University of Massachusetts - Amherst
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Masters Theses 1896 - February 2014
Dissertations and Theses
2012
RHYTHMIC GROWTH AND VASCULAR
DEVELOPMENT IN BRACHYPODIUM
DISTACHYON
Dominick A. Matos
University of Massachusetts - Amherst, dmatos@student.umass.edu
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Matos, Dominick A., "RHYTHMIC GROWTH AND VASCULAR DEVELOPMENT IN
BRACHYPODIUM DISTACHYON" (). Masters Theses 1896 - February 2014. Paper 929.
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Rhythmic Growth and Vascular Development in Brachypodium
distachyon
A Thesis Presented
By
Dominick A. Matos
Submitted to the Graduate School of the
University of Massachusetts Amherst in partial fulfillment
of the requirements for the degree of
MASTER OF SCIENCE
September 2012
Molecular and Cellular Biology
Rhythmic Growth and Vascular Development in Brachypodium
distachyon
A Thesis Presented
By
Dominick A. Matos
Approved as to style and content by:
___________________________________________________________
Samuel P. Hazen, Chair
___________________________________________________________
Tobias I. Baskin, Member
___________________________________________________________
Ana L.Caicedo, Member
__________________________________________
Barbara A. Osborne, Department Head
Molecular and Cellular Biology
ACKNOWLEDGEMENTS
I would like to thank my primary investigator, Samuel P. Hazen, for allowing me
the opportunity to conduct research in his lab and for being an outstanding and
knowledgeable mentor. Also, I would like to extend my gratitude to Benjamin J. Cole
from the University of California San Diego and Ian P. Whitney for their collaboration
during my research and helping with the data analysis. I want to thank current and past
members of the Hazen lab for the endless support and camaraderie. I would also like to
extend my appreciation to the members of my committee, Tobias I. Baskin and Ana L.
Caicedo, for agreeing to be a part of my committee and for all their help during this
process. I would like to acknowledge the Department of Energy for providing the funding
that made these projects possible. Lastly, I would like to thank the University of
Massachusetts Amherst for the opportunities I have had during my time as a student here.
iii
ABSTRACT
RHYTHMIC GROWTH AND VASCULAR DEVELOPMENT IN BRACHYPODIUM
DISTACHYON
SEMPTEMBER 2012
DOMINICK A. MATOS, B.S., UNIVERSITY OF MASSACHUSETTS AMHERST
M.S., UNIVERSITY OF MASSACHUSETTS AMHERST
Directed by: Professor Samuel P. Hazen
Plants reduce inorganic carbon to synthesize biomass that is comprised of mostly
polysaccharides and lignin. Growth is intricately regulated by external cues such as light,
temperature, and water availability and internal cues including those generated by the
circadian clock. While many aspects of polymer biosynthesis are known, their regulation
and distribution within the stem are poorly understood. Plant biomass is perhaps the most
abundant organic substance on Earth and can be used as feedstock for energy production.
Various grass species are under development as energy crops yet several of their
attributes make them challenging research subjects. Brachypodium distachyon has
emerged as a grass model for food and energy crop research. I studied rhythmic growth, a
phenomenon important to understanding how plant biomass accumulates through time,
and vascular system development, which has biofuel feedstock conversion efficiency and
yield. Growth rate changes within the course of a day in a sinusoidal fashion with a
period of approximately 24 hours, a phenomenon known as rhythmic growth. Light and
temperature cycles, and the circadian clock determine growth rate and the timing of rate
changes. I examined the influences of these factors on growth patterns in B. distachyon
using time-lapse photography. Temperature and, to a lesser extent, light influenced
growth rate while the circadian clock had no noticeable effect. The vascular system
transports important materials throughout the plant and consists of phloem, which
conducts photosynthates, and xylem, which conducts water and nutrients. The cell walls
of xylem elements and ground tissue sclerenchyma fibers are comprised of cellulose,
hemicelluloses, and lignin. These components are important to alternative energy
research since cellulose and hemicellulose can be converted to liquid fuel, but lignin is a
significant inhibitor of this process. I investigated vascular development of B. distachyon
by applying various histological stains to stems from three key developmental. My results
described in detail internal stem anatomy and demonstrated that lignification continues
after crystalline cellulose deposition ceases. A better understanding of growth cues and
various anatomical and cell wall construction features of B. distachyon will further our
understanding of plant biomass accumulation processes.
iv
TABLE OF CONTENTS
ACKNOWLEDGEMENTS.................................................................................................iii
ABSTRACT.........................................................................................................................iv
LIST OF FIGURES ............................................................................................................vii
CHAPTER
1. TEMPERATURE CYCLES DRIVE DAILY GROWTH RHYTHMS IN
BRACHYPODIUM DISTACHYON……….………………….……………………………………...……….... 1
1.1 Introduction ................................................................................................................ 1
1.2 Material and Methods ................................................................................................ 5
1.2.1 Plant material...................................................................................................... 5
1.2.2 Imaging system................................................................................................... 6
1.2.3 Image analysis .................................................................................................... 7
1.3 Results ........................................................................................................................ 8
1.3.1 Growth increases under warm light conditions .................................................. 8
1.3.2 Growth is arrhythmic under constant light or constant dark conditions ............ 9
1.3.3 Growth is arrhythmic under light dark cycles and constant temperature
conditions .................................................................................................................. 10
1.3.4 Temperature cycles induce rhythmic growth in constant light or constant dark
conditions……………....………………………………………………………..………………………..... 12
1.3.5 Growth follows temperature cycles even in inverted conditions………………… 13
1.4 Discussion ................................................................................................................ 14
2. VASCULAR TISSUE DEVELOPMENT AND DIFFERENTIAL DEPOSITION OF
LIGNIN AND CELLULOSE IN BRACHYPODIUM DISTACHYON ............................... 17
2.1 Introduction .............................................................................................................. 17
2.2 Material and Methods .............................................................................................. 21
2.2.1 Plant material.................................................................................................... 21
2.2.2 Whole plant measurements............................................................................... 21
v
2.2.3 Histochemistry.................................................................................................. 22
2.2.4 Image analysis and morphological measurements ........................................... 23
2.2.5 Statistical Analysis............................................................................................ 24
2.3 Results ...................................................................................................................... 24
2.3.1 Developmental stage and vascular anatomy identification .............................. 24
2.3.2 Cell wall biosynthesis in the stem occurs from elongation to senescence ....... 28
2.3.3 Lignin deposition continues from stem elongation to senescence……………….. 30
2.3.4 Crystalline cellulose deposition continues from stem elongation to
inflorescence emergence............................................................................................ 34
2.4 Discussion ................................................................................................................ 36
BIBLIOGRAPHY............................................................................................................... 40
vi
LIST OF FIGURES
Figure
Page
1. Leaf growth imaging system and leaf length analysis .....................................................7
2. Leaf growth in light dark and temperature cycle conditions ...........................................8
3. Leaf growth in constant conditions..................................................................................9
4. Leaf growth in light dark and constant temperature conditions ....................................11
5. Leaf growth in constant light or dark with temperature cycles .....................................13
6. Leaf growth in inverted light dark and temperature cycles ...........................................14
7. Three developmental stages for internode characterisation in Brachypodium
distachyon ..........................................................................................................................25
8. Whole plant characteristics of the three developmental stages in Brachypodium
distachyon ..........................................................................................................................26
9. Brachypodium distachyon vascular tissue anatomy ......................................................27
10 Internal characteristics of the three developmental stages in Brachypodium
distachyon ..........................................................................................................................28
11. Cell wall thickness increases throughout stem internode development.......................29
12. Cell wall thickness increases throughout stem internode development.......................30
13. Lignification increases throughout stem internode development ................................31
14. Lignin deposition increases throughout stem internode development.........................32
15. Lignin deposition increases throughout stem internode development.........................33
16. Crystalline cellulose deposition in Brachypodium distachyon stem internode
development.......................................................................................................................35
17. Crystalline cellulose deposition of the three developmental stages.............................36
vii
CHAPTER 1
TEMPERATURE CYCLES DRIVE DAILY GROWTH RHYTHMS IN
BRACHYPODIUM DISTACHYON
1.1 Introduction
Primary growth in plants is the product of cell division and elongation. Typically,
cell division occurs in the meristems where pluripotent undifferentiated cells are located.
