an Eppendorf 5415D® centrifuge. The
resulting bacterial pellet is kept and the liquid media is discarded. The pellet is resuspended in a
1.5 mL microcentrifuge tube with 250 l of P1 buffer, which contains RNase-A to degrade any
RNA present, and LyseBlue® which turns the solution blue upon successful cell lysis. Once fully
resuspended, 250 l of P2 lysis buffer is added. The mixture should be inverted 4-6 times to
ensure a thorough reaction, which can be observed as a transition from a white cloudy solution to
a clear blue solution. Immediately after the solution turns completely blue, 350 l of N3
neutralization buffer is added to halt the lysis reaction, which will cause the solution to turn
50
colorless and produce a white precipitate. This white precipitate is primarily cell lipids, which
are separated from the plasmid DNA by centrifuging the sample at 13,000 rpm for 10 minutes.
A white pellet will form at the bottom of the microcentrifuge tube. Being careful not to
disturb this pellet, the supernatant is pipetted from the tube and added to a QIAprep® spin
column. Using an Eppendorf 5415D® centrifuge, spin the column at 13,000 rpm for 60 seconds.
This spin step binds the plasmid DNA to the column silica-gel membrane. The supernatant from
the spin is discarded and 500 l of PB wash buffer is added to the column to remove residual
endonucleases from the membrane. The column is spun again at 13,000 rpm for 60 seconds and
the supernatant is discarded. 750 l of the PE wash buffer is added to the column, which
effectively removes salts from the membrane. The column is spun at 13,000 rpm for 60 seconds
and the supernatant is discarded, followed by an additional 1 minute spin at 13,000 rpm to
remove any lingering PE buffer. The QIAprep® column is then inserted into a clean 1.5 mL
microcentrifuge tube, and 50 l of elution buffer is added directly to the membrane surface. The
solution is allowed to soak into the membrane for one minute, and then centrifuged at 13,000
rpm for 60 second. The resulting supernatant will contain the desired purified plasmid.
DNA sequencing
Cloned PCR fragments in pCR2.1® were sequenced to check for mutations. 5 l of
purified plasmid DNA samples (10 ng/l) were submitted to the Core Sequencing Facility at the
University of Illinois at Champaign-Urbana for sequencing. DNA sequences were aligned using
the on-line resource CLUSTALW (http://www.genome.jp/tools/clustalw/) (Thompson, JD. et al.
1994).
51
Gateway™ subcloning
If a DNA fragment does not contain mutations after being sequenced in pCR2.1®, it is
subcloned into Gateway™ (Invitrogen) entry and destination vectors. This system allows for the
rapid characterization of protein expression and gene function by using site specific
recombination as opposed to restriction enzymes and ligase to easily transfer DNA fragments
from one vector to another. The first half to this process involves the Entry Clone
pCR®8/GW/TOPO® (Invitrogen). pCR®8 is a 2.8 kb vector carrying spectinomycin antibiotic
resistance. The insert site contains 3` poly-T overhangs and most importantly is flanked by attL1
and attL2 recombination sites, which allow for future recombination with any Gateway™
destination vector.
The first step taken to introduce a DNA fragment was to amplify the DNA fragment from
pCR2.1® using the high fidelity Platinum® Pfx DNA polymerase (Invitrogen) in PCR. The
Platinum® Pfx PCR solution consisted of 10 l 10x Pfx amplification buffer, 1.5 l dNTPs (2.5
mM), 1 l MgSO4 (50 mM), 1.5 l of both forward and reverse primers (10 M), 1 l DNA
template, 0.4 l of Platinum® Pfx DNA polymerase, and up to 50 l of water. Subsequent PCR
cycle information and electrophoresis were as outlined above.
After DNA fragment amplification, 5` poly-A over hangs are added by mixing the DNA
with a solution of 3 l 10x PCR buffer-mg2+, 0.5 l dNTPs (2.5 mM), 1 l MgCl (50 mM) and
0.2 l Native Taq polymerase (New England Bio Labs) and running the mixture on an Eppendorf
Mastercycler® pro for 10 minutes at 72° C. Once poly-A tails are added, 4 l of DNA are mixed
with 1 l salt solution and 1 l of pCR®8 entry vector and incubated at 23° C for 10 minutes.
52
The resulting plasmid is then transformed into competent E-coli cells (see E-coli transformation)
to be amplified and subsequently purified for the transfer into a destination vector.
Once the entry clone with the DNA fragment has been amplified, it can undergo site-
specific recombination with a destination vector using the Gateway® LR Clonase II™ kit. The
reaction is composed of 3 l of pCR®8 Entry Clone, 1 l of a destination vector (Table 2) and 1
l of Clonase™ incubated at 23° C for 1 hour. After this time, 1 l Proteinase K is added to the
mixture and heated to 37° C for 10 minutes to halt the LR reaction. The product can now be
transformed into E-coli competent cells for amplification.
Arabidopsis agrobacterium transformation via floral dip
Agrobacterium tumefaciens strain GV3101 was used for all Arabidopsis transformations.
Arabidopsis ecotype Columbia-0 were sown in 5-inch pots at about 10 individual plants per pot
and grown under LD conditions (16 hrs of light) until all of the plants bolted. The bolted main
stem was cut near the rosette leaves to allow uniform growth of future stems and facilitate
increased stem branching to produce the maximum amount of flowers for the floral dip. 10 days
after the main stems were cut, the plants were used for agrobacterium transformation.
Agrobacterium strain GV3101 was transformed by a binary vector carrying a gene of
interest. 5-9 l of plasmid was added to 50 l of GV3101 competent cells into a 1.5 mL
microfuge tube and left on ice for 30 minutes. The sealed 1.5 mL tube was exposed to liquid
nitrogen for 1 minute and immediately moved to a 37° C water bath for 3 minutes. 1 mL of Luria
broth (LB) media (20 g of LB mixture per liter of water) was added to the 1.5 mL tube, followed
by a 2 hour culturing period at 28° C with constant agitation (about 200 rpm). After this
incubation, 50 l and 150 l of cell mixture was spread evenly onto two separate LB-ager plates
53
(1.5% agar) containing 80 g/mL gentamycin, 50 g/mL rifampicin as well as the appropriate
antibiotic resistance contained within the plasmid. The LB-agar plates were incubated in the dark
at 28° C for 2 - 3 days. Once colonies appeared, single colonies were picked and incubated in
100 l of LB media with the previously mentioned antibiotics for 2 hours at 28° C with constant
agitation (200 rpm). The resulting culture was analyzed for the presence of the plasmid-insert by
polymerase chain reaction (PCR) (see section “Polymerase chain reaction” for cycle details).
Once both the Arabidopsis pots and agrobacterium were ready, the transformation
proceeded. 0.6 mL of a GV3101 culture harboring the correct plasmid-insert was added to 30 mL
of LB media containing 80 g/mL gentamycin, 50 g/mL rifampicin as well as the appropriate
plasmid antibiotic and was incubated at 28° C overnight with constant agitation (200 rpm). The
next day 300 mL of LB media was inoculated with 6 mL (1:50) of the incubated culture. The 300
mL culture was then incubated overnight at 28° C with constant agitation (200 rpm). The
following day the agrobacterium cells were harvested in 250 mL FALCON® centrifuge flasks by
centrifugation at 4000 rpm for 15 minutes in the Eppendorf 5810R®. The resulting supernatant
was discarded and the bacteria pellet was resuspended in 250 mL of transformation solution,
prepared as described in Clough and Bent. 1998. The flowers of the Arabidopsis plants were then
dipped into the agrobacterium-transformation solution and held for 15 seconds. After all the
plants were dipped, they were covered and placed in the dark overnight. The plants were then
uncovered and grown under LD conditions until the seeds are matured and ready for harvesting.
Arabidopsis transgenic screening
Screening for transgenic Arabidopsis seeds has three steps. The first step is checking
generation one transformants (T1) for successful transgene insertion. T1 seeds are collected from
54
Arabidopsis plants that have undergone Agrobacterium transformation. Seeds from individual
plants are harvested by mechanically removing the mature (brown) siliques from the stem on a
square piece of paper. The harvested seeds and cell debris were poured through sieve so that only
seeds are collected. These seeds are then stored in a labeled paper coin pouch at room
temperature. From this bag, about 500 T1 seeds are removed and placed in a 1.5 mL tube. The
following process sterilizes the seeds: 800 l of 70% ethanol is added to the 1.5 mL tube, which
is then inverted 6-8 times to mix. After the seeds have settled at the bottom of the tube, the
ethanol solution is removed, and is replaced by 400 L of 20% bleach. The seeds remain in the
bleach solution for 15 minutes with mild mixing every 1 or 2 minutes. After the 15 minute
incubation the bleach solution is removed. 800 l of water is then added to the seeds, and the
tube is inverted 6-8 times. After the seeds settle at the bottom of the tube, the water is removed.
This water washing process is then repeated 2 additional times. After the third consecutive
washing step, 800 l of water is added to the seeds, and the tube is placed in a 4° C refrigerator
overnight to promote germination.
After the overnight 4° C incubation, the seeds are pipetted equally onto two 4 inch
diameter circles of filter paper under a sterilization hood and allowed to dry. While the seeds dry,
Murashige and Skoog (MS) plates are prepared (4.4 g/L MS basil media (Sigma Aldrich), 16 g/L
sucrose, 8 g/L agar, pH adjusted to 5.8 with 1 M KOH) with the appropriate antibiotics and cast
into 25 mm petri dishes (Fisher). Once the seeds are dry and the plates are solidified, the seeds
are spread evenly over two MS plates giving each plate about 250 seeds. The plates are then
placed in a LD growth chamber for 14 days. By this time successfully transformed seeds with
antibiotic resistance will be easily distinguishable from non-transformed seeds, which will be
mostly dead. These transformed T1 seedlings are then individually transferred to LC1 soil (Peat
55
moss: coarse perlite: dolomitic limestone: starter nutrients) in plastic insert wells. The seedlings
are then allowed to grow under LD conditions until T2 seeds are ready for harvest.
The second step of seed screening involves checking for multiple DNA insertions in T2
seeds. T2 seeds are produced from the self-fertilization of a T1 plant, which will be heterozygous
for the DNA insert introduced through agrobacterium transformation. Using simple Mendelian
genetics we can predict that T2 seeds have a 3 out of 4 chance of acquiring at least one DNA
insert conferring antibiotic resistance. This translates to about 75% of T2 seeds surviving
exposure to the antibiotic in question. Sometimes agrobacterium-mediated transformation inserts
multiple copies of DNA insert into a genome. If a F1 transgenic plant contains multiple inserts, it
will have a much higher survival rate (greater than 75%) in the T2 generation. Multiple gene
inserts are often undesired because they introduce additional variation into transgenic lines, so
single insert transgenic are selected for. To accomplish this T2 seeds are harvested from T1
plants and sterilized as described above. After sterilization, T2 seeds for each original T1 line are
placed one at a time in rows onto individual MS plates with the appropriate antibiotics. Each
plate contains about 60 seeds. These plates are grown in an LD chamber for about 10-14 days.
