CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
IMMUNOLOCALIZATION AND CHARACTERIZATION OF
BETA-GLUCOSIDASE IN COLD-STRESS AND
COLD-STRESS RECOVERED Zea mays ROOT TIPS
A thesis in partial fulfillment of the requirements
for the degree of Master of Science in
Biology
By
Laura Feucht
December 2013
The thesis of Laura Feucht is approved by:
___________________________________________
Dr. Maria Elena De Bellard
______________
Date
___________________________________________
Dr. Sean Murray
______________
Date
___________________________________________
Dr. Maria Elena Zavala, Chair
______________
Date
California State University, Northridge
ii
ACKNOWLEGEMENT
I would like to take this small space to thank some of the people who helped significantly
on this journey. I would like to thank my committee members, Dr. Maria Elena De
Bellard and Dr. Sean Murray who helped shared their expertise and encouragement with
me.
I would like to also thank all of my lab mates who became friends, Leslie Tirado who
showed me the ropes around lab, Raghed Rabadi-Goldstein who was my partner in crime
for many years as we toiled over Arabidopsis and graduate classes, Ivan Correa who
always lent a word of encouragement and positive outlook. I would especially like to
thank Theavy Budyano who never let me give up and truly showed me how to be a
scientist.
I thank my family, my mom and dad and sister, Carol, who put up with my crazy lab
schedules and encouraged me all the way. I would also like to thank my husband, Brian
Apodaca, who also supported me all of these years and was always ready to accompany
me to lab even in the wee hours of the morning. I love you.
Finally, I owe a great deal of gratitude to Dr. Maria Elena Zavala, who despite her better
judgment, never changed the locks on me and handled me with what seems like an
infinite amount of patience. Whenever I have a particularly difficult time with a student,
I remember how patient you were with me and I try my best to model this kindness in the
best possible manner. I consider your stories about life invaluable. You are an amazing
role-model to thousands of people and I feel so privileged to know you and count you as
one of my mentors and friends. Thank you!
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TABLE OF CONTENTS
Signature Page…………………………………………………………………… ii
Acknowledgement…………………………………………………………….....
iii
List of Figures………………………………………………………………….… iv
List of Abbreviations………………………………………………………….…. vii
Abstract………………………………………………………………………..…. viii
Introduction……………………………………………………………………….
1
Materials and Methods…………………………………………………………… 14
Results……………………………………………………………………………. 20
Discussion………………………………………………………………………… 42
References………………………………………………………………………… 52
iv
LIST OF FIGURES
Figure 1
Diagram of root tip……………….………………………........
3
Figure 2
β-glucosidase amino acid sequence……………………………
5
Figure 3
Diagram illustrating the interconversion of zeatin to ZOG…....
6
Figure 4
β-glucosidase activity from seedlong roots exposed to various
Cold stress durations…………..………………..…..………….
7
Northern blot of β-glucosidase gene expression under various
cold stress and cold stress recovered conditions in maize……..
9
Tertiary structure of β-D-Glucosidase Zm-p60.1 wild-type
and derived mutant forms………………………………………
11
Immunolocalization of 3C, 4C and 4CS conditions in Zea
mays……………………………………………………………
21
Figures 8a-f
Immunolocalization of 5C and 5CS conditions in Zea mays….
23
Figures 9a-f
Immunolocalization of 6C and 6CS conditions in Zea mays…..
24
Figures 10a-f Immunolocalization of 7C and 7CS conditions in Zea mays….
26
Figures 11a-i Immunolocalization of 8C, 8CS and 8CSR conditions in Zea
mays……………………………………………………………
27
Figures 12a-I Immunolocalization of 9C, 9CS and 9CSR conditions in Zea
mays……………………………………………………………
29
Figures 13a-I Immunolocalization of 10C, 10CS, and 10CSR conditions in
Zea mays……………………………………………………….
30
Figure 5
Figure 6
Figures 7a-i
Figure 14
Figure 15a
Figure 15b
Immunolocalization of 3C, 4CS and 8CSR groups showing the
translocation of β-glucosidase within parenchyma cells at 40X
magnification…………………………………………………...
33
Western blot of β-glucosidase under various cold stress and
cold-stress recovered conditions 3C-7CS in maize………….....