Subsequently to cell division, some of the daughter cells replenish the meristem pool
while others differentiate and elongate. The elongation process is largely caused by
changes in turgor pressure via water intake and storage in the central vacuole accompanied
by loosening of the cell wall. This process results in increases in leaf, stem, and root
length. Interestingly, growth rate can vary throughout the day, manifested as a circadian
growth rhythm. As with myriad behavioral and physiological traits in plants, growth
rhythms are initiated by endogenous mechanisms and external cues (Harmer, 2009; Walter
et al., 2009; Farré, 2012). Two external cues implicated in driving growth rhythms are
daily light dark cycles and temperature cycles.
In Arabidopsis thaliana, light is perceived by phytochrome and cryptochrome
photoreceptors that directly interact with growth promoting factors as well as entrain the
circadian clock (Devlin & Kay, 2001; Nozue et al., 2007; Nusinow et al., 2011). In
constant light and temperature conditions, A. thaliana hypocotyl growth exhibits a growth
rhythm with a peak rate at subjective dusk (Dowson-Day & Millar, 1999). Several wellcharacterized clock mutants exhibit no such rhythm and thus this behavior is evidently the
result of circadian clock function (Dowson-Day & Millar, 1999; Nozue et al., 2007). On
1
the other hand, in the presence of light dark cycles, growth rhythms peak at dawn rather
than dusk (Nozue et al., 2007; Wiese et al., 2007). The concurrent effects of the circadian
clock that are revealed in the absence of external cues and light regulated events dictate
the timing of regulated growth processes (Nozue et al., 2007; Michael et al., 2008;
Nusinow et al., 2011). The mechanism underlying this behavior is similar to that of the
coincidence of external and internal cues that control photoperiodic flowering in A.
thaliana (Imaizumi, 2010). Expression of the growth activating basic helix-loop-helix
(bHLH) transcription factors PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and
PIF5 is repressed in the early evening by the circadian clock. Subsequent expression
results in late night growth that is ultimately repressed by light regulated degradation of
PIF4 and PIF5 proteins (Nozue et al., 2007). The components and behavior of the
circadian clock in numerous plant species appears to be relatively conserved and similar to
A. thaliana. This does not look to be the case for photoperiodic mechanisms and perhaps
this should come as no surprise considering responses to seasonal day length vary greatly
across species (Song et al., 2010). Daily growth rhythms are no exception (Walter &
Schurr, 2005; Farré, 2012).
Light dark cycles are clearly an important external cue that determines specific
time of day growth rhythms. Like A. thaliana, in the presence of light dark and
temperature cycles, Ricinus communi, Nicotiana tabacum, Flaveria bidentis, and Solanum
lycopersicum growth oscillations peak at dawn (Bertram & Lercari, 1997; Schmundt et al.,
1998; Wiese et al., 2007; Poire et al., 2010). On the other hand, other eudicot species such
as Populus deltoides and Vitis vinifera exhibit a maximum growth rate at dusk (Shackel et
al., 1987; Walter et al., 2005). The growth rates of some eudicot species peak during the
2
day as seen for Phaseolus vulgaris, Betula pendula, and Salix viminalis or during the night
as observed for Glycine max (Davies & Van Volkenburgh, 1983; Taylor & Davies, 1986;
McDonald et al., 1992; Ainsworth et al., 2005). Timing of peak growth rate persists in A.
thaliana, N. tabacum, R. communis, and Dendrantherna grandiflorum when temperature
is held constant in the presence of light dark cycles (Tutty et al., 1994; Nozue et al., 2007;
Poire et al., 2010). In constant light with temperature cycles, D. grandiflorum growth
rhythms were similar to growth in light dark cycles (Tutty et al., 1994). When light and
temperature are both held constant, A. thaliana, R. communis, and D. grandiflorum exhibit
a circadian clock regulated growth rhythm (Tutty et al., 1994; Nozue et al., 2007; Poire et
al., 2010). While the timing within a day may vary, light dark and temperature cycles as
well as the circadian clock regulate daily growth rhythms in eudicot species.
The mechanisms that regulate rhythmic growth in grasses are likely distinct in
some ways from eudicots. Rapid growth during the day has uniformly been observed in
numerous grasses including Zea mays, Oryza sativa, Sorghum bicolor, Sorghum vulgare,
Triticum aestivum, Hordeum vulgare, Agropyron desertorum, and Pseudoroegneria
spicata (Johnson, 1967; Gallagher & Biscoe, 1978; Acevedo et al., 1979; Cutler et al.,
1980; Kemp & Blacklow, 1980; Busso & Richards, 1992; Poire et al., 2010). While the
number of species described is limited to two, Z. mays and O. sativa, above ground growth
in monocots is arrhythmic in constant conditions (Poire et al., 2010). When temperature is
held constant, growth oscillations can still be seen in light dark cycles in both Z. mays and
Fescue arundinacea, but growth rate is greatest during the night and the rhythms are much
less robust (Durand et al., 1995; Poire et al., 2010). On the other hand, growth behavior in
constant light and temperature cycles mimics growth when both cues cycle (Poire et al.,
3
2010). Cell division and elongation increase with temperature in grass leaves (Watts,
1971; Sadok et al., 2007; Parent et al., 2010; Poire et al., 2010). One possible mechanism
is that temperature dependent enzyme activity coordinates the networks that influence
growth. Another mechanism of coordination may be through a common regulator that
senses temperature change and initiates a signal cascade. This latter possibility appears to
be a more plausible model considering the variable activity of growth associated enzymes
and photosynthetic rates measured within A. thaliana, Z. mays, and O. sativa (Parent et al.,
2010). Regardless, temperature is the most important external cue influencing daily
growth rhythms while the circadian clock appears to have no role in this respect.
As plants are sessile and some leaves and stems are rather large, growth can be
measured with the simple use of a ruler. Another common method is to measure the
displacement of a device attached to a leaf, referred to as a linear variable displacement
transducer (Meijer, 1968). Today, time-lapse photography of leaf, root, and stem are
common with high-resolution CCD cameras (Miller et al., 2007; Poire et al., 2010; Cole et
al., 2011; Iijima & Matsushita, 2011). Plants lack the ability to sense wavelengths above
far-red; therefore, infrared lighting can be used to illuminate plants in otherwise complete
darkness (Nozue et al., 2007; Poire et al., 2010; Cole et al., 2011). Image sequence
analysis can be performed using morphometric, particle tracking, or optical flow based
approaches. Using these methods, total length or area can be calculated for each image
within the time-lapse sequence. Indeed, numerous phenomic platforms have both
automated image acquisition and analysis (Granier et al., 2006; Jansen et al., 2009; Walter
et al., 2012).