After this period seedlings are counted on each plate, and only those plates with a survival rate of
75% are chosen for continued selection. From each successfully selected T2 line, 9 seedlings
introduced into LC1 soil and grown under LD conditions until T3 seeds are ready for harvest.
The final step of transgenic seed selection involves identifying homozygous T3 lines.
Now it is known that the remaining lines have a single insert, it is necessary for the insert to be
present on both chromosomal pairs in the Arabidopsis diploid genome. T3 seeds that are
homozygous for the transformed insert will have a survival ratio of 100% on antibiotic plates.
Therefore, T3 seeds are harvested from T2 plants and sterilized as described above. After
56
sterilization, 9 sets of T3 seeds for each T2 line are placed one at a time in rows onto individual
MS plates with the appropriate antibiotics. Each plate contains about 60 seeds. These plates are
grown in an LD chamber for about 10-14 days. After this period seedlings are counted on each
plate, and only those plates with a survival rate of 100% are chosen. These homozygous T3 seeds
are now ready for experimentation.
Root gravitropism
Measuring root gravitropism involves observing the effect that gravity has on root
development and directionality. Normally, roots will always grow in the direction that gravity
pulls in, but there are some mutations in plants that lead to the reduced or heightened ability to
sense and respond to gravity. To measure root gravitropism in Arabidopsis, seedlings were
vertically grown in rows on square Murashige and Skoog (MS) plates under LD conditions for 9
days. For each Arabidopsis line 30 seedlings were randomly distributed among 6 designated
areas (top left, top right, middle left, middle right, bottom left, bottom right) on each plate. After
9 days, the plates were wrapped in foil to prevent any competing heliotropic effects and inverted
180° so that the roots would be oriented opposite the pull of gravity. Measurements of root
curvature were then measured at 4, 6, 8, and 10-hour intervals. Measurements were recorded by
rapidly taking pictures of the plates at each time point under white light in a Gel Doc™ XR+
(BIO-RAD) using the program Image Lab™. The images were then analyzed using the program
Image-J™. The root tips of each seedling were traced using the angle function producing
individual measurements of root curvature. These measurements were then input in Microsoft
Excel™ where they were organized and analyzed for averages, standard deviation, standard
error, and student’s t-test in comparison to wild type for each line.
57
Flowering time experiment
Flowering time can be measured in Arabidopsis by counting the number of rosette leaves
and cauline leaves at different developmental stages. Rosette leaves form around the base of the
plant before floral transition occurs, and the number of rosette leaves accumulated before bolting
(stem formation) is indicative of the amount of time spent in the vegetative stage. Therefore
rosette leaves were counted before bolting as a measure of time spent in the juvenile phase.
Likewise, the number of cauline leaves, which generally grow at branching nodes on the stem, is
indicative of the amount of time a plant has spent in the reproductive phase. Therefore, the
number of cauline leaves was counted after bolting on the primary stem to measure time spent in
the reproductive phase. Lines selected for Arabidopsis flowering experiments were randomly
assigned locations using the Excel™ “random function” on an 8-6 (8 sections consisting of 6
wells each) plastic insert tray filled with LC1 soil (Peat moss: coarse perlite: dolomitic
limestone: starter nutrients). Each line had between 25-30 wells. Once randomized, 3 seedling of
a particular line were sown in each well and grown under LD conditions for 14 days. After this
time, all but one of the seedlings were removed from each well in an effort to ensure all of the
plants were of similar sizes and growth stages before measurements are taken. Measurements
were taken in 2-3 day intervals and continued until leaf formation on the primary stem halted.
Measurements were recorded in Microsoft Excel™ and analyzed for averages, standard
deviation, standard error and students T-test.
Confocal microscopy
Confocal microscopy was conducted using a Zeiss™ LMS 700 Confocal Laser Scanning
Microscope at the Institute for Genomic Biology Core Facilities at the University of Illinois
58
Champaign-Urbana. The imaging program used during microscopy was the Zeiss™ ZEN
software. Post-imaging was accomplished using Image J™.
Arabidopsis root samples were grown vertically on square MS plates under LD
conditions for 9 days. The entire seedlings were then moved to a 22x50 mm (Corning) coverslip
and the roots were covered in 50 l of water to avoid drying out. A 22x50 mm (Corning) glass
slip was then placed on top of the water-covered roots, making sure not to trap any air bubbles
under the slide. The slide was then ready for imaging.
Nicotiana benthamiana leaf samples were taken 3 days after infiltration (see transient
expression in Nicotiana benthamiana). 1x2 cm tissue sections were excised from the infiltrated
leaves, and placed on a 22x50 mm (Corning) coverslip containing a 50 l bead of water. To
account for the increased thickness of the tissue section, two 22x50 mm (Corning) glass slips
were placed in positions flanking the tissue sample on the water drop, and a third 22x50 mm
(Corning) glass slip was placed above the flanking glass slips. This ensured that the leaf tissue
was encapsulated in water without being crushed. The slide was then ready for imaging.
For samples containing a GFP-fusion tag, the 488 nm excitation laser was used producing
a 509 nm emission. In addition to the 488 nm laser, the emission filter LP560 was used to block
any emissions above 560 nm from being captured, thus eliminating much, but not all, of the
auto-florescence in the leaf. For samples containing an RFP-fusion tag, the 555 nm laser was
used producing a 605 nm emission. For all images taken, Airy Units (AU) was always set to 1,
laser strength was anywhere between 3% and 80% power depending on the fluorescent signal
intensity, master gain was set at 850, and digital gain was set to 1.
59
Transient expression in Nicotiana benthamiana
Nicotiana benthamiana seeds were sown in 5-inch pots containing LC1 soil (Peat moss:
coarse perlite: dolomitic limestone: starter nutrients) and grown under long day conditions. Once
plants have mature leaves about 4 inches long, but have not yet begun flowering, they are ready
for agrobacterium infiltration.
Agrobacterium tumefaciens strain GV3101 was used for all N. benthamiana infiltrations.
Plasmid-insert (i.e. plasmids containing a desired DNA fragment) introduction into GV3101
competent cells was accomplished as follows. 5-9 l of plasmid was added to 50 l of GV3101
competent cells into a 1.5 mL microfuge tube and left on ice for 30 minutes. After which time the
sealed 1.5 mL tube was exposed to liquid nitrogen for 1 minute and immediately moved to a 37°
C water bath for 3 minutes. 1 mL of Luria broth (LB) media (20 g of LB mixture per liter of
water) was added to the 1.5 mL tube, followed by a 2 hour culturing period at 28°C with constant
agitation (about 200 rpm). After this incubation, 50 l and 150 l of cell mixture was spread
evenly onto two separate LB-ager plates (1.5% agar) containing 80 g/mL gentamycin, 50
g/mL rifampicin as well as the appropriate antibiotic resistance contained within the plasmid.
The LB-agar plates were incubated in the dark at 28° C for 2-3 days. Once colonies appeared,
single colonies were picked and incubated in 100 l of LB media with the previously mentioned
antibiotics for 2 hours at 28° C with constant agitation (200 rpm). The resulting culture was
analyzed for the presence of the plasmid-insert by polymerase chain reaction (PCR) (see section
“Polymerase chain reaction” for cycle details).
50 l of culture from a single agrobacterium colony is then added to 10 mL of fresh LB
with appropriate antibiotics, and incubated for 24 hours at 28° C under constant agitation (200
rpm). Afterwards, the culture was centrifuged for 15 minutes at 4000 rpm in 50 mL greiner
60
tubes. The excess media was discarded and the resulting pellet was resuspended in 10 mL of AS
medium (1% 1 M magnesium chloride, 1% 1 M MES-KOH-pH 5.6, 0.1% 150 mM
acetosyringon (Sigma-Aldrich) dissolved in DMSO, dilute with water). After resuspension, the
culture is incubated at room temperature for 4 hours with moderate agitation (100 rpm). Once the
incubation is complete, the solution is ready for infiltration into N. benthamiana leaves. The
culture is taken up in a 3 mL syringe without the needle. Gentle pressure is applied to the adaxial
side of the leaf and the syringe is placed on the abaxial side of the leaf, opposite the applied
pressure. The plunger of the syringe is then slowly depressed to infiltrate the leaf. Infiltrated
tissue will take on a dark green hue. Due to tissue vascularity, it may be necessary to repeat this
process several times to inoculate the whole leaf. Once the infiltration is complete, the plants
were returned to a long day growth chamber for 3 days. After this time, the tissue is ready for
harvest.
Bimolecular fluorescent complementation
Constructs acquired for bimolecular fluorescent complementation (BiFC) were attained
through Stanton Galvin from the Arabidopsis Research Center (ACR). Two types of constructs
were acquired for this experiment. The first type of BiFC construct contained yellow fluorescent
protein (YFP) tags and used the Gateway™ (Invitrogen) for plasmid cloning (Table 2). The
second type of BiFC constructs contained multicolor fluorescent tags, cyan fluorescent protein
(CFP), Venus, and Cerulean, and required traditional restriction digest and T4 ligations for
plasmid cloning (Table 2). For both construct types successful BiFC expression required TFL1
and TIL3/5PTase13 to be fused with either an N-terminal fluorescent fragment or a
61
complimentary C-terminal fluorescent fragment. Both constructs were then transiently expressed
in N. Benthamiana leaves, and observed under the Zeiss™ LMS 700 microscope.
For BiFC constructs compatible with the Gateway™ cloning system, TFL1 and TIL3/
5PTase13 DNA sequences were where introduced into the pCR8® (Invitrogen) entry vector, and
subcloned into the appropriate BiFC destination vectors as described in the “Gateway™
subcloning” section of materials and methods, transformed into agrobacterium as described into
the “Agrobacterium transformation” section of the materials and methods, and introduced into N.
Benthamiana leaves as described in the “Transient expression in Nicotiana benthamiana” section
of materials and methods.
BiFC constructs that involved traditional cloning steps required the selection of proper
restriction enzymes. To accomplish this the TFL1, TIL3/5PTase13, and multicolor BiFC vector
DNA sequences were searched for suitable cut sites. Ultimately, the restriction enzymes HindIII-
HF (CAAGCTT) (NEB) and XmaI-HF (CCCGGG) (NEB) were selected for 5` and 3` ends of
TFL1 respectively, and SalI-HF (GTCGAC) (NEB) and XmaI-HF (CCCGGG) (NEB) were
selected for 5` and 3` ends of TIL3/5PTase13 respectively. In order to introduce these cut sites
onto the 5` and 3` ends of TFL1 and TIL3/5PTase13, primers were designed with partial 3`
complementarity to the genes and a 5` region containing the cut sites (Table 2). PCR using high
fidelity Platinum® Pfx DNA polymerase (Invitrogen) was then used to incorporate the primers on
to the ends of the TFL1 and TIL3/5PTase13 full-length fragments. The Platinum® Pfx PCR
solution consisted of 10 l 10x Pfx amplification buffer, 1.5 l dNTPs (2.5 mM), 1 l MgSO4
(50 mM), 1.5 l of both forward and reverse primers (10 M), 1 l DNA template, 0.4 l of
Platinum® Pfx DNA polymerase, and up to 50 l of water. Subsequent PCR cycle information
and electrophoresis methods were outlined in the Polymerase chain reaction and Electrophoresis
62
sections in the materials and methods. In the case of TIL3/5PTase13, the region of DNA
encoding the C-terminal domain (810 bp from the 3` end) was isolated using PCR and protein
information from the Arabidopsis information resource (TAIR) database
(http://www.arabidopsis.org/). This C-terminal DNA fragment was used in addition to full length
TIL3/5PTase13 for BiFC multi-color constructs.