38
Western blot of β-glucosidase under various cold stress and
cold-stress recovered conditions 8C-10CSR in maize…………
38
v
Figure 16
Figure 17
Figure 18
Figure 19
Table 1
Table 2
Average Relative densities of Western blot bands of control,
cold stress and cold stress recovery root tips………..…………
39
Putative N-glycosylation sites as predicted by NetNGlyc
Program……………………………….………………………..
40
Putative glycosylation sites and proposed potentials as
predicted by NetNGlyc…………………………………….….
41
Model of relocalization of active β-glucosidase according to
activity levels……………………………………………….….
50
Hydrolytic velocities for tZOG, cZOG, Z7G and Z9G by wild
-type and engineered Zm-p60.1 β-D-Glucosidases…………….
12
Summary of localization patterns of β-glucosidase in 3C, 4C,
4CS, 8C, 8CS, 8CSR, 10C, 10CS, 10CSR groups……………..
34
vi
LIST OF ABBREVIATIONS
BGAF:
β- Glucosidase Aggregating Factor
BSA:
Bovine Serum Albumin
C:
Control
CS:
Cold Stress
CSR:
Cold Stress Recovered
PBS:
Phosphate Buffered Saline
TBS:
Tris Buffered Saline
Z:
Zeatin
ZOG:
Zeatin-O-Glucoside
vii
ABSTRACT
IMMUNOLOCALIZATION AND CHARACTERIZATION OF
BETA-GLUCOSIDASE IN COLD STRESSED AND
COLD STRESS RECOVERY ROOT TIPS IN Zea mays
By
Laura Feucht
Master of Science in Biology
Cytokinins are plant hormones that regulate meristem activity in roots and shoots by
initiating mitotic divisions. Such cytokinins are subject to environmental stresses such as
light and nutrient availability and pH and temperature change. When temperature drops
below optimum for metabolic processes a cytokinin, zeatin, levels decrease and ZeatinO-glucoside (ZOG) increase, mitotic divisions arrest and growth ceases. Studies
implicate that cold stress causes the enzyme zeatin-O-glycotransferase to glycosylate
Zeatin and convert it into its storage form, ZOG. Return to 25oC causes β-glucosidase to
hydrolyze the glycosidic linkage in ZOG N6 side chains and liberate the active form,
zeatin. Zea mays β-glucosidase activity peaks within the first 24 hours of return to 25oC
after cold stress. Zea mays β-glucosidase was localized to the plasma membrane in
parenchyma cell wall regions of the cortex within 24 hours of recovery at 25oC after 4
viii
days of cold stress. In addition, accumulation of the β-glucosidase protein was observed
according to development, but not according to stress.
ix
INTRODUCTION
A variety of tropic behaviors result from response to abiotic stimuli. Due to their sessile
nature, plants must maintain homeostasis by varying hormone quantities and their
distributions in response to changes in the environment. One class of enzymes involved
in this process is β-glucosidase. Βeta-glucosidase catalyzes the hydrolysis of the βglucoside linkages in di- and poly-saccharides. This suggests that β-glucosidases play key
roles in initiating developmental processes such as cell wall formation, pigment
metabolism and defense. In addition to these well-described roles, in normal plant
growth, β-glucosidases regulate plant development by releasing cytokinins from their
inactive cytokinin-O-glucoside forms (Taiz and Zeiger 1998).
Environmental factors hinder or accelerate development in plants. Environmental cues
can signal a plant to cease growth until conditions become more favorable, allowing
optimal growth to resume. Environmental cues include desiccation, light and nutrient
availability, pH changes and temperature. For example, drops in temperature signal the
down-regulation of hormones associated with plant maturation as a means to decrease
metabolic activity. These hormones include ethylene, auxin, gibberellin, abscisic acid and
cytokinin. Beta-glucosidases have been implicated in the regulations of all of these
hormones (Brzobohaty et al. 1993).
Seasonally, temperature drops coincide with shorter photoperiods of the winter months.
The drop in temperature reduces water uptake and inhibits primary growth (Treshow
1
1970). Morphological and physiological adaptations include the leaf drop in deciduous
trees, the hindrance of seed germination and the cessation of root growth. A monocot root
is composed of several tissue types including the root cap, quiescent center, apical
meristem, cortex, pericycle and pith (Figure 1). The cortex and pith are composed of
spongy parenchyma cells that have thin primary cell walls. These cells undergo the
highest number of cell divisions when compared to the other tissue types. These tissues
are most susceptible to abiotic stresses that include availability of water, nutrients and
temperature.