4
It is essential to translate mechanistic understanding of growth regulation to energy
crops where total biomass accumulation is the most vital attribute. Currently, several
grasses are in various stages of development as energy crops including switchgrass,
Miscanthus sp., Z. mays, S. bicolor, and prairie grass mixtures (Tilman et al., 2006;
Dhugga, 2007; Rooney et al., 2007; Schmer et al., 2008; Dohleman & Long, 2009).
Several relevant differences between these species and the model system A. thaliana
dictate that one or more grass species should serve as the focus of in depth study. Yet,
energy crops have many features unfavorable to laboratory research, namely large and
redundant genomes, large stature and long lifecycles, and inadequate genetic and genomic
tools. On the contrary, Brachypodium distachyon exhibits many model system attributes
including a sequenced genome, notable transformation efficiency, small stature, and a
rapid life cycle (Initiative, 2010; Brkljacic et al., 2011). Here I investigated the effects of
light, temperature, and the circadian clock on daily growth rhythms in B. distachyon.
1.2 Material and Methods
1.2.1 Plant material
Dry seed of Brachypodium distachyon accession Bd21-3 was imbibed and
stratified in a wet paper towel at 6°C for sixteen days. Seeds were then sown towards one
edge of 10 cm pots containing potting mix (#2; Conrad Fafard Inc., Agawa, MA, USA).
Half of the soil was covered with 0.508 mm thick infrared absorbing paper followed by
0.254 mm infrared light absorbing paper (Edmund Optics; Barrington, NJ, USA). Plant
were cultivated and imaged in a Percival model CU36L6 Growth Chamber (Percival
5
Scientific, Perry, IA, USA). Light conditions were 62 to 74 µmol of photons.m-2.s-1. The
hot cold temperature parameters correspond to 28 ± 0.6°C and 12 ± 0.6°C, respectively.
Percival conditions were monitored and recorded using a HOBO U12 Data Logger (Onset
Computer Corporation, Bourne, MA, USA).
1.2.2 Imaging system
Time-lapse photography was conducted using modified Canon SD870 IS cameras
(Canon, Lake Success, NY, USA). This 8 megapixel digital camera has a 3.8x wide-angle
optical image stabilized zoom and a 28 mm lens. The camera was modified with a deep
black white infrared filter by Life Pixel (Mukilteo, WA, USA) in order to capture only the
infrared spectrum ranging from 750 to 1000 nm. The Canon Hack Development Kit
(CHDK) advanced operating system file on an 8 GB standard SHDC memory card along
with a DiskBoot BIN file allowed the Canon SD870 IS camera to boot into the CHDK
operating system via the firmware enhancement option found in the camera operating
system menu. A script was adapted from the freely available lapse.lua script written by
Fraser McCrossan to provide the camera with time-lapse capability featuring an internal
tick clock, fixed focus, camera flash disablement, and infinite capturing ability (File 1).
This new script was used to capture an image every 30 minutes with a -2 exposure and
macro and black and white settings enabled. The cameras were placed 50 mm away from
the pots (Fig. 1A). A 100 by 100 mm LED backlight emitting 880 nm infrared light
(Edmund Optics, Barrington, NJ, USA) was placed 90 mm away from the pots to the right
of the camera to illuminate the growing plants (Fig. 1A-B). The cameras and LED
backlight were placed on a horizontal plane as the soil surface (Fig. 1B).
6
Figure 1. Leaf growth imaging system and leaf length analysis. (A-B) Two black and white
infrared filtered CCD cameras each placed 0.5 cm from a (C) pot illuminated by an 880 nm
infrared light. (D) Each leaf in each frame was (E) converted to a binary image and (F)
skeletonized for automated length measurement. Visualization of sequentially cropped (G)
leaf images and (H) skeletonization from a time course experiment.
1.2.3 Image analysis
The images were compiled into movies using Quicktime Pro (Apple, Cupertino,
CA, USA) at a rate of 24 frames per second and captions were added to the movies using
Windows Live Moviemaker (Microsoft, Redmond, WA, USA). ImageJ was used to
analyze the image sequences (Abramoff et al., 2004). The first leaf image was converted
to a binary image (Fig 1C-E). Any gaps present in the foreground were colored with white,
and 2 to 3 morphological erosions were implemented (Fig 1E). Afterwards, plant images
were skeletonized and measured to determine leaf length (Fig 1F). This process was done
for every frame in which the leaf was visible (Fig 1G-H). Growth rate was calculated as
the leaf length measurement minus the leaf length from the previous measurement and
plotted as the average of every two hours.
7
1.3 Results
1.3.1 Growth increases under warm light conditions
I first characterized growth
under diurnal conditions of 12 hr
light, 28°C and 12 hr dark, 12°C
(LDHC) by measuring leaf length
(Fig. 2A-B). Growth rhythms were
observed with a 24 hr period. Growth
rate dramatically increased within the
first hour of day and peaked at an
average of 8.5 hr after lights
whereupon it decreased rapidly
following the transition from light to
dark (Fig. 2B, Movie 1-2). The
length of the first leaf increased by an
average of 5.2 mm during hot light
Figure 2. Leaf growth in light dark and
temperature cycle conditions. (A) Temperature
(black) and light intensity (gray) of a 12 hr
light:12 hr dark temperature cycle time course.
(B) Leaf length (gray) and growth rate (black) of
the first leaves. Leaf length data are means ± SEM,
n = 6 - 7.
conditions and 1.4 mm during cold
dark conditions. Accordingly, the
average growth rate was considerably
greater during simulated day
conditions, 432 µm/hr, than nighttime condition,120 µm/hr.
8
1.3.2 Growth is arrhythmic under constant light or constant dark conditions
To elucidate the effect of the circadian clock on growth rhythms, I measured leaf
length under constant light and temperature conditions. After entrainment in 5 days of
LDHC, growth condition were changed to constant light and 28°C, LLHH (Fig. 3A).
Growth remained constant, similar to what was observed during daytime condition in the
diurnal time course (Fig. 3B, Movie 3-4). Leaf length increased by an average of 7.2 mm
during subjective day and 7.3 mm during subjective night. Likewise, growth rate was
continuous with a subjective day average of 589 µm/hr and subjective night average of
Figure 3. Leaf growth in constant conditions. (A) Temperature (black) and light intensity
(gray) of a 12 h light:12 h dark temperature cycle time course transferred to constant (A)
28°C light or (C) 12°C dark conditions. (B, D) Leaf length (gray) and growth rate (black) of the
first leaf. Leaf length data are means ± SEM, n = 4 - 7.
9
603 µm/hr (Fig. 3B).
To test if constant light drives growth and masks a circadian clock regulated
growth oscillation, I measured leaf length under constant dark and 28°C conditions,
DDHH, following LDHC entrainment (Fig. 3C). Initially, growth persisted similar to
daytime conditions (Fig 3D, Movie 5-6). Leaf length increased by an average of 4.4 mm
during subjective day and 4.7 mm during subjective night. Within the course of the first
day of constant darkness, growth rate began to diminish to a rate of 250 µm/hr likely the
effect of an absence of photosynthetic activity. The average growth rate in DDHH was 364
µm/hr during the subjective day and 389 µm/hr during the subjective night. As might be
expected, growth was slower in DDHH than in LLHH. Nonetheless, the similar and
constant growth rate observed under 28°C regardless of constant light or constant dark
conditions suggests that the circadian clock does not influence growth rate in B.
distachyon.