Once the TFL1 and TIL3/5PTase13 DNA fragments had the needed cut sites, double
restriction digests were performed on the DNA fragments and the intended BiFC vectors to
create the appropriate hanging ends for subsequent cloning and ligation. For TFL1 the restriction
digest consisted of 0.5 l of 100x Bovine Serum Albumin (BSA) (NEB), 5 l 10x NEbuffer4
(NEB), 1.5 l HindIII-HF (NEB), 1.5 l XmaI-HF (NEB), 6 l TFL1 DNA, and 35.5 l water.
For TIL3/5PTase13 the restriction digest consisted of 0.5 l of 100x Bovine Serum Albumin
(BSA) (NEB), 5 l 10x NEbuffer4 (NEB), 1.5 l SalI-HF (NEB), 1.5 l XmaI-HF (NEB), 6 l
TIL3 DNA, and 35.5 l water. Similar conditions were used for the multicolor BiFC vectors
intended for each DNA fragment. All the restriction digests were incubated at 37° C for 1 hour,
followed by a 60° C incubation for 20 minutes.
The resulting solution was run on an electrophoresis gel (outlined in the
“Electrophoresis” section in the materials and methods). Proper sized DNA bands (TFL1= 0.5
kb, full-length-TIL3/5PTase13=3.5 kb, C-terminal TIL3/5PTase13= 0.8 kb) were then excised
from the gel using a scalpel and a FOTO/convertible® UV-lamp (Fotodyne) to visualize the
DNA. The DNA fragments were purified using the QIAquick® Gel extraction kit (Qiagen).
Using reagents supplied by the QIAquick® kit, the excised gel fragments were weighed and
deposited into 1.5 mL tubes. 3 volumes of QG buffer are added for every 1 volume of gel. The
solution is then heated at 50° C for 10 minutes (or until gel has completely dissolved). Once
63
heated, 1 gel volume of 100% isopropanol is added to the sample and vortexed for several
seconds. The sample is then placed in a QIAquick® spin column with accompanying collection
tube and centrifuged at 12,000 rpm using an Eppendorf 5415D® centrifuge for 1 minute. The
flow through is discarded and 500 l of QG buffer is added to the column. This is then
centrifuged at 12,000 rpm for 1 minute. Once again the flow through is discarded and 750 l of
PE wash buffer is added to the column. This is centrifuged at 12,000rpm, for 1 minute, and
followed by an additional 3 minutes after the PE flow through is discarded. Once the column
filter is dry, replace the collection tube with a 1.5 mL microfuge tube. 50 l of EB elution buffer
is added directly to the column filter and allowed to sit for 2 minutes. After this time, the column
is centrifuged for 1 minute at 12,000 rpm, and the resulting flow through contains the purified
DNA.
This purified DNA is then used in the ligation step. The ligation solution consists of 2 L
10x T4 buffer (NEB), 9 l of digested TFL1 or TIL3/5PTase13 DNA, 3 l of the appropriate
digested BiFC vector, 1 l T4 ligase (NEB), and 5 l of water. This solution is incubated for 2
hours at room temperature to allow for complete integration and ligation of the DNA fragment
into the BiFC vector. The ligation solution is then transformed into competent E-coli cells as
described in the “E-coli transformation” section in the materials and methods. Once the cloned
BiFC vector is amplified and purified it is transformed again into Agrobacterium and infiltrated
into N. Benthamiana as described in the “Agrobacterium transformation” and “Transient
expression in Nicotiana benthamiana” sections of materials and methods.
64
Arabidopsis mutant accessions
Arabidopsis mutant lines were ordered from the Arabidopsis biological research center
(ABRC). T-DNA mutant lines used in this work were 5PTase13 mutant (til3-1/5ptase13-1)
GARLIC 350F1 (Ananieva, E. et al. 2008), 5PTase14 mutant (til3l1-1/5ptase14-1) GABI-KAT
309D03, and 5PTase12 mutant (til3l2-1/5ptase12-1), SALK 065920 (Figure 6). The tfl1-1
mutant (accession CS6167), which was the product of a single nucleotide substitution G to A at
the 5'-end of the fourth exon leading to a missense mutation Gly to Asp at residue 105, was also
used.
2.4 Results
TFL1 affects root gravitropism
In an effort to clarify the role of TIL3/5PTase13 in TFL1 function, we sought a functional
connection between TIL3/5PTase13 and TFL1. Previous studies suggested that TIL3/5PTase13
was involved in root gravitropism through vesicle trafficking (Wang, Y. et al. 2009), thus we
measured root gravitropic response of Arabidopsis wild type (Col-0), til3-1/5ptase13-1, tfl1-1,
35S:TFL1, and tfl1-1 til3-1/5ptase13-1 double mutant plants (Figure 7). While all genotypes
showed increased root curvature following time, we observed variation in root curvature among
genotypes at hour 4 after 180° inversion of gravity. As previously observed, til3-1/5ptase13-1
showed increased sensitivity to gravity compared to wild type (Figure 8). Both 35S:TFL1 and
tfl1-1 mutant plants showed stronger gravitropic response than til3-1/5ptase13-1, with 35S:TFL1
displaying the highest response throughout the time course (Figure 7), demonstrating that TFL1
affects root gravitropism as shown in TIL3/5PTase13. The gravitropic response of the tfl1-1
65
til31/5ptase13-1 double mutant was similar with tfl1-1 (Figure 8), suggesting that TFL1 and
TIL3/5PTase13 may act in the same genetic pathway in root gravitropism
til3-1/5ptase13-1, til3l1-1/5ptase14-1, and til3l2-1/5ptase12-1 mutants suppress
35S:TFL1 late flowering
tfl1-1 mutants are known to display early flowering (Shannon, S. and Wagner, DR. 1991),
while ectopic expression of TFL1 shows a delay in flowering (Ratcliffe, OJ. et al. 1998). To
further explore the role of TIL3/5PTase13 and its homologs, TIL3L1/5PTase14 and
TIL3L2/5PTase12 in TFL1 function, we thus examined their effects in flowering control. The
number of rosette leaves in til3-1/5ptase13-1, til3l1-1/5ptase14-1, and til3l2-1/5ptase12-1 single
mutants was similar to that in wild type (Figure 9). When combined with 35S:TFL1, however,
til3-1/5ptase13-1, til3l1-1/5ptase14-1, and til3l2-1/5ptase12-1 displayed significant suppression
of the late flowering phenotype of 35S:TFL1 (Figure 10). While 35S:TFL1 showed a high
accumulation of both rosette (23± 0.7) and cauline (23± 1.7) leaves as compared to wild type
(8.8± 0.2 and 2.7± 0.2 respectively), til3-1/5ptase13-1, til3l1-1/5ptase14-1, and til3l2-
1/5ptase12-1 mutants showed significant reductions in the number of rosette leaves (19 ± 0.8, 13
± 0.4, 10 ± 0.6, respectively) and cauline leaves (17 ± 1.1, 4 ± 0.3, 3 ± 0.3 respectively). The
early flowering effects of til3l1-1/5ptase14-1, and til3l2-1/5ptase12-1in 35S:TFL1 late flowering
were particularly severe, especially in the number of cauline leaves. This observation indicates
that TIL3/5PTase13 and its homologs are important for proper function of TFL1 in flowering
control.
66
TFL1 and TIL3/5PTase13 proteins co-localize in nucleus and cytoplasm
To verify the interaction between TFL1 and TIL3/5PTase13 observed in yeast, we
examined the subcellular localization of TFL1 in transgenic Arabidopsis plants expressing TFL1
fused with an N-terminal green fluorescent protein (GFP) tag under the control of a 35S
promoter (35S:GFP:TFL1). Strong GFP signal was observed in the nucleus of the root cells as
well as the cytoplasm, as previously reported by (Hanano, S. and Goto, K. 2011) (Figure 11).
Arabidopsis plants containing transgenic TIL3/5PTase13 with fluorescent tags are currently
under development.
Transient expression in Nicotiana benthamiana was chosen as an alternative method for
observing TFL1 and TIL3/5PTase13 subcellular localization. Consistent with the result observed
in Arabidopsis root cells, TFL1 localization was observed in the nucleus and cytoplasm (Figure
12). Similar to the cellular localization of TFL1, TIL3/5PTase13 was observed in the nucleus and
the cytoplasm (Figure 13). These results indicate that both TFL1 and TIL3/5PTase13 proteins
localize in the same subcellular organelles.
To further verify these results, 35S:RFP:TFL1 and 35S:GFP:TIL3 were co-infiltrated
into N. benthamiana leaf epidermal cells with overexpressed TIL3/5PTase13 protein fused with
an N-terminal GFP tag (35S:GFP:TIL3). TFL1 and TIL3/5PTase13 co-localized in the nucleus
and to a lesser degree in the cytoplasm (Figure 14). Notably, we observed that TFL1 and
TIL3/5PTase13 localization did not perfectly overlap. While TFL1 localized in nucleus,
TIL3/5PTase13 localization appeared to be expanded to outside of the nucleus, creating a
corona-like structure around the nucleus (Figure 15). This structure may illustrate the nuclear
membrane, which would be consistent with the role of TIL3/5PTase13 in lipid signaling, or the
67
endoplasmic reticulum. These observations demonstrate that TIL3/5PTase13 and TFL1 are
predominately co-localized.
TFL1 and TIL3/5PTase13 interact in vivo
To confirm the TFL1 and TIL3/5PTase13 interaction in vivo, the bimolecular fluorescent
complementation (BiFC) approach was used. BiFC is a reliable method to demonstrate protein-
protein interactions in living plant tissue. The N-terminal half of YFP fused to the C-terminal end
of TIL3/5PTase13 (35S:TIL3:nYFP) and the C-terminal half of YFP fused to the N-terminal end
of TFL1 (35S:cYFP:TFL1) were co-infiltrated into N. benthamiana epidermal cells. The YFP
fluorescent signal, which signifies direct protein-protein interactions, was observed to localize in
the nucleus and the cytoplasm (Figure 16). These results support the hypothesis that TFL1 and
TIL3/5PTase13 proteins physically interact with each other, and strengthen the functional
connection between TFL1 and TIL3/5PTase13 described above.