Plants respond to changes in photoperiods by altering phytohormones which issue
physiological changes in plants. Previous studies suggest that β-glucosidases hydrolyze
inactive phytohormones into active conjugates in order to trigger metabolic pathways
(Palme and Schell 1993). Βeta-glucosidase is not only linked to the presence of
phytochemicals involved in the defense of the plant against herbivory, but can be
artificially added to elicit the predation of the herbivorous caterpillars by the parasitic
wasps. In cabbage (Brassica oleracea) evidence indicates that β-glucosidase found in
caterpillar regurgitant attracts a predatory wasp (Mattiacci 1994). Moreover, other
studies suggest the importance of β-glucosidase especially in young plants where high
production rates of the enzyme generate a drop in aphid numbers. In both cases, βglucosidase plays an important role in the defense mechanisms of the plant by initiating
the production of volatile phytochemicals in the presence of a threat to plant health. It
may be due to a limited ability to initiate specific immune responses that β-glucosidases
2
FIGURE 1:
FIGURE 1: Diagram of a primary root showing the root cap, apical meristem,
elongation zone, epidermis, cortex and quiescent zone from http://www.biosci.ohiostate.edu/~plantbio/
3
activate according to certain stresses and elicit defensive responses accordingly (Cairns
and Esen 2012).
Maize β- glucosidases derive from 2 genes, Gluc1 and Gluc2 (Esen 1992). It is a
homodimer of approximately 130 kDa and consists of 566 amino acids ( Figure 2
Bandaranayake and Esen 1996). Βeta-glucosidases that release cytokinin from inactive
cytokinin were isolated from maize coleoptiles (Brzobohaty et.al. 1993). cDNA was
synthesized and used to identify the a gene as Zm-p60.1. Further investigation of this
gene found that it directly regulated β-glucosidase located in plastids through the
hydrolysis of cytokinin-O and N3-glucosides. This was shown through a correlation of
the distribution of Zm-p60.1 and the onset of meristemetic activity (Kristoffersen et al
1998). The hydrolysis of cytokinin-O-glucosides results in a reversible process whereas
the hydrolysis of N3-glucosides results in an irreversible process. The increase in
meristemtic activity in relation to the distribution of Zm-p60.1 suggests a link between an
increase of the enzyme and a change in growth pattern. Moisture, light availability and
temperature contribute to altered growth patterns by lowering germination rates. Low
moisture, high light and low temperature delay or impede germination.
Previous studies indicate that primary root growth also relies on the active cytokinin,
zeatin. During cold stress, zeatin decreases while the inactive form (ZOG) zeatin-Oglucoside increases (Figure 3 Brandon, Zavala 1992). The activation of ZOG is
modulated by β-glucosidase which de-glycosylates zeatin-O-glucoside into its active
form during cold stress recovery (Allen 2002). β-glucosidase activity was found to be
4
FIGURE 2:
1 mapllaaamn haaahpglrs hlvgpnnesf srhhlpsssp qsskrrcnls fttrsarvgs
61 qngvqmlsps eipqrdwfps dftfgaatsa yqiegawned gkgesnwdhf chnhperild
121 gsnsdigans yhmyktdvrl lkemgmdayr fsiswprilp kgtkegginp dgikyyrnli
181 nlllengiep yvtifhwdvp qaleekyggf ldkshksive dytyfakvcf dnfgdkvknw
241 ltfnepqtft sfsygtgvfa pgrcspgldc ayptgnslve pytaghnill ahaeavdlyn
301 khykrddtri glafdvmgrv pygtsfldkq aeerswdinl gwflepvvrg dypfsmrsla
361 rerlpffkde qkeklagsyn mlglnyytsr fsknidispn yspvlntdda yasqevngpd
421 gkpigppmgn pwiymypegl kdllmimknk ygnppiyite ngigdvdtke tplpmedaln
481 dykrldyiqr hiatlkesid lgsnvqgyfa wslldnfewf agfterygiv yvdrnnnctr
541 ymkesakwlk qfnaakkpsk kiltpa
FIGURE 2: β-glucosidase amino acid sequence (Acession number NP_001105454)
(Bandaranayake and Esen 1996)
5
FIGURE 3:
FIGURE 3: Diagram illustrating hypothesis that β-glucosidase is converting zeatinO-glucoside to zeatin in cold-stressed recovered conditions (Brandon and Zavala
1992)
6
FIGURE 4:
FIGURE 4: β-Glucosidase activity in control (CC), cold stress (CS), and cold-stress
recovery (CSR) root tips (Allen 2002)
Control seedlings (CC) were kept at 25°C throughout the experimental period of ten
days. Treated seedlings were grown at 25°C for days 1-3 “normal” conditions, placed in
4°C for days 4-7 for ‘cold-stress’ CS, days 8-10 are ‘cold stressed recovered’ CSR.