1.3.3 Growth is arrhythmic under light dark cycles and constant temperature
conditions
I next investigated the effects of light dark cycles on growth rhythms. Following
LDHC entrainment, growth conditions were changed to a constant temperature of 28°C
with 12 hr light and 12 hr dark, LDHH (Fig. 4A). Growth was arrhythmic and remained
somewhat constant, similar to what was observed during daytime condition in the diurnal
time course (Fig. 4B, Movie 7-8). Leaf length increased by an average of 4.6 mm during
the subjective day and 4 mm during the subjective night. Accordingly, the average growth
10
rate was greater during subjective day, 391 µm/hr, than in subjective night, 351 µm/hr
(Fig. 4B).
To further elucidate the effects of light on growth, I measured leaf length under a
constant temperature of 12°C with 12 hr light and 12 hr dark, LDCC (Fig. 4C). Growth
rate was similar to the rate observed in the nighttime conditions of LDHC conditions with
no discernible rhythm to growth (Fig. 4D, Movie 9-10). Leaf length increased by 0.8 mm
with an average rate of 65 µm/hr at subjective day by 1.1 mm with an average of 95 µm/hr
Figure 4. Leaf growth in light dark and constant temperature conditions. (A)
Temperature (black) and light intensity (gray) of a 12 hr light:12 hr dark temperature
cycle time course transferred to constant (A) 28°C or (C) 12°C conditions with
continued light dark cycles. (B, D) Leaf length (gray) and growth rate (black) of the first
leaf. Leaf length data are means ± SEM, n = 5 - 9.
11
during subjective day (Fig 4D). Total growth was much slower during LDCC, 80 µm/hr,
than LDHH, 378 µm/hr (Fig 4B, D). The arrhythmic growth observed in these growth
conditions indicates that light dark cycles do not influence growth rhythms in a noticeable
manner.
1.3.4 Temperature cycles induce rhythmic growth in constant light or constant dark
conditions
Considering the waveform properties observed in LDHC were not emulated in the
four constant temperature conditions, LLHH, DDHH, LDHH, or LDCC, I measured leaf
growth in constant light with a 12 hr 28°C and 12 hr 12°C temperature cycle, LLHC (Fig.
5A). Growth was similar to that observed in LDHC (Fig. 5B, Movie 11-12). Leaf length
increased by an average of 5.4 mm during the day and 1.5 m during the subjective night.
Accordingly, the growth rate was considerably greater during the day, 453 µm/hr,
than nighttime condition,124 µm/hr. Similar to LDHC, daytime growth rate increased
gradually and peaked 3 hr prior to subjective night (Fig. 5B).
To test if temperature could drive growth rhythms in the absence of light, I
measured leaf length under constant darkness with a 12 hr 28°C and 12 hr 12°C
temperature cycle, DDHC (Fig. 5C). As in LLHC, growth patterns were similar to LDHC
(Fig. 5D, Movie13-14). The length of the first leaf increased by an average of 6.5 mm
during the subjective day and 1.8 mm during subjective night. The average subjective
day and night growth rate was 542 and 152 µm/hr, respectively. While growth rate was
faster during DDHC than LLHC, the peak rate occurred at three hours before subjective
12
dusk in both conditions (Fig. 5B, D). Taken together, these results strongly suggest that
temperature cycles can drive rhythmic growth.
Figure 5. Leaf growth in constant light or dark with temperature cycles. (A)
Temperature (black) and light intensity (gray) of a 12 hr light:12 hr dark temperature
cycle conditions transferred to constant (A) light or (C) dark conditions with continues
temperature cycles. (B, D) Leaf length (gray) and growth rate (black) of the first leaf. Leaf
length data are means ± SEM, n = 4 - 6.
1.3.5 Growth follows temperature cycles even in inverted conditions
Since growth rhythms were observed in conditions with either temperature or light
dark cycles, I tested the effect of inverting the treatments. Following growth in LDHC for
three days, temperature cycles were inverted to 12°C during the light period and 28°C
13
during the dark period, thus LDCH
(Fig. 6A). Leaf length increased by 5.3
mm in response to 28°C dark
conditions and only by 1.1 mm in 12°C
light conditions (Fig. 6B, Movie 1516). Appropriately, growth rate was
greater at the daytime temperature in
the dark, 431 µm/hr, than the nighttime
temperature in the light, 90 µm/hr.
Interestingly, peak growth rate occurred
3 hr into the 28°C dark period similarly
to what was seen in LDHH and LDCC
(Fig 4B, 4D, 6B). Also, amplitude
Figure 6. Leaf growth in inverted light dark
and temperature cycles. (A) Temperature
(black) and light intensity (gray) of a 12 h
light:12 h dark temperature cycle conditions
transferred to 12°C days and 28°C nights. (B)
Leaf length (gray) and growth rate (black) of
the first leaf. Leaf length data are means ±
SEM, n = 5.
changes when conditions changed from
nighttime to daytime and vice versa
were sharp and occurred within 5 hr
evoking what is typically seen when
conditions are changed from 28°C to 12°C in LDHC, LLHC, and DDHC (Fig 5B, 5D,
6B).
1.4 Discussion
Growth rhythms of B. distachyon under diurnal growth conditions were typical of
other grass species grown both in the field and controlled environments with a peak
growth rate observed during the day (Acevedo et al., 1979; Busso & Richards, 1992; Poire
14
et al., 2010). In the absence of external cues, oscillations were lost, demonstrating that the
circadian clock had no influence on daily growth rhythms. This observation has also been
noted in both Z. mays and O. sativa (Poire et al., 2010). Interestingly, the lack of growth
rhythms in constant conditions is not due to an absence of a circadian clock in monocots
(McClung, 2010; Song et al., 2010). Growth was arrhythmic in light dark cycles and
constant temperature conditions, a result unlike what was observed in Z. mays and F.
arundinacea (Durand et al., 1995; Poire et al., 2010). It was suggested that plant water
relations may have played a role as evaporative demand increases over the course of a day
and decreases at night; growth responds inversely in Z. mays (Sadok et al., 2007; Poire et
al., 2010). In our study, growth was fastest during the subjective day.
Temperature is clearly a major driver of growth rate in B. distachyon, a result also
observed in other monocots such as Z. mays O. sativa, and F. arundinacea (Watts, 1971;
Durand et al., 1995; Parent et al., 2010; Poire et al., 2010). Growth oscillations during
temperature cycles in constant light and darkness were very similar to the ones seen
during the diurnal time course suggesting that temperature was an important cue driving
growth oscillations. Also, in constant 28°C and light, dark, or light dark conditions,
average growth rate during the first and second 12 hr period of a day followed the faster
pace seen during daytime conditions of the diurnal time course. Plants grown in constant
12°C and light dark cycles had a slow growth rate similar to the diurnal nighttime
conditions. Taken together, growth rate appears to be influenced by temperature. The
importance of the effect of temperature was more evident when light dark and temperature
cycles were inverted and growth was always greatest at 28°C regardless of light
conditions. The stems of two eudicot species, D. grandiflorum and L. esculentum, have
15
been shown to exhibit similar behavior in inverted light dark and temperature cycles
(Bertram & Karlsen, 1994; Tutty et al., 1994). To our knowledge, this observation has not
been made previously in monocots.