Further observation specified a domain of TIL3/5PTase13 required for interaction with
TFL1. An 813 bp region containing the TIL3/5PTase13 C-terminal domain fused with the C-
terminal half of cyan fluorescent protein (CFP) (35S:cTIL3:cCFP) and the N-terminal half of
Cerulean florescent protein (35S:TFL1:nCer) resulted in strong fluorescent signal in both the
nucleus and the cytoplasm (Figure 17), as observed in the interaction of full-length
TIL3/5PTase13 with TFL1.
Together, these results demonstrate the protein-protein interaction between TFL1 and
TIL3/5PTase13 and the involvement of TIL3/5PTase13 in TFL1 function.
68
2.5 Discussion
In this study we reported the role of TIL3/5PTase13 in TFL1 function. TFL1 is known to
repress phase changes of the SAM and floral transition (Abe, M. et al. 2005; Wigge, PA. et al.
2005). Previous work has shown that TIL3/5PTase13, which encodes inositol polyphosphate 5-
phosphotase 13 (5PTase13), interacts with TFL1 at a critical amino acid for TFL1 function
(Hanzawa, Y. unpublished).
TIL3/5PTase13 has been shown to interact with the protein kinase SnRK1 through WD40
repeat regions (Ananieva, E. et al. 2008). It is proposed that TIL3/5PTase13 affects SnRK1
stability through recruitment of the proteasome. Though previous yeast two-hybrid results
indicate TIL3/5PTase13 does not interact with TFL1 through WD40 repeats (Hanzawa, Y.
unpublished), it is possible that TIL3/5PTase13 regulates TFL1 or FD stability in a similar
manner to SnRK1. Nevertheless, the role of TIL3/5PTase13 in TFL1 function had not yet been
confirmed nor characterized.
Mutations in both TFL1 and TIL3/5PTase13 are shown to affect vesicle trafficking (Sohn,
EJ. et al. 2007; Wang, Y. et al. 2009). In TIL3/5PTase13 this results in a notable root gravitropic
affect. To elucidate the role of vesicle trafficking in TFL1 root gravitropism, and thus create
functional link between TFL1 and TIL3/5PTase13, we characterized plants overexpressing
TFL1, containing tfl1-1, and til3-1/5ptase13-1 single mutations, and til3-1/5ptase13-1 tfl1-1
double mutants. As expected, til3-1/5ptase13-1 mutants displayed in increased sensitivity to root
gravitropism, as well as both overexpressed and mutant TFL1. Plants containing the tfl1-1 til3-
1/5ptase13-1 double mutant also showed sensitivity to root gravitropism, but the effect was not
observed to be additive, suggesting TFL1 and TIL3/5PTase13 function in the same pathway
when affecting root development. While the gravitropic effect on tfl1-1 mutants were not
69
significantly different from til3-1/5ptase13-1 mutants (P = 0.11), the phenotype of the tfl1-1 til3-
1/5ptase13-1 double mutant is more similar to the tfl1-1 single mutant that the til3-1/5ptase13-1
single mutant. While not conclusive, this result suggests that TFL1 epistatically affects root
gravitropism downstream from TIL3/5PTase13. Moreover, overexpression of TFL1 showed a
notably stronger effect than til3-1/5ptase13-1 mutants (P = 0.0003). We conclude from these
results that there is a functional connection between TFL1 and TIL3/5PTase13, most likely
mediated through vesicle trafficking.
The effects of TFL1 on floral repression have been well documented. Overexpression of
TFL1 results in significantly delayed floral transition (Shannon, S. and Wagner, DR. 1991),
whereas mutations in TFL1 induce early flowering and terminal flower buds (Ratcliffe, OJ. et al.
1998). In this work we demonstrate that knockout mutants of til3-1/5ptase13-1 and its homologs
til3l1-1/5ptase14-1 and til3l2-1/5ptase12-1 do not have significantly different flowering
phenotypes from wild type Arabidopsis. However, in an overexpressed TFL1 background, all
til3-1/5ptase13-1 mutants are shown to significantly reduce the late-flowering phenotype of
35S:TFL1. These observations suggest that TIL3/5PTase13 and its homologs redundantly
maintain TFL1 floral repression. However, since til3-1/5ptase13-1 mutants alone do not result in
early flowering, it may then be interesting to observe the flowering phenotype of til3-1/5ptase13-
1 til3l1-1/5ptase14-1 til3l2-1/5ptase12-1 triple mutants and determine their functional
redundancy. Alternatively, there may be other mechanisms that assist in TFL1 floral repression,
maintaining TFL1 protein levels at a certain threshold of wild type until environmental
conditions are ideal for flowering. Ultimately, these results indicate that TIL3/5PTase13 is
functionally connected to TFL1 floral repression, but the particulars of this interaction require
further corroboration.
70
To support the interaction between TFL1 and TIL3/5PTase13, the subcellular localization
of both proteins was visualized. GFP:TFL1 was shown to localize in the nucleus and cytoplasm
in both transgenic Arabidopsis root tissue and transiently infiltrated N. benthamiana leaf tissue,
which is in agreement with previous TFL1 localization studies (Hanano, S. and Goto, K. 2011).
Similarly, GFP:TIL3 was also shown to localize in the nucleus, as previously demonstrated
(Ananieva, S. et al. 2009), and cytoplasm in transiently infiltrated N. benthamiana leaf tissue.
This indicates that both TFL1 and TIL3/5PTase13 proteins predominately co-localize, through
TFL1 and TIL3/5PTase13 signal did not completely overlap around the nucleus, suggesting
independent functions for both TFL1 and TIL3/5PTase13. These results are consistent with the
functions of each protein. TFL1 has been shown to act as a transcriptional regulator in the
nucleus (Hanano, S. and Goto, K. 2011), as well as an agent of vesicle transport in the cytoplasm
(Sohn, EJ. et al. 2007). TIL3/5PTase13, as a member of the lipid-signaling pathway, has been
shown to act in cytoplasmic vesicle transport (Wang, Y. et al. 2009), as well as in nuclear
regulation of metabolism (Ananieva, S. et al. 2009). The nuclear co-localization of TFL1 and
TIL3/5PTase13 demonstrate that it is possible for TIL3/5PTase13 to act upon TFL1
transcriptional regulation of flowering.
Furthermore, FT, a homolog of TFL1, has been shown to interact with the endoplasmic
reticulum (ER) to facilitate intercellular movement (Liu, L. et al. 2012). Due to the similarity
between TFL1 and FT it would be interesting to clarify whether or not TFL1 interacts with the
ER when it propagates from cell to cell (Conti, L. and Bradley, D. 2007).
We confirmed a protein-protein interaction between TFL1 and TIL3/5PTase13 in the
nucleus and cytoplasm through a BiFC assay. Considering that both TFL1 and TIL3/5PTase13
have roles in vesicle transport, it is possible that TFL1 and TIL3/5PTase13 work together in
71
protein shuttling to and from the nucleus. Moreover, previous work has shown that the C-
terminal domain of TIL3/5PTase13, as opposed to the WD-40 repeats, is necessary for proper
binding to TFL1 (Hanzawa, Y. unpublished). Our BiFC results support this by demonstrating that
an 813bp region of the C-terminal domain of TIL3/5PTase13 is sufficient for proper binding to
TFL1. A more through investigation of the amino acid sequence in this region is needed to
isolate potential protein binding motifs.
With this information it is then possible to formulate a hypothesis on the mechanism of
TFL1-TIL3/5PTase13 interactions (Figure 18). TFL1 binds with the DNA-binding transcription
factor FD in the nucleus (Hanano, S. and Goto, K. 2011). FD has been shown to bind to the
promoter regions of meristem identity genes in combination with FT (Wigge, PA. et al. 2005); I
propose that TFL1 binds to the promoter regions of meristem identity genes in a similar manner.
This FD-TFL1 binding leads to repression of floral induction. While bound to the promoter
region I hypothesize TFL1 is modified by a ligand. This modification acts as a signal to an
uncharacterized removal or degradation complex, and facilitates binding to TFL1. TFL1 is then
removed from the promoter region of the floral meristem identity genes, and floral inhibition is
released. I propose it is the job of TIL3/5PTase13 to bind to TFL1 and remove the TFL1 bound-
ligand (Figure 19). Due to the role of TIL3/5PTase13 in phosphate hydrolysis (Zhong, R. and Ye,
ZH. 2004; Chen, X. et al. 2008), a reasonable candidate for TFL1 modification is
phosphorylation. Once the ligand is removed from TFL1, the removal complex is no longer
recruited to TFL1. Thus TFL1 floral repression is maintained through an interaction with
TIL3/5PTase13. Naturally this hypothesis makes several assumptions, such as TFL1
modification actually occurring in plant tissue, this modification acting as a signal facilitating
TFL1 removal, and that TIL3/5PTase13 acts to remove this modification. It is the aim of my
72
future research to address each of these uncertainties, and further elucidate the mechanisms
involved in TFL1-TIL3/5PTase13 interaction.
In conclusion, BiFC experiments reveal that TFL1 and TIL3/5PTase13 physically interact
in the nucleus and cytoplasm within plant cells. Furthermore, functional experiments that
measure the effects of root gravitropism suggest that TIL3/5PTase13 and TFL1 function in the
same pathway when affecting root development, potentially through vesicle trafficking.
Experiments measuring floral initiation show that TIL3/5PTase13 reduces that late flowering
phenotype of overexpressed TFL1, indicated that TIL3/5PTase13 plays a role in maintaining
TFL1 floral repression. Despite recent studies, the mechanism for TFL1-TIL3/5PTase13 protein
interaction is largely unknown. Further experiments are required to clarify the exact role of
TIL3/5PTase13 in TFL1 floral repression.
73
2.6 Figures
Figure 6. Locations of T-DNA insertions for 5PTase mutant Arabidopsis lines. Boxes represent
exons and lines represent introns. til3-1 has a T-DNA insert in its fourth exon, til3l1-1 has a T-
DNA insert in its ninth exon, and til3l2-1 has a T-DNA insert in the eleventh exon. The T-DNA
inserts are not to scale.
74
Figure 7. A10-hour time course observing the root gravitropic effects of TFL1 overexpression
and mutant plants and til3-1 mutants in Arabidopsis seedlings. 35S:TFL1, tfl1-1, til3-1, and tfl1-1
til3-1 double mutants all show increased sensitivity in root gravitropism compared to wild type,
with 35S:TFL1 showing the strongest sensitivity across all time points. The largest differences in
sensitivity between plant lines can be observed during time point 4.
0
20
40
60
80
100
120
140
0 4 6 8 10
Ro
ot
Cu
rva
ture
(D
eg
ree
s)
Hours
WT
35S:TFL1
tfl1-1
til3-1
tfl1-1 til3-1
75
Figure 8. The differences in root curvature during the fourth hour of the root gravitropic
experiments. The results demonstrate that 35S:TFL1 seedlings have the strongest root gravitropic
effect, with tfl1-1, til3-1, and tfl1-1 til3-1 double mutants having similar increases in gravitropic
sensitivity. Error bars indicate the standard error for each plant line. An asterisk indicates that a
plant line shows a significant (p < 0.05) difference in root curvature compared to the wild type.