Control day 3 was set as 100%, and each subsequent day of the experiment was
compared to control Day 3.
Data are mean +/- SD (n=3)
Students t-test were used in statistical analysis: (*) indicates statistical difference between
β-Glucosidase activity in treated and control seedlings on each day. From (Allen 2002)
7
greatest at the terminal root tip within the first 2mm at the apical meristem. This previous
study also correlates a higher β-glucosidase activity with cold-stress conditions until the
eighth day of cold stress recovery. After the eighth day, β-glucosidase levels plummet in
subsequent recovery stages (Figure 4 Allen 2002).
Previous findings in our laboratory analyzed RNA accumulation in cold stress, cold stress
recovery and control root tips. Northern blot analysis suggests that transcriptional activity
for the most part remains consistent regardless of age or stress (Silva 2004 Figure 5).
There is an exception with the eight day cold stress recovery condition, where a decrease
in mRNA expression was observed. However many other experiments indicate that
protein concentrations may vary depending on posttranscriptional modifications that limit
protein synthesis (Mann and Jenson 2003).
A recent study indicates that an overexpression of the β-glucosidase gene in Arabidopsis
results in increased drought resistance, and dwarf morphology (Han et al. 2012).
Transgenic plants that overexpressed β-glucosidase were found to have limited primary
growth with smaller leaf widths, but these mutants also had longer lifespans when
deprived of water. Wild type plants contained lower levels of β-glucosidase in the leaves
and also had lower retention of water. This suggests the important interplay between βglucosidases and the development of a plant’s shoot and root system under stress
conditions.
8
FIGURE 5:
FIGURE 5: Northern blot analysis of β-Glucosidase gene expression in Control, Coldstress and cold-stress-recovery Zea mays root tips (Silva 2004)
Control seedlings (CC) were kept at 25°C throughout the experimental period of ten
days. Treated seedlings were grown at 25°C for days 1-3 “normal” conditions, placed in
4°C for days 4-7 for ‘cold-stress’ CS, days 8-10 are ‘cold stressed recovered’ CSR.
The Northern blot illustrates a steady level of mRNA transcript during growth for up to
ten days. A decrease in transcript is observed at the eighth day cold stress recovery.
9
Other studies showed that different mutations in the active sites of β-glucosidase resulted
in an upset of cytokinin homeostasis in plants. The entrance to the active site determines
its substrate specificity and is identified as an aglycone binding pocket. It is 22 Ȧ long,
8Ȧ wide and is formed by amino acid residues including F193, F200, F461 and W373
(Fig. 6 Filipi et al. 2012). These residues are highly variable and indicate that certain
combinations of amino acids result in binding to different substrates. The stabilizing
aglycone part of the substrate influences catalytic activity. Mutations in this region
reduce catalytic activity (Table 1 Filipi et. al. 2012).
β-glucosidases activate zeatin-o-glucosides in Zea mays, hydrolyze dhurrins in Sorghum
bicolor. Previous research shows that mutations of the active site in the CK-specific βglucosidase Bgl4:1, confirmed that the crucial residue in the active site is W373 and
although the study showed impaired catalytic activity with artificial substrates, it retained
a limited ability to cleave tZOG (Table 1 Filipi et. al. 2012).