The effects of light, temperature, and the circadian clock on the daily growth
rhythms of B. distachyon and other monocots are distinct from eudicots. This may be the
result of differences in the exposure of the apical meristem to temperature (Walter et al.,
2009). The eudicot growth zone is typically located apically and relatively exposed to the
environment. In monocots, the growth zone is often surround by leaf sheathes. As it is not
exposed to light, the grass growth zone is not photosynthetically active and thus the
regulation of this process typically regulated by the circadian clock is unnecessary
(Harmer, 2009). Perhaps with buffered temperature exposure and restricted light exposure,
there is a limited or nonexistent role for growth signaling from the circadian clock in
grasses. An intriguing question is whether growth rate in monocots is a passive reaction
sensitive to temperature either through changes in enzyme activity or biomechanics of
stress physiology, or actively regulated similar to the action of photoreceptors by a
temperature sensing mechanism.
16
CHAPTER 2
VASCULAR TISSUE DEVELOPMENT AND DIFFERENTIAL DEPOSITION OF
LIGNIN AND CELLULOSE IN BRACHYPODIUM DISTACHYON
2.1 Introduction
Vascular tissue first emerged in land plants in the mid-Silurian period. This
development allowed vascular plants to grow large and colonize areas further away from
bodies of water. The vascular systems of early tracheophytes were organized in a prostele
pattern of a single column. Subsequent diversification included the appearance of
numerous types of arrangements of vasculature and among the most advanced and
diverse are the angiosperms (Worsdell, 1902; Ye, 2002). Angiosperms first appeared in
the fossil record in the early Cretaceous period and subsequently diversified 140-150
million years ago into an array of plant life including eudicots and grass (Chaw et al.,
2004). Along with numerous other distinctions, eudicots and monocots developed unique
vascular patterning and internal anatomy (Ye, 2002).
The vascular system transports water and nutrients throughout the plant and is
comprised of xylem and phloem found conjointly in bundles. The phloem consists of
bilateral pathways that conduct photosynthates produced mostly in leaves to the nonphotosynthetic tissues such as roots and seeds. Hormones, proteins, and nucleic acids, for
example, are also ferried by these cells. Living companion cells are interspersed
throughout interconnected conductive yet dead sieve elements to perform cellular
functions. Xylem stores and transports water, mineral nutrients and hormones from the
roots shootward. These cells are either conductive tracheids and vessel elements or non-
17
conducting xylary parenchyma cells. Both the tracheids and vessel elements have thick
secondary cell walls, a characteristic that provides the mechanical strength necessary to
withstand the negative pressure generated during water transport. After completing
secondary cell wall biosynthesis, vessel elements are generally thought to undergo cell
death and are perforated at both ends to form the final vessel, a continuous hollow
column where water transport occurs. On the other hand, tracheids do not form a vessel
(Carlquist & Schneider, 2002).
In the eudicot stem, vascular bundles are fused in a ring to create a pattern known
as eustele. In the grass stem, the vascular tissue is organized in an atactostele pattern
described as vascular bundles scattered throughout the ground tissue. Within the vascular
bundle, the phloem is outside of the xylem. The sieve elements and their companion cells
are generally smaller than their xylem counterparts. Within the xylem, the vessel
elements typically are larger and have thicker cell walls than tracheids (Ye et al., 2002).
The area between the vascular bundles, the interfascicular region, may be comprised of
two different cell types in both monocots and eudicots: parenchyma and sclerenchyma
(Zhong et al., 2001). Parenchyma cells typically have a large central vacuole to facilitate
storage of polysaccharides, fats, and even proteins. These very large cells are
predominantly found in the pith although in some species they can be found in vascular
bundles and in the interfascicular region. They remain alive throughout the life cycle of
the living plant and have only a primary cell wall. On the other hand, sclerenchyma cells
undergo cell death following secondary cell wall biosynthesis and provide mechanical
support. In monocots, sclerenchyma fibers form the bundle sheath, a layer of protective
fibers that surround the vascular bundle and separate phloem from xylem. In dicots, the
18
cortex, which is the layers of cells found between the epidermis and vascular tissue, is
made up of collenchyma and parenchyma cells. Collenchyma cells have thick primary
cell walls and provide support to the stem. In monocots, these cells tend to be absent
although the cortex may still refer to layers of ground tissue found just below the
epidermis and above the outermost vascular bundle. Chlorenchyma cells can also be
found in both dicots and monocots, and are characterized as having chloroplasts and thin
primary cell walls. These photosynthetic cells tend to be located near the epidermis when
present.
A defining aspect of plant cell function is the wall. Following cell division,
primary cell walls are formed and thicken during cell elongation. Once a cell has taken
final shape, some specialized cell types, which include tracheary elements and
sclerenchyma cells, undergo further wall thickening by secondary cell wall biosynthesis
(Cosgrove, 2005). Cell walls are mostly comprised of three different components:
cellulose, hemicellulose, and lignin. The most abundant polysaccharide in the majority of
tissues is the glucan cellulose, which exists as an unbranched chain containing up to
15,000 β-1,4-linked glucose molecules (Somerville, 2006). The glucan chains are crosslinked to each other via hydrogen bonds and in turn are thought to spontaneously
assemble to form cellulose microfibrils that provide tremendous tensile strength to plant
cell walls and can exist in crystalline, para-crystalline, and non-crystalline states (Tomme
et al., 1995; Genet et al., 2005; Harris & DeBolt, 2008). In contrast, the shorter
hemicelluloses are chemically and physically more complex and their monomer
compositions vary among species, tissues and cell types within an individual plant
(Scheller & Ulvskov, 2010). For example, eudicots contain large amounts of pectin and
19
xyloglucan, while commelinoid monocotyledons, including the grasses and cereals, have
little pectin, large amounts of glucuronoarabinoxylan and a unique hemicellulose, mixed
linkage (1,3;1,4)-β-glucan (Vogel, 2008; Scheller & Ulvskov, 2010). Lignin is built from
three monolignols: p-coumaryl, coniferyl, and sinapyl alcohols that polymerize to form phydroxyphenyl, guaiacyl, and syringyl phenylpropanoid units (Bonawitz & Chapple,
2010). Crosslinking lignin with hemicellulose in secondary cell walls of vascular tissue
increases hydrophobicity and thus gives these functional tissues the capacity to efficiently
conduct water (Donaldson, 2001). Lignin also provides the structural rigidity needed to
keep the plant continuously erect as it grows.
In addition to a long history of serving as sources of fiber for paper making and
other applications, plant cell wall polysaccharides can be saccharified and fermented by
some microorganisms that make byproducts capable of functioning as fuel (Carroll &
Somerville, 2009). Therefore, the biosynthesis of plant cell walls and the relative
efficiencies with which they can be converted to sources of energy is of keen interest.
Directly working with cultivated species or emerging bioenergy crops would be ideal, but
several attributes make them challenging subjects. In general, food and energy crops are
large, requiring considerable space for cultivation, and have relatively long life cycles.
Crops also tend to have large and redundant genomes (Bennett & Leitch, 1995). Recently,
Brachypodium distachyon has emerged as a model species for various food and
bioenergy crops (Draper et al., 2001). It exhibits most of the model system properties of
Arabidopsis thaliana, but as a grass B. distachyon serves as a model for potential energy
crops such as Panicum vergatum, Sorghum bicolor, and Miscanthus sp., as well as for the
cereal crops that constitute a large part of the world’s diet (Catalán & Olmstead, 2000;
20
Brkljacic et al., 2011). Here I investigate the deposition of lignin and cellulose and
describe the anatomy of the B. distachyon stem.