76
Figure 9. The number of rosette and cauline leaves counted for til3-1, til3l1-1, and til3l2-1
mutants in Arabidopsis. The results show that til3-1 and its homologs do not significantly deviate
from the leaf number of wild type plants. Error bars indicate the standard error for each plant
line.
77
Figure 10. A comparison of the number of rosette and cauline leaves counted in overexpressed
35S:TFL1 and til3-1, til3l1-1, and til3l2-1 mutants in a 35S:TFL1 background. The results show
that til3-1, til3l1-1 and til3l2-1 all display a decease in both rosette and cauline leaf number when
compared to 35S:TFL1. An asterisk indicates that a plant line shows a significant (p < 0.05)
difference in leaf count compared to 35S:TFL1 plants. Error bars indicate the standard error for
each plant line.
78
Figure 11. A confocal image of mature Arabidopsis root tissue, 9 days after germination.
Fluorescent localization of GFP:TFL1 is observed in both nuclear and cytoplasm. Image taken
with a 488 nm laser at 20x magnification.
79
Figure 12. A confocal image of mature N. benthamiana leaf tissue, 3 days after agrobacterium
infiltration. Fluorescent localization of GFP:TFL1 is observed in both nuclear and cytoplasm.
Image taken with a 488 nm laser at 20x magnification. BF= Bright field. White arrows indicate
chloroplasts imaged with a 555 nm laser.
80
Figure 13. A confocal image of mature N. benthamiana leaf tissue, 3 days after agrobacterium
infiltration. Fluorescent localization of GFP:TIL3 is observed in both nuclear and cytoplasm.
Image taken with a 488 nm laser at 20x magnification. BF= Bright Field. White arrows indicate
chloroplasts imaged with a 555 nm laser.
81
Figure 14. A confocal image of mature N. benthamiana leaf tissue, 3 days after agrobacterium
co-infiltration of 35S:GFP:TIL3 (green) and 35S:TFL1:RFP (red). Fluorescent localization of
GFP and RFP is observed in both nuclear and cytoplasm. Co-localization is represented by
yellow fluorescence. Image taken with a 488 nm and 555 nm laser for GFP and RFP
fluorescence respectively at 20x magnification. White arrows indicate chloroplasts imaged with a
555 nm laser.
82
Figure 15. A confocal image of mature N. benthamiana leaf tissue, 3 days after agrobacterium
co-infiltration of 35S:GFP:TIL3 (green) and 35S:TFL1:RFP (red). Fluorescent localization of is
observed in both nuclear and cytoplasm. Co-localization is represented by yellow fluorescence.
Image taken with a 488 nm and 555 nm laser for GFP and RFP fluorescence respectively at 40x
magnification. White arrows indicate chloroplasts imaged with a 555 nm laser. The white box
highlights the cell nucleus in which the TFL1:RFP fluorescence signal does not perfectly overlap
with GFP:TIL3.
83
Figure 16. A confocal image of mature N. benthamiana leaf tissue, 3 days after agrobacterium
co-infiltration of BiFC constructs 35S:TIL3:nYFP and 35S:cYFP:TFL1. Protein-protein
interactions are observed in both nuclear and cytoplasm. Image taken with a 488 nm at 20x
magnification. White arrows indicate chloroplasts imaged with a 555 nm laser. BF= Bright Field.
84
Figure 17. A confocal image of mature N. benthamiana leaf tissue, 3 days after agrobacterium
co-infiltration of BiFC constructs 35S:cTIL3:cCFP and 35S:TFL1:nCerulean. cTIL3 is
composed of the c-terminal 813 bp of full length of TIL3. Protein-protein interactions are
observed in both nuclear and cytoplasm. Image taken with a 405 nm at 20x magnification. White
arrows indicate chloroplasts imaged with a 555 nm laser. BF= Bright Field.
85
Figure 18. A hypothetical model explaining control of the TFL1 floral repression. TFL1 (red
oval) interacts with FD (grey circle) at the promoter region of meristem identity genes to repress
floral initiation. TFL1 becomes modified (“M” contained within a blue hexagon). Modified
TFL1 is the target of an unknown removal complex (green drop), and is removed from the
meristem identity gene promoter. With floral repression relaxed, RNA polymerase (yellow circle)
is now able to transcribe meristem identity genes
86
Figure 19. A hypothetical model illustrating the role of TIL3 in maintaining TFL1 floral
repression. TFL1 (red oval) interacts with FD (grey circle) at the promoter region of meristem
identity genes to repress floral initiation. TFL1 becomes modified (“M” contained within a blue
hexagon). TIL3 (green diamond) then binds to TFL1 and acts to cleave the modification from
TFL1, which prevents the removal complex (green drop) from binding. Thus through interaction
with TIL3, TFL1 floral repression is maintained.
87
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93
CHAPTER 3
Identification of novel flowering QTLs in Glycine max
3.1 Abstract
Flowering response to seasonal photoperiod changes is a critical trait to environmental
adaptation and productivity of cultivated soybean, and a major target for domestication. To explore
genes underlying the evolution of photoperiodic flowering response during the domestication
process of soybean, we conducted QTL mapping using a population of 115 F6 recombinant inbred
lines (RILs) that were created from a backcross between cultivated soybean, G. max, and its
ancestor, G. soja.
Agriculturally important traits of: flowering time (R1), maturity time (R8), height, yield,
lodging, and stem vining were measured in two years in four field locations. When compared to
G. max, trait distribution in the F6 RIL population showed overall trends towards later flowering,
earlier maturity, increased height, decreased yield, and increases in both lodging and stem vining,
which is consistent with the allelic influence of G. soja. Principle component analysis revealed
three distinct groups in the traits: maturity and flowering; stem vining, height, and lodging; and
yield.
QTL mapping analysis identified 32 loci for flowering and 31 loci for maturity. Of these
loci, 16 overlapped with each other. One of these loci coincided with a previously identified
maturity locus, E6. 37 known flowering genes were identified near the flowering and maturation
QTLs that included circadian clock genes, floral integrators, and meristem identity genes. For
height, yield, stem vining and lodging, 28, 33, 32, and 33 QTLs were identified, respectively.
94
Stem vining QTLs and lodging QTLs overlapped at 20 discrete loci. We found four QTLs that
affected 5 or more traits and showed the phenotypic effects of the G. max alleles for all traits,
suggesting that directional selection might have acted on these loci. These QTLs, therefore, may
have been targets of human selection and influenced the domestication process of cultivated
soybean, G. max.
3.2 Background
The initiation of flowering is a major developmental event that occurs in the life cycle of
all angiosperms. Successful floral transition ultimately dictates the survival and reproductive
success of a plant. However, unlike model species, the mechanisms of flowering are not well
characterized for the majority of cultivated species. Therefore, there is a pressing need to explore
the basic mechanisms of flowering in crop species.
Soybean (Glycine max [L.] Merr.) is a dicot native to East Asia that flowers under short-
day conditions. It is a major staple in both human and animal diets, and is primarily grown for its
high oil or protein content. Like many other crop species flowering is not well defined in
soybean, however some elements controlling floral initiation have been identified.
Currently, nine major QTLs have been identified that have control over flowering and
maturity in soybean, the eight E-maturity loci (E1-E8) and J (Bernard, R. 1971; Buzzell, RI.
1971; Buzzell, RI. and Voldeng, H. 1980; McBlain, BA. and Bernard, R. 1987; Bonato, ER. and
Vello, NA. 1999; Cober, E. and Voldeng, H. 2001; Cober, E. et al. 2010; Ray, J. et al. 1995). Of
the E-loci, E1 has been observed to have the strongest effect on flowering in soybean. The causal
gene of E1 has been isolated to a 17.4kb region on chromosome 6 (Xia, Z. et al. 2012a). It
encodes a transcription factor that does not have close homologs in Arabidopsis and acts as a
repressor of both floral initiation and seed maturation (Xia, Z. et al. 2012a). E2 has been
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identified as a homolog to Arabidopsis GIGANTIA (GI) (Watanabe, S. et al. 2011), and E4 and
E3 have been identified as homologs to the Arabidopsis photoreceptor PHYTOCHROME A
(PHYA), GmphyA2 and GmphyA3 respectively (Liu, B. et al. 2008; Watanabe, S. et al. 2009).
Furthermore, ten orthologs to Arabidopsis FLOWERING LOCUS T (FT) have been isolated in
soybean (Kong, FJ. et al. 2010) in addition to eight CONSTANS (CO) orthologs. Moreover,
comparative genome studies between soybean, Arabidopsis, and other plant species have
revealed a plethora of flowering orthologs within soybean that have yet to be characterized but
provide many opportunities for further research (Jung, CH. et al. 2012, Hecht, V. et al. 2005;
Ehrenreich, IM. et al. 2009).
Despite such new information, thorough understanding of soybean flowering requires
further discoveries of genes and their function. In addition, previously identified maturity loci are
observed to affect both flowering and maturity phenotypes simultaneously (Xia, Z. et al. 2012a).
As a consequence, we currently have limited genetic resources to optimize soybean life cycle for
diverse maturity zones in the U.S. Therefore, there remains a critical need to identify novel loci
or alleles that would modify flowering time independently of seed maturation, to achieve higher
environmental adaptation and productivity. One obstacle in the identification of these loci is the
lack of genetic variation inherent in cultivated soybean (Brown-Guedira, GL. et al. 2000). This
limited variation is a result of North American soybean varieties originating from only a few
landraces during the original cultivar development. To overcome this hindrance, it is essential to
explore sources of wider genetic diversity. The wild ancestor of soybean, Glycine soja, is a
promising candidate to address this issue. G. soja was believed to be domesticated into G. max
5,000 years ago in China (Guo, J. et al. 2010; Guo, J. et al. 2013). However, notwithstanding the
close relationship between G. max and G. soja, they have notably different phenotypes (Figure
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20), including the stringent short day requirement for G. soja, resulting in late flowering
compared to G. max under North American environments.
To explore the underlying mechanisms of this flowering response and apply them to
benefit G. max, a mapping population of 115 BC2F6 recombinant inbred lines (RILs) was
developed from a backcross between G. max and G. soja. Quantitative trait loci (QTL) mapping
was used to identify novel loci affecting flowering time (R1) and plant maturity (R8) along with
other agronomic traits such as height, yield, stem vining, and lodging. Here I report the results of
our initial experiments. Multiple factor analysis reveals that our traits form 3 groups with
maturity and flowering in one, height, stem vining and lodging in another, and yield composing
the final group. In the QTL analysis 32 and 31 loci were detected for flowering and maturity
respectively, in addition to similar numbers of QTLs for height, lodging, stem vining and yield.