It has been previously reported that the active form zeatin, is O-linked glycosylated and is
converted into the inactive storage form, zeatin-O-glucoside (ZOG), by zeatin-Oglycotransferase (Martin, et al. 1999). The inactive ZOG has been shown to accumulate
during periods of decreased root growth (Brandon, et al. 1992). Additionally, it has been
shown that Phalainopsis leaves accumulated cytokinin-glucosides when exposed to high
temperature stress (Chou et al. 2000). In cold-stressed maize root tips, the active
cytokinin, zeatin, is converted to ZOG (Sosa, et al. 2000). When seedlings are returned to
the permissive temperature, ZOG levels decrease and there is a concomitant increase in
zeatin levels (Brandon, et al. 1992).
10
FIGURE 6:
FIGURE 6: Tertiary structure of β-D-Glucosidase Zm-p60.1 wild-type and derived
mutant forms.
(A) Tertiary structure of Zm-p60.1; wild-type with colored loop III. (B) Detailed
representation of loop II of wild type Zm-p60.1. (C) Amino-acid alteration W373K. (D)
Amino-acid alteration P372S/W373K/M376L. (E) Amino-acid alteration
P372T/W373K/M376L. Amino-acid structures at mutated positions were inferred using
PyMOL 1.1 (Molecular Graphic System; Schrodinger, LLC). (Filipi et al. 2012)
11
TABLE 1:
TABLE 1: Hydrolytic velocities for tZOG, cZOG, Z7G and Z9G by wild-type and
engineered Zm-p60.1 β-D-Glucosidases. Hydrolytic velocity for 9mM substrate is
expressed as v/[E] (s-¹). (Filipi et al. 2012)
12
Previous research also indicates decreased activity of β-glucosidase after the third day in
cold stressed and control root tips. Within 24 hours of return to permissive (25°C)
conditions after cold stress (4°C), root tips demonstrated increased β-glucosidase activity
(Allen 2002). However, previous research also reports no difference in transcription
regardless of exposure to cold-stress conditions (Silva 2004).
HYPOTHESIS
We hypothesize that β-glucosidase activity correlates with relocation in root tissues that
have increased growth in the maize root tip, primarily in the parenchyma cells where
growth, expansion and cell division occur(Gibson 2012). We also predict a change in
subcellular localization that reflects β-glucosidase proximity to zeatin-o-glucoside.
Furthermore, we hypothesize that increases in concentration of β-glucosidase will
correspond to activity levels.
13
MATERIALS AND METHODS
PLANT GROWTH AND GERMINATION
Zea mays seeds obtained from Burpee (Early Sunglow Yellow) were sterilized in 50%
bleach, containing 20uL of Tween-20 detergent and mixed for 20 minutes under constant
agitation. Seeds were then rinsed with sterile deionized water 6 times before they were
aseptically planted on sterile aluminum pans lined with moistened sterile paper towels.
Seeds were grown in the dark while covered with a double layer of aluminum foil at
25°C. Three groups of seeds were maintained, Control (C), Cold stressed (CS) and cold
stress recovery (CSR). They were grown for a period of 3-10 days. Control maize (C)
was grown in the dark at 25°C for ten days. CS maize was grown three days at 25°C,
placed at 4°C for up to seven days. CSR was grown three days at 25°C, placed at 4°C for
up to four days, then returned to 25°C for the recovery period for up to 3 days. Root tips
(2-3mm) were collected for control and experimental groups days aseptically. Visible
root caps were removed before freezing the root samples in liquid nitrogen and stored at 80°C (Esen 1992).
SAMPLE FIXATION AND EMBEDDING
After root tips from each group were collected, they were pre-fixed in 20mL sodium
metaperiodate dissolved in 50mM carbonate-bicarbonate buffer pH9.6 for two hours.
They were then washed in 50mL PBS pH7.2 at room temperature on a rotary shaker.
They were placed in 2mM sodium borohydride in 50mM Tris-HCl buffer pH 7.6 for one
14
hour. After washing in PBS, sections were fixed in 2.5% paraformaldehyde in 50mM
PBS pH 7.2 overnight at 4°C. After rinsing, sections then underwent a dehydrated
ethanol series from 20%-100% for 15 minutes at each step. Tissues were placed through
a tert-butyl alcohol ethanol series with ratios of 1:3, 1:1, 3:1 and 100% tert-butyl
respectively for one hour each. Sections were poured onto filtered paraffin, covered with
tert-butyl and then left overnight at 60°C. Paraffin was poured off and replaced with new
paraffin and incubated for 4 more hours. Samples were arranged in melted paraffin then
cooled on an ice bath for 5 to 10 minutes and stored at 4°C.