2.2 Material and Methods
2.2.1 Plant material
Dry seed of B. distachyon accession Bd21-3 was imbibed and stratified in a wet
paper towel at 6°C for seven days. Seeds were then sown in 10 cm pots containing
potting mix (#2; Conrad Fafard Inc., Agawa, MA, USA). Growth chamber temperature
was maintained at 20 °C with 20 h light:4 h dark cycles at a fluence rate of 220 µmol of
photons.m-2.s-1 and relative humidity of 67 to 69. For further histochemical analysis, the
first internode of the tallest stem was removed from plants of three different stages. The
first stage, stem elongation, corresponded to when the first internode above the crown of
the tallest stem was elongating. The second stage, inflorescence emergence, was when the
first internode was completely elongated and the inflorescence began to emerge from the
flag leaf. The third stage, senescence, was when the first internode has senesced and the
stem reached its maximum height with all of the leaves showing signs of senescence.
2.2.2 Whole plant measurements
Tiller count was recorded as the number of stems per plant and height as the
tallest tiller per plant to the tip of the inflorescence of fully senesced plants. Biomass
accumulation was quantified as the weight of the total above ground biomass. Internode
length was determined by removing the leaf sheath and imaging the tissue using a stereo
21
dissecting Leica MZ16 F microscope (Leica Microsystems, Buffalo Grove, IL, USA) and
the line measurement feature of ImageJ (Abramoff et al., 2004).
2.2.3 Histochemistry
Cross-sections of the first internode were made manually using a No. 11 scalpel
blade and a Nikon SMZ445 dissecting microscope (Nikon, Melville, NY, USA). Sections
were transferred to 1.5 mL Eppendorf microcentrifuge tubes containing distilled water.
Cross-sections were subjected to two different histological stains: toluidine blue or the
Wiesner reagent. Following a 30 second treatment with 7.4 µM toluidine blue, sections
were rinsed twice with distilled water. To stain and visualize lignin, sections were treated
with the Wiesner reagent (79 µM phloroglucinol-ethanol in 13.7 mM HCl) for 2 minutes.
Stained cross-sections were mounted on microscope slides and visualized using a Nikon
Eclipse E200MV R microscope (Nikon, Melville, NY, USA) attached to a PixeLINK 3
MP camera (PixeLINK, Ottawa, Canada). Images were captured using the associated
PixeLINK uSCOPE software (PixeLINK, Ottawa, Canada) and further processed with
Adobe Photoshop CS5.5 (Adobe, Waltham, MA, USA). The cellulose-binding module
CBM3a (PlantProbes, Leeds, England) was used to detect crystalline cellulose content
(Blake et al., 2006). Stem cross-sections were rinsed twice with phosphate-buffered
saline (PBS; 33 mM Na2HPO4, 1.8 mM NaH2PO4 and 140 mM NaCl, pH 7.2) prior to
incubation with 100 µL of 10 µg/mL CBM3a diluted in PBS for 1 hour. The solution was
then removed, and two 5 minute long washes were followed by two rapid changes of
PBS. The cross-sections were then treated with 100 mL of anti-His antibody produced in
mouse (Sigma-Aldrich, St. Louis, MO, USA) and diluted in PBS at a 1:1000 ratio for 1
22
hour. The solution was then removed and two five minute long washes followed by two
rapid changes of PBS. The cross-sections were then treated with 100 µL of rabbit antimouse antibody conjugated to Texas Red fluorophore (Invitrogen, Grand Island, NY,
USA) and diluted in PBS at a 1:100 ratio for 1 hour. Afterwards, the solution was
removed and two 5 minute washes were followed by two changes of PBS. Fluorescence
microscopy was performed using a Leica MZ16 F microscope equipped with a mercury
bulb attached to a Leica DFC300FX 1.4 MP camera (Leica Microsystems, Buffalo
Grove, IL, USA). The violet filter (425/40 nm) was used to visualize lignin
autofluorescence, and the Texas Red filter (560/40 nm) was used to visualize Texas Red
fluorescence. Images were captured using the Image-Pro Plus imaging software (Media
Cybernetics, Bethesda, MD, USA) and further processed using Adobe Photoshop CS5.5.
2.2.4 Image analysis and morphological measurements
ImageJ was used to analyze images by automatically measuring selected areas of
interest after scale calibration (Abramoff et al., 2004). Stained whole stem images were
used to observe total vascular bundle count. To measure stem cross-section and vascular
bundle area, the ImageJ freehand selection tool was used to trace the appropriate
anatomy. For interfascicular region measurements, the area in between vascular bundles
was traced using the polygon tool. For vascular bundle cell wall thickness, lines was
drawn across the cell walls of vessels and four adjacent bundle fibers and then averaged.
For interfascicular region cell wall thickness, lines were drawn across the adjacent cell
walls of the first and second row of cells outside the bundle sheath of a vascular bundle
and then averaged. The adjacent cell walls of the second and third row of cells were also
23
measured using the previous technique. For the various fluorescence quantification
measurements, the corrected total fluorescence formula was used (Burgess et al., 2010).
The freehand selection tool was used to trace vascular bundles, interfascicular regions,
and the whole stem cross-section with the square select tool being used to select
background regions to quantify the background fluorescence of each image.
2.2.5 Statistical Analysis
For each measurement, 10 to 25 independent plants were sampled. Analysis of
variance and Dunnett’s contrasts were performed in R v2.15.0.
2.3 Results
2.3.1 Developmental stage and vascular anatomy identification
I selected three developmental stages to characterize internode and vascular
development in B. distachyon: stem elongation, inflorescence emergence, and
senescence. For all three stages, the first stem internode above the crown was sampled.
During the first developmental stage sampled, the first and only internode was elongating
at the base of the tallest tiller (Fig. 7A-B). This was the first appearance of stem tissue,
which occurred 18 to 22 days after germination. At the second stage, the inflorescence
first emerged from the flag leaf sheath (Fig. 7C-D). This developmental stage occurred 25
to 30 days after germination. These first two stages are roughly equivalent to stages 30
and 51 of the BBCH-scale for cereals (Lancashire et al., 1991). The third stage sampled
was when the plants had reached their maximum height and the first internode and basal
24
Figure 7. Three developmental stages selected for internode characterization in
Brachypodium distachyon. (A, C, E) Intact and (B, D, F) dissected plants when (A-B) the
first internode was elongating, (C-D) the first inflorescence was emerging, and (E-F) the
first internode was senesced. FI, first internode; S, inflorescence. Bars = 5 cm. Remaining
internodes are marked with an asterisk.
leaves were completely senesced (Fig. 7E-F). This stage, which occurred 39 to 45 days
post germination, is more difficult to equate with the BBCH crop phenology system as it
describes grain development characteristics at senescence with developmental stages. The
number of stems per plant from the elongation stage to senesce did not change (Fig. 8A).
Total stem height significantly increased at each stage by nearly 10 cm, and fresh weight
significantly increased from 144 mg to 394 mg (Fig. 8B). The length and width of the
25
first internode increased significantly
before inflorescence emergence, but not
after (Fig. 8C). These three stages
encompass nearly the complete
development of a stem internode.
I characterized numerous
anatomical features of the stem at all
three developmental stages.