Of the 32 flowering loci, 10 have been previously identified in other analyses. Furthermore, 26
candidate genes related to flowering and maturity are located within our QTLs. QTL clusters on
chromosomes 1, 9, and 11 are shown to be highly significant according to phenotypic and
statistic observations, with chromosome 9 in particular containing a homolog of GIGANTEA,
the E2 maturity loci. These clusters in general are consistent with the pattern of domestication
QTLs and may present valuable insight in into the domestication process of G. max.
3.3 Materials and methods
Mapping Population
The initial cross between the G. max variety Williams 82 and the G. soja accession PI
549046 was made in 1996. PI 549046 was selected because it was one of the lines that were most
genetically distinct form the G. max based on genotyping results using RAPD markers among G.
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max and G. soja accessions selected from four Chinese provinces (Li and Nelson, 2002).
Backcrosses were made with F1 plants in 1997 and the BC1F1 plants were grown in 1998.
Approximately 2% of the 3000 F2 plants were selected and 87 plants were selected from within
17 F3 families in 2000. In the F4 generation, nine lines were bulk harvested based on agronomic
appearance. LG01-7909 and LG01-7919 both flowered 10 to 12 days later than Williams 82 and
matured 7 to 13 days earlier than Williams 82. These lines were among those retested in 2002
and again they flowered 9 to 11 days later than Williams 82 and matured 11 to 12 days earlier. In
2003 these two lines were backcrossed again to Williams 82 and the F1 plants were grown the
following winter in Puerto Rico. These populations were advanced through single seed descent
from the F2 to the F4 generation. In the F4 generation, 79 plants were harvested from the
population with LG01-7919 as a parent and 109 plants were harvested from the population with
LG01-7909 as a parent. When these lines were planted in 2007 there was still considerable
phenotypic segregation, so a single plant was harvested from each row. From the 166 F5 rows
planted in 2008, we selected 115 inbred lines that had maturity dates between September 18 and
September 30 for our mapping population 78 had the pedigree of F5 Williams 82 x LG01-7909
and 47 had the pedigree of F5 Williams 82 x LG01-7919.
Field Evaluation
The RILs, the parent line Williams 82, and maturity checks IA2023 and LD00-3309 were
evaluated at Urbana and Stonington, Illinois in 2009 and at Urbana, Villa Grove, Bellflower, and
Stonington, Illinois in 2010. All field plots were four rows wide with 0.76 m spacing and 3.1 m
long and were planted at a rate of 30 seeds m-1 The agronomic data collected include flowering
date (R1) which was recorded when approximately 50% of the plants had at least one flower
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(Fehr et al., 1971), plant maturity date (R8) recorded when approximately 95% of the pods had
reached mature pod color (Fehr et al., 1971), plant lodging scored at maturity based on a 1 to 5
scale (1 = all plants are erect, 5 = all plants are prostrate), plant height (cm) measured from the
soil surface to the top node of the main stem and seed yield (kg ha-1) recorded at 13% moisture,
and a stem vininess score between 1 (typical indeterminate cultivar) and 5 (viney stem
termination).
The test was divided by maturity based on the data collected in 2008. Lines that matured
between September 18 and 24 were blocked together and those that matured between September
25 and 30 were blocked together. The test was replication twice at each location.
Statistical analysis of phenotypic data
Statistical analysis of phenotypic distribution and multiple factor analysis (MFA) were
carried out using a home-developed R script. Box plots were created using the Psych package,
multiple factor analysis (MFA) was conducted with the FactominR package, and the best linear
unbiased predication (BLUP) analysis used the lme4 package.
Genotyping
1536 single nucleotide polymorphisms (SNPs) from the 1,536 Universal Soy Linkage
Panel (USLP 1.0) developed by Hyten, DL. et al. (2010) were used for genotyping assay. Single
leaf from single plant was collected for each line. DNA samples from 115 F5 RILs and Williams
82 were obtained using DNeasy Plant Mini kit (QIAGEN) and sent to (Perry Cregan, USDA-
ARS, Maryland) and genotyping was carried out using the Illumina GoldenGate® Assay.
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SNP selection
SNPs of interest were selected using Illumina’s Genome Studio® 1.0. Initial screening
excluded markers with low call numbers (below 56) or where no segregation was observed. In
addition, SNPs that both parents shared the same allele type were removed. Of the original 1,536
SNPs used, only 545 were selected for QTL mapping.
QTL mapping
Marker locations in Williams 82 (G. max) were obtained from Hyten, DL. et al. 2010 and
a genetic map were constructed based on the marker location information using JoinMap® 4.0
QTL analysis was conducted using MapQTL® 4.0. The genetic linkage map for Williams 82 was
used, and Kruskal-Wallis test was performed. P-value of less than 0.01 was used as a threshold to
declare significance for Kruskal-Wallis test. The directionality for individual QTLs was
determined by subtracting the value of the G. max allele from the G. soja allele and dividing by
2. It was then determined if this value was consistent with the desirable traits in cultivated
soybean (early flowering, late maturity, decreased height, increased yield, and decreases in both
stem vining and lodging)
In silico candidate gene selection
A list of flowering related genes was obtained from (Kim, MY. et al. 2012). Using the
SoyBase database (http://soybase.org/), the physical location of significant flowering and
maturity SNPs were compared to the location of the flowering genes. Candidate genes were
selected if they located less than 500 kb away from QTL-associated SNPs.
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3.4 Results
Population distribution
The population of 115 F6 RILs showed a large range of trait distribution in all traits
(Figure 21). Notably, the interquartile range of flowering time was above the mean value of
Williams 82 in each year, likely due to polymorphisms from G. soja genome in the population.
The interquartile range of stem termination also was higher than Williams 82. Whereas, the
interquartile range of seed maturity time and yield was below Williams 82. These results are
consistent with our observation of G. soja phenotypes with late flowering, early maturity, vining,
and low yield.
Comparison of trait distribution ranges showed a strong year effect in flowering time and
seed maturity time (Figure 22). Flowering time was distributed from 43.5 to 63.8 days with the
mean of 55.6 in 2009, and from 23.0 to 39.3 days with the mean of 30.8 in 2010. Seed maturity
time showed a similar year effect, however smaller than that of flowering time, ranging from
118.8 to 133.5 days with the mean of 126.0 in 2009, and from 105.5 to 114.4 days with the mean
of 110.3 in 2010. The difference of mean values between 2009 and 2010 was significant in all
traits.
Aiming at identification of flowering time QTLs independent of seed maturity, the 115
RILs were classified into two sets based on their seed maturity time: Early maturity (n=64) and
late maturity (n=51) and trait distribution within each maturity set was observed (Figure 22).
Ranges of trait distribution in the maturity set 1 and the maturity set 2 were similar in all traits,
but different in seed maturity, with a shift to later maturity in the maturity set 2 (ranging from
118.0 to 124.2) than the maturity set 1 (ranging from 111.6 to 120.5).
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The phenotypic distribution differed between maturity sets 1 and 2 in most of the traits,
whereas no significant difference was observed between subpopulations 1 and 2 in all traits. The
maturity sets 1 and 2 showed a difference of 4.8 days in the mean values of R8 (P = 8.19E-27).
The maturity sets also showed 4.6 days difference in R1 (P = 2.12E-7), 9.2 cm difference in
height (P = 1.48E-3), and 20% difference in winding (P = 0.287). The observed difference in the
maturity distribution between maturity sets 1 and 2 was expected, as the sub-populations were
selected for early maturity and late maturity sets during the process of creating the population.
The effects in R1, height, and winding suggest that the selection for maturity also affects these
traits.
Multiple factor analysis
Multiple Factor Analysis (MFA) demonstrates the different behavior among the traits
(Figure 23). Variation is divided into Dimension 1, which explains 45.3% of total variation,
Dimension 2, which explains 23.1% of total variation, and Dimension 3, which is not represented
in Figure 23, but defined in (Table 3) and makes up 16.7% of the total variation. The traits are
observed to clustered into three groups. Height, stem vining, and lodging make up major
components in Dimension 1 (0.84, 0.80, and 0.86 respectively) flowering and maturity are
represented together in Dimension 2 (0.82 and 0.56), which is consistent with flowering and
maturation behavior of E-loci (Buzzell, R. and Voldeng, H. 1980; McBlain, B. et al. 1987;
McBlain, B. and Bernard, R. 1987; Cober, E. and Voldeng, H. 2001). Finally, yield alone is the
primary component in Dimension 3 (0.83).
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QTL mapping
In total, our analysis has uncovered 189 QTLs distributed among 6 traits. Among them,
32 loci were shown for flowering and stem vining. Furthermore, 33 loci were identified for both
yield and lodging, and 28 and 31 loci for height and maturity respectively. QTLs that affect
flowering and maturity specifically are represented on (Figure 24), and QTLs that affect traits in
addition to flowering and maturity are shown on (Figure 25). In both figures, exhibited QTLs
have a p-value of at least 0.01 determined through Kruskul Wallis single marker analysis (SMA).
Particularly significant (p ≤ 0.005) SNPs identified in SMA are listed in (Table 4). Additionally,
we represent directionality in relation to phenotypic effect of the G. max allele; for example, an
upward arrow on a flowering or maturity QTL would suggest early flowering or late maturation
respectively. SNPs with a significant (p ≤ 0.05) phenotypic difference between the G. max and G.
soja allele are represented in (Table 5). The location of 8 previously identified E-loci is portrayed
on each map (Figure 24, Figure 25).
Flowering and maturity QTLs
In figure 24, flowering and maturity QTLs are observed on all 20 soybean chromosomes,
most of which do not overlap with previously identified E-loci. 16 QTLs affecting flowering are
shown to occur independently of maturity, 13 QTLs affecting maturity are shown to occur
independently of flowering, and the remaining 16 QTLs affect both flowering and maturity. In an
attempted to further characterize these QTLs, a list of over 100 flowering genes was consulted
(Kim, MY. et al. 2012). The chromosomal location of these flowering genes was then compared
with physical location of SNPs associated with flowering or maturity loci. The physical location
of SNPs was determined through use of the SoyBase database (http://soybase.org). 6 genes were
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shown to associate with QTLs that affect maturity independent of flowering, 6 genes were shown
to associate with QTLs that affect maturity independent of flowering, and 3 genes were shown to
associate with QTLs affecting both flowering and maturity (Table 6).
Of particular interest are the GI homolog on chromosome 9, and the FT homologs found
on chromosome 16 and 19. A BLAST comparison reveals that the GI homolog of chromosome 9
shares 76% amino acid identity with the previously identified causal gene of E2 locus, GIa
(Watanabe, S. et al. 2011). Furthermore, the marker (BARC-026035-05236) closest to the GI
homolog resides on a QTL showing notable difference in flowering time (5 days) between G.
max allele and G. soja allele (Table 5). Similarly, the marker (BARC-016775-02320) closest to
the FT homologs on chromosome 16 also resides on a QTL showing a notable difference in
flowering time (7 days) (Table 5). These loci are interesting due to their critical role as floral
inducers in both long day (Kobayashi, Y. et al. 1999, Kardailsky, I. et al. 1999) and short day
model species (Kojima S. et al 2002). Other phenotypically significant QTLs containing
candidate genes are located on chromosomes 5, 10, and 20, consisting of SPA2, ATC, and CRY2,
respectively. Several QTLs are revealed to have highly significant SNPs according to SMA
(Table 4), particularly the QTL associated with flowering on chromosome 1. None of these QTLs
are shown to harbor corresponding candidate genes.