ANTIBODY SCREENING
The primary antibody, rabbit anti-almond β-glucosidase (Agrisera #AS09 449) was tested
for cross-reactivity with crude extracts of protein extracted from Zea mays using dot blot
analysis at concentrations of 1:10, 1:100 and 1:500. 10uL of crude protein extracts were
blotted onto a nitrocellulose membrane and incubated for 1 hour at room temperature.
The membranes were then blocked in a 0.1% BSA solution in 0.01M TBS pH7.2 for 1
hour at room temperature. Blocking solution was poured off and membrane was
incubated with primary antibody and 0.01M TBS for 1 hour at room temperature. The
membrane was washed three times in TBS and Tween (TTBS) for 10 minutes per wash
step. The membrane was incubated with secondary antibody (goat anti-rabbit Agrisera
#AS31210) for 1 hour at room temperature in TTBS. Membrane was then washed in
TTBS with constant agitation and visualized with Vector Laboratories Alkaline
Phosphatase Substrate Kit BCIP/NBT (SK-5400).
15
IMMUNOLOCALIZATION
Embedded root tips were sliced lengthwise at a thickness of 9µm using a microtome,
collected on coated slides and dried. Mounted sections were rehydrated using a graded
ethanol series with concentrations ranging from 100%-20% for 15 minutes at each. They
were incubated in PBS for 20 minutes then blocked in 0.1% bovine serum albumin
(BSA) in 0.01M PBS pH 7.2 at 37°C for 1 hour. Samples were washed for 5 minutes in
0.01M PBS pH 7.2 then incubated in 1:200 dilution of primary antibody, rabbit antialmond β-glucosidase dissolved in 0.1% BSA in 0.01M TBS pH 7.2. Sections were
washed twice in 50mM 0.01M TBS pH 7.2 with 0.1% Tween 20 for 5 minutes. Samples
were then incubated in 1:2000 dilution of secondary antibody, goat-anti rabbit alkaline
phosphatase antibody from antibodies online (#ABIN101985) dissolved in 0.1% BSA in
0.01M TBS pH 7.2. Sections were washed once more in TBS before they were stained
using the Vector Red Substrate kit (SK-5100). Sections were dehydrated using an ethanol
gradient from 20% - 100% and cleared with xylene. Sections were than mounted with
Permount and visualized using the Alexa Red filter at 650nm on Zeiss AxioObserver.Z1
inverted microscope and the Zen Imaging software. Lighting and contrast were set to
“Best fit parameters”. Control tissue was incubated in primary antibody alone, secondary
antibody alone, and in the absence of both primary and secondary antibody.
16
ENZYME EXTRACTIONS
Approximately 60-100 root tips were collected for each experimental condition and
ground into a fine powder using liquid nitrogen. Crude protein was weighed and proteins
were extracted using a 1:1 ratio of 1M DTT, 55mM Tris-HCl and 0.5M extraction buffer
then centrifuged at 13000rpm at 4°C for 6 minutes. Supernatant was removed and
precipitated through the slow addition of a 60% Ammonium Sulfate solution at a 1:1 ratio
of dry weight to ammonium sulfate volume. Homogenates were left to incubate on ice for
30 minutes and vortexed every 5 minutes to prevent spatial nonconformity of the
precipitation. Samples were then centrifuged for 30 minutes at 13000rpm at 4°C.
Supernatant was removed and pellet was resuspended in fresh extraction buffer. Samples
were dialyzed in fresh extraction buffer for 6 to 24 hours with constant stirring at 4°C
(Esen 1992). Halt Protease Inhibitor cocktail (Thermo scientific #87786) was added to
prevent degradation of proteins. Samples were stored at 4°C.
PROTEIN QUANTIFICATION
Protein extracts were quantified using the Bradford assay and Thermo Scientific
Nanodrop 2000c spectrophotometer. 10uL of dialyzed proteins were removed and added
to 300uL of Coomassie Plus Protein Assay Reagent (Thermo Scientific # 1856210). A
BSA standard was made from a 2mg/mL stock solution. Standard samples were loaded
onto the Bradford assay test program which established a standard curve at an
absorbance of 570nm. 2uL of coomassie blue/ crude protein mixtures were vortexed and
tested in duplicate. Average value quantifications were used to normalize the amount of
protein used for Western blotting analysis. Each lane was loaded with 5µg of dialyzed
17
sampleper lane total protein. Dot blot analysis was used to determine optimal ratios of
protein to primary and secondary antibodies.