Brachypodium distachyon possesses an
atactostele arrangement of vascular
bundles with two circles at the periphery
of the stem (Fig. 9A). The innermost
vascular bundles are considerably larger
than the outer ring of bundles. The cell
types within the vascular bundles exhibit
a pattern typical of other monocots with
Figure 8. Whole plant characteristics of
the three developmental stages in
Brachypodium distachyon. (A) Stem
count. (B) Stem height (black) and stem
weight (gray). (C) Internode length (black)
and stem area (gray). Growth stages
correspond to stem elongation (E),
inflorescence emergence (F), and
senescence (S). Data are means ±
standard deviation, n = 13 - 25. Points
annotated with the same letter are not
significantly different at P < 0.05.
the phloem at the outside, and the xylem
oriented towards the center of the stem
(Fig. 9C). Each vascular bundle is
surrounded by bundle sheath cells that
also separate the xylem and phloem. The
xylem is comprised of large vessels located at the interior end and both sides of the
bundle with tracheids located in between the vessels. While not very
26
Figure 9. Brachypodium distachyon vascular tissue anatomy. (A) Cross section of
whole stem and (B) higher magnification of the first stem internode. Red, inner vascular
bundles; pink, outer vascular bundles; cyan, interfascicular region compromised mostly
of sclerenchyma fibers; gray, pith; lime green, chlorenchyma and sclerenchyma cells
comprise the cortex; brown, epidermis. (C) High magnification of a vascular bundle.
Green, bundle sheath (B); dark purple, phloem (P); blue, xylem vessels (XV); orange,
xylem tracheids (XT); magenta, lacuna (Lc); yellow, xylem parenchyma cells (XP). (A-B) Bar
= 0.1 mm, (C) bar = 0.01 mm.
pronounced, some bundles have a lacuna, which is a region of variable size and shape
where protoxylem may have existed. The areas in between the vascular bundles, also
known as the interfascicular region, are mostly comprised of sclerenchyma fibers (Fig.
9B). These fibers along with chlorenchyma are also located in the layer of cells between
the epidermis and outer vascular bundle, a region referred to as the cortex. The center of
the stem, sometimes referred to as the pith, is populated with parenchyma cells that are
larger than surrounding cell types (Fig. 9B). A few parenchyma cells are located in the
interfascicular region since the outermost exterior of the pith can be located in between
inner vascular bundles (Fig. 9B). Interestingly, all of the vascular bundles were formed at
or before the point of stem elongation, as the number did not significantly change (Fig.
10A). Similarly, the size of the vascular bundles did not change over time with an
average size of 3100 µm2 for outer bundles and 7800 µm2 for inner bundles (Fig. 10B).
On the other hand, the area between the vascular bundles significantly increased by 5300
µm2 from stem elongation to inflorescence emergence (Fig. 10C). Therefore, the increase
27
in interfascicular region size accounts for
the increase in stem area between
elongation and flowering and not a
change in vascular bundle size.
2.3.2 Cell wall biosynthesis in the stem
occurs from elongation to
senescence
First internode cross-sections were
treated with a polychromatic dye,
toluidine-blue, that stains polysaccharides
violet and lignin turquoise allowing for
the visualization of plant cell walls. At
stem elongation, the cells of the vascular
bundle appear darker and have thicker
cell walls than other cell types (Fig.11AB). At inflorescence emergence, the cells
Figure 10. Internal characteristics of the
three developmental stages in
Brachypodium distachyon. (A) Vascular
bundle count. (B) Inner (black) and outer
(gray) vascular bundle area. (C)
Interfascicular region area. Data are means ±
standard deviation, n = 20 - 38. Points
annotated with the same letter are not
significantly different at P < 0.05.
of both the vascular bundle and
interfascicular region appear slightly
darker and have noticeably thickened cell
walls (Fig. 11C-D). At senescence, these
same cell types appear darker, and their cell walls are substantially thicker (Fig. 11E-F).
For both inner and outer vascular bundles, the total cell wall size of adjacent xylem
28
Figure 11. Cell wall thickness increases throughout stem internode
development. Whole stem (A, C, E) and higher magnification (B, D, F) of
Brachypodium distachyon cross-sections stained with toluidine-blue. (A-B)
Elongating, (C-D) inflorescence emergence, and (E-F) senescenced stem
internode transverse cross-sections. Images were taken using brightfield
microscopy. Bars = 0.1 mm.
29
vessels and bundle sheath significantly increased from stem elongation to senescence
(Fig. 12A). This growth was quite dramatic with cell wall thickness increasing from 2.7
to 4.6 µm for inner vascular bundles and from 2.0 to 3.3 µm for outer vascular bundles.
For the interfascicular region, wall thickness of neighboring sclerenchyma also increased
significantly from stem elongation to senescence (Fig. 12B). The sclerenchyma closest to
the bundle sheath thickened from 1.7 to
5.0 µm while the sclerenchyma second
nearest to the bundle thickened from
1.7 to 4.1 µm. Thus, cell wall
biosynthesis appears to continue
throughout development in both the
vascular bundle and interfascicular
region.
2.3.3 Lignin deposition continues
from stem elongation to senescence
Stem cross-sections were
treated with the Wiesner reagent to
Figure 12. Cell wall thickness increases
throughout stem internode development.
(A) Cell wall thickness of adjacent vessel
and bundle fibers of inner (black) and outer
(gray) vascular bundles. (B) Cell wall
thickness of adjacent sclerenchyma nearest
(black) and second nearest (gray) to the
bundle sheath. Data are means ± standard
deviation, n = 96 - 100. Points annotated
with the same letter are not significantly
different at P < 0.05.
30
observe changes in lignin deposition
across development. The Wiesner
reagent stains lignin yellow at low
concentrations and becomes
increasingly red at higher
Figure 13. Lignification increases throughout stem internode development.
Whole stem (A, C, E) and higher magnification (B, D, F) of Brachypodium
ditachyon cross sections of all three developmental stages stained with the
Wiesner reagent. (A-B) Elongating, (C-D) inflorescence emergence, and (E-F)
senescenced stem internode transverse cross-sections. Images were taken
using brightfield microscopy. Bars = 0.1 mm.
31
Figure 14. Lignin deposition increases throughout stem internode
development. Whole stem (A, C, E) and higher magnification (B, D, F) of
Brachypodium distachyon cross sections of all three developmental stages
observing the autofluorescence of lignin. (A-B) Elongating, (C-D)
inflorescence emergence, and (E-F) senescenced stem internode transverse
cross-sections. Images were taken using wide field epifluorescence
microscopy. Bars = 0.1 mm.
32
concentrations. At stem elongation,
the vascular bundles stained a pale
red while the interfascicular region
appeared bright yellow (Fig. 13A-B).
At inflorescence emergence, the
vascular bundles stained a more
distinctive red and the interfascicular
region a darker yellow (Fig. 13C-D).
At senescence, the vascular bundles
and interfascicular region both
stained a dark red (Fig. 13E-F). Since
lignin is autofluorescent, fluorescence
microscopy was used to further
observe and quantify lignin content
within stems. When elongating, the
vascular bundles and interfascicular
region faintly appeared and was more
Figure 15. Lignin deposition increases
throughout stem internode development.
(A) Corrected total autofluorescence of
whole stem, (B) inner (black) and outer (gray)
vascular bundles, and (C) interfascicular
region. Data are means ± standard deviation,
n = 16 - 27. Points annotated with the same
letter are not significantly different at P <
0.05.
apparent at higher magnifications
(Fig. 14A-B). At inflorescence
emergence, both the vascular bundles
and interfascicular region were
noticeably more fluorescent (Fig. 14C-D). At senescence, the vascular bundles were
especially bright, and the interfascicular region became strikingly more fluorescent (Fig.