QTLs affecting multiple traits
QTLs that affect yield, height, stem vining and lodging were widely distributed on all 20
chromosomes (Figure 25). Many of these QTLs affected multiple traits. Among these, clusters of
QTLs found on chromosomes 1, 9, and 11 between 35-4cM, 34-59cM, and 31-55cM respectively
are particularly interesting. The cluster found on chromosome 1 contains highly significant SNPs
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(Table 4) for flowering, maturity, lodging, and stem vining traits. The cluster on chromosome 11
is shown to be notable in both single marker analysis (Table 4) for flowering, maturity, and stem
vining, and in phenotypic value (Table 5) for height and stem vining. The cluster on chromosome
9 is phenotypically significant in flowering, height, lodging, and stem vining, and also contains
two candidate flowering genes, PHYE and GI (Table 6), the GI homolog already having been
identified as a target of future interest for its homology with the E2 maturity locus. The only
other candidate gene to associate with a QTL affecting multiple traits is AGL20 on chromosome
9 between 91-93cM.
QTLs that may influence Glycine max domestication
When looking at the phenotypic effect of the Glycine max allele among the QTL clusters
(Figure 25) three QTL clusters with multiple traits that show consistent positive direction can be
found on chromosome 10 between 105-118cM, chromosome 8 between 9-16cM, and on
chromosome 1 between 35-45cM. It is possible that these loci had a desirable effect on the
domestication of G. max. Much more common however are loci that contain traits with
predominantly negative phenotypes with respect to G. max. The directionality for the majority of
QTLs among individual traits are negative with the exception of height which has about equal
number of positive and negative QTLs and maturity, which has slightly more positive QTLs than
negative QTLs. This general display of undesirable phenotypes from the G. max alleles presents
the opportunity to examine wild soybean, and search the G. soja alleles for regions that may
improve the observed phenotype in cultivated soybean.
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3.5 Discussion
In this study we investigate novel flowering and maturity loci in soybean, by exploring
the wider genetic variation found in the wild ancestor of Glycine max, Glycine soja. This was
accomplished by generating 115 BC2F6 RILs from an initial cross between G. max and G. soja,
followed by QTL mapping.
In our mapping population, RILs were used to create a mosaic of G. max and G. soja
genomes. 115 RILs were selected to capture the entire G. soja genome, thus allowing us to more
fully observe the allelic affects of G. soja in a G. max background.
A major obstacle for our QTL mapping emerged, however, after genotyping using the
1,536 USLP 1.0 (Hyten, DL. et al. 2010). Significant segregation distortion was observed, in
which the distribution of G. soja alleles at segregating markers was less than expected for many
loci. This likely suggests that during the selfing stage during the process of the RIL population
creation, plants containing the G. soja allele were blindly selected against, resulting in most loci
containing an overabundance of G. max alleles. Similar phenomenon has been previously
observed in crosses between cultivated crops and their wild progenitors (Keim, P. et al. 1990;
Yamanaka, N. et al. 2001). As a result, construction of a linkage map generated by our
population was exceedingly difficult. As a substitute previously identified marker locations for
the 1,536 USLP 1.0 (Hyten, DL. et al. 2010) were used to align our markers in the correct
linkage group. A consequence of this is that the portrayed position of certain SNPs may not
reflect their actual location in our population. Accordingly, the results of interval mapping were
difficult to interpret. In addition, opposing effects of neighboring SNPs hindered our interval
mapping analysis. To reduce these problems, single marker analysis (SMA) was chosen as a
viable alternative.
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Despite the previously mentioned challenges, our QTL mapping analysis identified many
loci of interest in our population. Particularly, we identified many flowering and maturity QTLs
that have not been previously characterized. Moreover, several of these flowering QTLs appear
to act independently of maturity QTLs, and vise versa for maturity QTLs. This independent
action of flowering and maturity QTLs has not yet been greatly studied in soybean since
flowering and maturity QTLs are usually linked, a prime example being the E-loci. A possible
explanation for the presence of these novel QTLs in our population is that we use G. soja as a
parental line. In identification of the E-loci, two G. max parents were crossed to create the
mapping populations (Buzzell, R. and Voldeng, H. 1980; McBlain, B. et al. 1987; McBlain, B.
and Bernard, R. 1987; Cober, E. and Voldeng, H. 2001). In using G. soja we gain access to the
genetic variation therein, allowing for the identification of a plethora of new loci. Additionally,
selection for early or late maturity groups within our 115 lines may have helped isolation of
flowering QTLs independent of maturity.
Out of the flowering QTLs we identified, 10 overlap with previously identified flowering
QTLs (Table 7). Of these, two overlap with the candidate flowering genes AGL8 and SRF6 on
chromosomes 5 (Tasma, IM. et al. 2001) and 13 (Orf, JH. et al. 1999) respectively. Moreover, a
phenotypically significant flowering QTL we identified on chromosome 9 at 43cM locates near
the previously characterized QTL, FT3-3 (Reinprecht, Y. et al. 2006). Similarly, a highly
significant (p< 0.005) flowering QTL we identified on chromosome 11 at 46cM locates near the
previously characterized QTL, Fflr 11-1 (Gai, J. et al. 2007). Interestingly, the SoyBase database
shows that no flowering QTLs have been identified on chromosome 1, which contains our
strongest identified flowering QTL according to SMA. Furthermore, suitable matches for the
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remaining 22 flowering QTLs could not be found, suggesting that these loci may be novel QTLs
originating from our population’s G. soja parentage.
While identification of flowering and maturity QTLs was a focus of this study, an
important second objective was the characterization of potential domestication loci. According to
previous studies (Ross-Ibarra, J. 2005), domestication QTLs commonly have several reoccurring
patterns. The first pattern is that the distribution of domestication QTLs appears non-random, and
multiple traits usually form linked-clusters at a single locus. (Cai, HW. and Morishima, H. 2002).
An additional trend is that domestication related traits are usually controlled by a small number
of strong loci (Ross-Ibarra, J. 2005), though exceptions have been noted such as in the case of
sunflower (Burke, JM. et al. 2002) where a large number of weak-effect QTLs control
domestication traits. In our QTL mapping analysis, we observed multiple QTLs that contain
clusters of domestication traits on chromosomes 1, 9, 11, 13, and 18, which is in agreement with
the trend that domesticated related traits form groups. However, our population deviates from
pre-existing patterns of domestication in that there are multiple weak QTLs that contribute to
domestication traits in addition to strong loci, such as those identified in table 4 and table 5.
Despite the fact that all the QTL examined in this study have a SMA significance of p≤ 0.01, it is
possible that a more stringent threshold is necessary. Conversely, as in the case of sunflower
(Burke, JM. et al. 2002), genetic architecture of soybean domestication may deviate from the
general pattern and consist of multiple weak - moderate domestication loci.
Along with positional patterns, the phenotypic directionality of domestication-related
QTLs is often insightful as to the role of the QTL. It is expected that alleles from a domesticated
parent in loci underlying key domestication traits are associated with the phenotypes observed in
the domesticated parent, indicating those allele types are selected over the course of
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differentiation from non-domesticated ancestral varieties (i.e. if a plant is found to be
domesticated for early flowering, the allele type of the domesticated variety on QTLs underlying
domestication may likely convey early flowering) (Rieseberg, LH. et al. 2002). There are several
clusters of domestication-related QTLs in our population that show positive direction with
domestication, such as on chromosome 8 at 16cM and chromosome 10 at 117cM. However,
many other domestication-related QTLs in our population are directionally opposite of G. max
domestication phenotypes. A similar instance was reported in domesticated sunflower (Burke,
JM. et al. 2002) where multiple domestication QTLs were revealed to have opposite the expected
direction. It is suggested that these negative QTLs may have been maintained in domesticated
crops due to their high linkage disequilibrium with positive QTLs.
Moreover, studies in bean (Koinange, EMK. et al. 1996), peanut (Fonceka, D. et al.,
2012), barley (Pillen, K. et al. 2004) and rice (Xiao, JH. et al. 1998; Xiong, LX. et al. 1999)
demonstrate that wild progenitors of cultivate crops contain multiple beneficial alleles that are
not present in the cultivate varieties. This implies that positive traits can be lost during the
domestication process, and that wild progenitors can be a source of agriculturally significant
variety.
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3.6 Tables
Trait Dimension 1 Dimension 2 Dimension 3
Flowering 0.414 0.822 0.019 Maturity 0.541 0.555 0.500 Height 0.842 -0.302 0.167 Lodging 0.806 -0.423 0.018 Yield -0.372 -0.312 0.838 Stem Vining 0.866 -0.186 -0.141
Table 3. This table displays three eigan-vectors (i.e. principle components) for each trait, and
demonstrates the strength of trait association within a dimension of the MFA. Height, lodging,
and stem vining are major components of dimension 1, flowering and maturity make up
dimension 2, and yield makes up dimension 3.
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Marker Chromosome Position (cM) Trait P-value
BARC-030973-06982 1 33.542 Stem Vining 0.005
BARC-011057-00831 1 39.921 Flowering 0.001
Maturity 0.0005
BARC-061099-17047 1 41.578 Lodging 0.005
Maturity 0.001
BARC-058135-15106 1 41.58 Flowering 0.005
Maturity 0.005
BARC-065079-19090 1 41.58 Maturity 0.005
BARC-054393-12560 1 42.007 Flowering 0.005
BARC-063183-18261 1 45.061 Stem Vining 0.001
Flowering 0.005
BARC-038331-10034 1 45.071 Stem Vining 0.001
Flowering 0.001
BARC-032679-09011 2 88.855 Yield 0.001
BARC-021647-04164 2 103.895 Height 0.005
BARC-046796-12751 3 27.779 Yield 0.005
BARC-017957-02482 3 38.175 Stem Vining 0.001
BARC-014699-01621 3 85.18 Height 0.005
BARC-049091-10809 5 47.268 Maturity 0.005
BARC-042853-08438 5 49.474 Maturity 0.005
BARC-059081-15595 5 55.158 Maturity 0.005
BARC-019085-03298 5 57.233 Maturity 0.005
BARC-029341-06154 5 64.367 Yield 0.005
BARC-056069-14029 6 16.388 Flowering 0.005
BARC-047995-10452 7 68.69 Stem Vining 0.001
BARC-021937-04237 8 9.566 Yield 0.005
BARC-022031-04262 9 5.329 Yield 0.001
BARC-055141-13084 9 6.863 Yield 0.001
BARC-020735-04704 10 80.177 Yield 0.005
BARC-040851-07854 11 44.651
Stem Vining 0.005
Flowering 0.005
Maturity 0.001
Table 4. A list of SNPs that Kruskal-Wallis single marker analysis listed as notably significant
(p≤ 0.005).