33
14E-F). I then quantified the total corrected lignin autofluorescence of the whole stem,
vascular bundles, and interfascicular region. The whole stem significantly increased in
total autofluorescence from stem elongation to senescence (Fig. 15A). Accordingly, both
inner and outer vascular bundles increased significantly in total autofluorescence over
time (Fig. 15B). The interfascicular region had the most substantial change in total
autofluorescence where it nearly increased by three-fold from stem elongation to
senescence (Fig. 15C). These observations demonstrated that lignification occurred
throughout development in both the vascular bundle and interfascicular region.
2.3.4 Crystalline cellulose deposition continues from stem elongation to inflorescence
emergence
Stem cross-sections were immunostained with the CBM3a recombinant protein
probe and Texas Red fluorophore in order to observe crystalline cellulose content
throughout development. At stem elongation, the xylem was more fluorescent than the
interfascicular region (Fig. 16A-B). The interfascicular region appeared slightly more
luminous at inflorescence emergence but the vascular bundles did not increase (Fig. 16CD). Interestingly, neither the interfascicular region nor the vascular bundles seemed more
fluorescent at senescence (Fig. 16E-F). I then quantified the total corrected crystalline
cellulose fluorescence of the whole stem, vascular bundles, and interfascicular region.
The total crystalline cellulose fluorescence of the whole stem significantly increased from
stem elongation to inflorescence emergence where it appeared to plateau (Fig. 17A). This
same trend was observed for inner vascular bundles and the interfascicular region (Fig.
17B-C). For outer vascular bundles, there was no change in fluorescence throughout
34
Figure 16. Crystalline cellulose deposition in Brachypodium distachyon
stem internode development. Whole stem (A, C, E) and higher
magnification (B, D, F) images of the Texas red fluorescence of
Brachypodium distachyon cross sections immunostained with the CBM3a
probe. (A-B) Elongating, (C-D) inflorescence emergence, (E-F) and
senescenced stem internode transverse cross-section. Images where taken
using wide field epifluorescence microscopy. Bars = 0.1 mm.
35
development (Fig. 17B). Thus,
crystalline cellulose appears to be
deposited in vascular bundles and the
interfascicular region from stem
elongation to inflorescence emergence
and then seemingly ceases afterwards.
2.4 Discussion
Here I have described and quantified
growth and cell wall deposition of three
stages of B. distachyon stem internode
development. Sampling was conducted
based on important developmental
milestones rather than time after
germination, thus allowing for simple
application by different investigators to
Figure 17. Crystalline cellulose
deposition of the three developmental
stages. (A) Corrected total crystalline
cellulose fluorescence of the stem. (B)
Corrected total crystalline cellulose
fluorescence of inner (black) and outer
(gray) vascular bundles. (C) Corrected total
crystalline cellulose fluorescence of the
interfascicular region. Data are means ±
standard deviation, n = 15 - 26. Points
annotated with the same letter are not
significantly different at P < 0.05.
diverse accessions grown under a variety
of conditions.
Interestingly, the size and number
of vascular bundles did not change after
the point of stem elongation. Therefore,
any increase in stem area was due to
changes in the interfascicular region. On the other hand, changes in fresh weight were the
36
result of secondary cell wall thickening, which again, did not result in a change in
vascular bundle size. While I cannot rule out changes at earlier stages of growth, vascular
bundle structure appears to be established at the point of meristem differentiation.
The term for grass vascular patterning is atactostele; atacto meaning disarranged.
However, the arrangement is clearly not random and scattered. For example, the two
circles of vascular bundles, the inner bundles being considerably larger than the outer,
observed in B. distachyon are regularly spaced and of fairly predictable number. This
design is similar to closely related C3 crop species such as Oryza sativa and Triticum
aestivum with some subtle differences (Patrick, 1972; Li et al., 2003). In O. sativa and T.
aestivum, the inner and outer vascular bundles are located directly across from each other
and the inner bundles are surrounded by parenchyma cells while outer bundles are
surrounded by sclerenchyma fibers. In B. distachyon, inner and outer vascular bundles
are diagonal to each other, and both types of bundles are surrounded by sclerenchyma
fibers. For this reason, the interfascicular region, a term commonly used to describe
dicots yet applicable to monocots, is mostly comprised of sclerenchyma fibers in B.
distachyon. Larger grass species such as Zea mays and S. bicolor have many more
vascular bundles with a spiral pattern from the center of the stem (Kiesselbach, 1949;
Wilson et al., 1993; Sindhu et al., 2007). The rind is the region near the epidermis with
small vascular bundles surrounded by highly thickened sclerenchyma fiber. Interestingly,
this area has all the hallmarks of the interfascicular region of B. distachyon. In C4 grasses,
bundle sheath cells contain chloroplast and thin walls (Sage et al., 2012). I use the term
bundle sheath in C3 grass B. distachyon as an anatomical term, rather than a functional
label. These cells appear to be thick walled fibers without chloroplasts in B. distachyon.
37
The cell types and organization of the ground tissue outside of vascular bundles is typical
of other monocots. Chlorenchyma cells also are found within the stem adjacent to the
epidermis and collenchyma cells are not observed in the cortex, an observation typical of
monocots.
The cell walls of the vascular bundle and interfascicular region continued to
thicken throughout development indicating that cell wall biosynthesis continued up until
plant senescence. Previous transcriptomic analysis at the resolution of whole tissue types
revealed a strong correlation among lignin and cellulose structural gene expression
(Brown et al., 2005; Persson et al., 2005; Ruprecht et al., 2011). Histological observation
of different cell types revealed differences in both the temporal and special deposition of
lignin and crystalline cellulose in B. distachyon stem. Lignification continued from stem
internode elongation to senescence in sclerenchyma fibers and tracheary elements, cells
known to undergo exclusive secondary cell wall biosynthesis, a process typified by high
lignin deposition (Ye, 2002). Continued lignification was also observed in parenchyma
cells, which do not undergo secondary cell wall biosynthesis. While primary cells walls
are often not considered lignified, this observation is not unusual (Chesson et al., 1997;
Lin et al., 2002). Unlike lignin, crystalline cellulose stained content did not increase in
the first internode following inflorescence emergence and was most abundant in xylem
vessels and tracheids. While relatively little crystalline cellulose was observed in the
bundle sheath cells, they were heavily lignified. Thus, differences in timing of deposition
and cell type were apparent through the analysis of three stages of B. distachyon
internode development.
38
Xylem cells undergo apoptosis before becoming functional for water transport
(Roberts & McCann, 2000; Bollhoner et al., 2012). As the cell matures, secondary wall
biosynthesis occurs while the autolytic factors associated with cell death such as
hydrolytic enzymes are suppressed by inhibitors and by storage in the central vacuole
(Obara et al., 2001). After a critical level of serine protease is reached in the extracellular
space, an influx of calcium ions into the cytoplasm triggers a rupture of the central
vacuole releasing the autolytic factors that lead to rapid cell death (Groover & Jones,
1999; Obara et al., 2001). Remarkably, even after death, the cell walls of tracheary
elements continue to lignify (Stewart, 1966; Pesquet et al., 2010). Although this process
is not well understood, xylem parenchyma cells may deposit lignin monomers into the
walls of dead cells and provide the hydrogen peroxide needed for polymerization
(Hosokawa et al., 2001; Ros Barceló, 2005; Tokunaga et al., 2005). In the developing B.
distachyon stem internode, cell death likely occurs following the biosynthesis of
cellulose, sometime between elongation and inflorescence emergence. After this point,
lignification of xylem and fiber cells continues. These mechanisms could explain the
differential deposition of crystalline cellulose and lignin throughout development since
lignification would continue even after cellulose and lignin deposition during secondary
cell wall biosynthesis has concluded.
39
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