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BARC-042515-08280 13 54.922 Stem Vining 0.005
BARC-017917-02456 13 57.158 Stem Vining 0.005
BARC-065411-19443 14 13.099 Yield 0.001
BARC-044201-08645 14 89.965 Lodging 0.001
BARC-022009-04249 15 91.304 Flowering 0.005
BARC-042521-08287 16 24.374 Lodging 0.005
BARC-064455-18689 16 71.559 Yield 0.005
BARC-063551-18386 17 57.33 Maturity 0.005
BARC-040479-07752 18 9.437 Stem Vining 0.005
BARC-047516-12955 18 55.603 Yield 0.005
BARC-065047-19054 20 1.029 Yield 0.001
Table 4. Continued.
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SNP marker Chromosome Position (cM) Trait sub set G. max Allele G. soja Allele
BARC-021647-04164 2 103.895
Flowering 43.5008 37.25
Height 123.823 99.3333
Stem Vining 3.54472 1.83333
BARC-038333-10036 3 61.887 Height 123.643 98.4375
Stem Vining 3.53125 1.5625
BARC-046758-12733 3 63.125 Height 123.484 91.25
BARC-013865-01261 3 66.302 Height 123.643 98.4375
Stem Vining 3.53125 1.5625
BARC-029787-06340 5 86.746 Maturity 118.208 113.062
BARC-023203-03824 6 106.465 Yield 42.9448 49.575
BARC-038861-07350 6 132.414
Yield 42.9608 49.575
Lodging M1 4.97603 2.5
Stem Vining 3.51881 1
BARC-039923-07610 9 21.428 Height 122.628 91.25
BARC-047743-10393 9 21.503 Height 123.484 91.25
BARC-055301-13192 9 23.331 Height 123.484 91.25
BARC-028040-06720 9 23.81 Height 123.484 91.25
BARC-053591-11917 9 29.323 Height 123.484 91.25
BARC-028249-05804 9 39.511
Flowering 43.3892 37.3958
Lodging 4.99219 2.83333
Stem Vining 3.53683 1.25
BARC-026143-05278 9 39.513
Flowering 43.4242 37.3958
Height 123.682 94.3125
Lodging 4.98611 2.83333
Stem Vining 3.53829 1.25
BARC-042559-08304 9 91.916 Lodging 4.94707 3.16667
Stem Vining 3.48986 1.5
BARC-035255-07160 10 41.762 Maturity 118.246 113.062
BARC-051149-11016 10 47.348 Maturity M1 118.246 111.625
BARC-040851-07854 11 44.651 Stem Vining 3.55682 1.84375
BARC-042999-08498 11 55.607 Height 123.484 91.25
Table 5. A list of SNPs that were found to have a significant (p≤ 0.05) phenotypic difference,
determined by a T-test, between the G. max and G. soja allele as compared the average difference
between the G. max and G. soja alleles. M1= early maturation set, M2 = late maturation set. The
G. max and G. soja allele values represent raw trait values (flowering =days, maturity = days,
height = centimeters, yield = kg/hectare, stem vining= 1-5 score, lodging 1-10 score).
113
BARC-059773-16088 11 80.992 Lodging 4.99662 3.38889
Stem Vining 3.54167 1.83333
BARC-044711-08768 11 111.022 Yield 43.1071 33.05
Lodging 4.93105 7.58333
BARC-019775-04370 12 49.406 Flowering 43.4468 37.2639
BARC-025943-05179 12 55.638 Flowering 43.4468 37.2639
BARC-014411-01355 13 28.209 Lodging 4.97013 3.16667
BARC-028907-06042 15 25.476 Height 123.501 90.125
BARC-028159-05778 16 41.631 Flowering 43.3423 36.7083
BARC-059867-16171 16 46.048 Flowering M2 43.4022 36.7083
BARC-062135-17666 16 46.657 Flowering 43.4588 36.9167
Height 123.849 99.75
BARC-020357-04569 17 5.07 Stem Vining 3.5473 1.5
BARC-060011-16286 17 7.521 Stem Vining 3.5279 1.5
BARC-062213-17705 17 74.476 Yield M2 43.1425 38.0083
BARC-014395-01348 18 19.482 Yield 43.1377 36.3625
BARC-047516-12955 18 55.603 Flowering 43.1747 49.4861
Yield 43.1352 37.1917
BARC-050455-09643 20 49.925 Height 123.484 91.25
BARC-060361-16629 20 92.027 Lodging 4.92001 6.875
BARC-055173-13105 20 97.406 Flowering 43.3881 37.4583
BARC-048955-10759 20 111.847 Flowering 43.3881 37.4583
Table 5. Continued.
114
SNP Marker Chromosome QTL location Trait Gene Name SoyID
BARC-025955-05182 2 47cM Flowering TEM1 Glyma02g11060
BARC-016079-02059 2 48cM Flowering FEZ Glyma02g11900
BARC-020101-04452 3 62cM Flowering RAP2.7 Glyma03g33470
BARC-053497-11882 5 17cM Flowering AGL8 Glyma05g07380
BARC-059073-15592 5 77cM Maturity FKF1 Glyma05g34530
BARC-058647-17392 5 80cM Maturity SPA2 Glyma05g37070
BARC-048543-10663 6 80cM Flowering LFY Glyma06g17170
BARC-039223-07476 8 98cM Flowering AGL8 Glyma08g27680
BARC-021937-04237 8 9cM Maturity SPA2 Glyma08g02490
BARC-026035-05236 9 35cM Maturity
GI Glyma09g07240 Flowering
BARC-058901-15494 9 40cM Maturity PHYE Glyma09g11600
BARC-051771-11237 9 93cM Maturity AGL20 Glyma09g40230
BARC-051149-11016 10 47cM Maturity ATC Glyma10g08340
BARC-042953-08476 13 102cM Maturity TOE2 Glyma13g40470
BARC-041649-08056 13 68cM Flowering SFR6 Glyma13g31480
BARC-025915-05157 13 98cM Maturity ATC Glyma13g39360
BARC-016775-02320 16 27cM
Flowering FT5a Glyma16g04830
FT3a Glyma16g04840
Maturity FT5a Glyma16g04830
FT3a Glyma16g04840
BARC-063377-18348 16 8cM Flowering
LHY Glyma16g01980 Maturity
BARC-020069-04425 18 96cM Flowering COL2 Glyma18g51320
BARC-020457-04632 19 35cM Maturity FT3b Glyma19g28390
FT5b Glyma19g28400
BARC-010719-00713 20 109cM Flowering RF12 Glyma20g38050
BARC-055173-13105 20 97cM Flowering CRY2 Glyma20g35220
Table 6. A list of flowering related genes was obtained from (Kim, MY. et al. 2012). Using the
SoyBase database (http://soybase.org/), the physical location of significant flowering and
maturity SNPs were compared to the location of the flowering genes. Candidate genes were
selected if they located less than 500kb away from QTL-associated SNPs.
115
Chromosome Position (cM) Previously Identified QTL Name
5 17-22 R7 2-21, R3 2-21
6 16 Reprod 4-12
6 106-108 R3 2-11, Fflr 4-12
7 41.4 Reprod 4-32, Fflr 2-25
9 34-44 FT 3-33
11 44-45 Fflr 11-14
13 68 Reprod 4-42
16 27-47 R3 1-21, R7 1-21
18 0-8.4 FT 3-43
18 55-56 Fflr 9-21
Table 7. 10 previously identified flowering QTLs that overlap with currently identified
flowering QTLs. Tasma, IM. et al. 20011, Orf, JH. et al. 19992, Reinprecht, Y. et al. 20063, Gai, J. et al. 20074, Mansur, LM. et al. 19935
116
3.7 Figures
Figure 20. (A) A row of Glycine max compared to (B) a pot containing a single Glycine soja
plant. Image courtesy of Dr. Randall Nelson USDA at the University of Illinois Champaign-
Urbana.
117
Figure 21. Distribution of traits in the 115 F6 RIL population by year and by maturity set. (A)
Distribution of traits in 2009 and 2010 (n=115). Yield were measured only in 2009. The black
horizontal line indicates the distribution median and the cross indicates the distribution mean.
The box indicates the interquartile range, and black circles represent outliers. The red horizontal
line represents the mean of Williams 82. P-values were obtained using a Students T-Test.
Flowering time shows the time when 50% of plants have at least one flower on any node (R1),
and maturity time shows when 95% of seed pods reach full mature color (R8). Lodging is shown
in 1 – 10 scales where 1 represents Williams 82 and 10 reflects lodging in the G. soja parent.
Vining is shown in 1 – 5 scales where 1 is no vining as in Williams 82 and 5 is vining
represented by the G. soja parent.
118
Figure 22. Distribution of traits in the early (Matset1) and late (Matset2) maturation groups (n =
64 and n = 51, respectively). The black horizontal line indicates the distribution median and the
cross indicates the distribution mean. The box indicates the interquartile range, and black circles
represent outliers. The red horizontal line represents the mean of Williams 82. P-values were
obtained using a Students T-Test. Flowering time shows the time when 50% of plants have at
least one flower on any node (R1), and maturity time shows when 95% of seed pods reach full
mature color (R8). Lodging is shown in 1 – 10 scales where 1 represents Williams 82 and 10
reflects lodging in the G. soja parent. Vining is shown in 1 – 5 scales where 1 is no vining as in
Williams 82 and 5 is vining represented by the G. soja parent.
119
Figure 23. A multiple factor analysis (MFA) displaying the two-dimensional (dimension 1 and 2
represented by the horizontal and vertical dashed lines respectively) correlation structure among
the 115 F6 RILs. The blue arrows demonstrate the strength of trait association within a
dimension. The first dimension accounts for 45.26% of the total variation and the second
dimension accounts for 23.13% of the total variation.
120
Figure 24. Quantitative trait loci (QTL) and linkage map of BC2F6 RILs showing flowering and maturity QTLs in red and blue
respectively. Single marker analysis of QTLs was performed using the Kruskal-Wallis test (p≤ 0.01). The black arrows on these QTLs
directionally indicate the phenotypic affect of the Glycine max allele. Grey arrows within QTLs represent individual SNPs that have
an opposite phenotypic direction as compared to the general direction of the QTL. Black boxes within the linkage groups represent the
8 previously identified e-loci and potential candidate genes.
121
Figure 25. Quantitative trait loci (QTL) and linkage map of BC2F6 RILs showing flowering, maturity, height, yield, stem vining, and
lodging QTLs in red, blue, pink, light blue, green, and yellow respectively. Single marker analysis of QTLs was performed using the
Kruskal-Wallis test (p≤ 0.01). The black arrows on these QTLs directionally indicate the phenotypic affect of the Glycine max allele.
Grey arrows within QTLs represent individual SNPs that have an opposite phenotypic direction as compared to the general direction
of the QTL. Black boxes within the linkage groups represent the 8 previously identified e-loci and potential candidate genes.
122
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