SDS PAGE AND WESTERN BLOT
5ug of total protein extracts were used for analysis in Western blotting.10uL samples
were denatured by boiling for 5 minutes at 95°C in 2x mercaptoethanol buffer. Samples
were then kept on ice until loaded onto a 12% acrylamide gel. A control of purified
almond β-glucosidase was included as a reference point. Proteins and Full-range
Rainbow marker (Amersham #RPN 800E) were separated by gel electrophoresis in a
denaturing Tris-Glycine running buffer containing 0.1% SDS. They were run on 12%
Mini-PROTEAN TGX precast gels from Bio-Rad (#456-1045) at 15V for 24 hours.
Gels were removed, soaked in Towbin’s transfer buffer and layered onto Hybond-C
Nitrocellulose 0.45 micron paper (#64198). They were then electroblotted at 70V for 2
hours. The uniform transfer of the rainbow marker was used to determine the
effectiveness of the transfer. The nitrocellulose membrane was removed, rinsed in
deionized water and blocked in 1% BSA/TBS and 0.01% Tween for 1hour at room
temperature under constant agitation.
Nitrocellulose was then incubated overnight at 4°C in primary antibody at a 1:2000
dilution. Nitrocellulose was washed in 1% BSA 0.01M TBS solution and incubated in a
secondary antibody rabbit anti-alkaline phosphatase dilution of 1:5000 for 2 hours.
18
Nitrocellulose was washed three times for 10 minutes and developed for 20 minutes
using Vector Laboratories Alkaline Phosphatase Substrate Kit BCIP/NBT. Band
intensities were measured using the Image J program. Bands were normalized by
dividing percent values by background intensity and plotted on a graph using Excel.
19
RESULTS
We hypothesized that the increase in activity observed in previous studies is accompanied
by relocalization of β-glucosidase root tip tissues that demonstrate increased mitotic
activity, primarily parenchyma tissue located in the cortex and pith. To determine
whether β-glucosidase is localized differently in the various treatments in the Zea mays
root tip, immunolocalizations were conducted to visualize presence and relative
abundance of β-glucosidase proteins.
IMMUNOLOCALIZATION
The immunolocalization of the maize root tips shows a strong positive signal (red
fluorescence) of β-glucosidase in the three-day control. In the four-day control, βglucosidase signal declines significantly and remains consistently low. The presence of βglucosidase signal peaks in the eight-day cold-stress-recovery root tips 24 hours after the
roots are returned to the permissive (25°C) temperature.
Seeds grown at 25°C had peak levels at the 3 day control and showed a high signal of
labeling in the cytoplasm at the lateral root tip. Additional labeling was observed in the
cell wall. The signal was localized to the cortex and pith tissue. The epidermis, pericycle
and quiescent center were not labeled (Figures 7a, 7b, and 7c). In 4 day control root tips
signaling declines significantly. Localization is still observed in the cortex but it is
observed farther away from the epidermis. Labeling in the pith decreases to the point of
bare visibility and appears closer to the root apical meristem. The root cap is clearly
20
FIGURE 7:
FIGURE 7: Immunolocalizations of 3C, 4C and 4C
Control seedlings (C) were kept at 25°C for three (3C) and four (4C) days. 4CS Treated
seedlings were grown at 25°C for 3 days and placed in 4°C for one day. Samples
incubated in 1° rabbit anti β-glucosidase antibody and 2° goat anti-rabbit. Samples were
labeled using Vector Red Substrate kit. Β-glucosidase is labeled in red. Green color is
autofluorescence. Sections were visualized at 10X, 20X and 40X magnification using the
Alexa Red filter at 650nm on Zeiss AxioObserver.Z1 inverted microscope and the Zen
Imaging software.
AM= Apical Meristem
PC= Pith
C= Cortex
RC= Root Cap
E= Epidermis
QC= Quiescent Center
P= Pericycle
21
labeled (Figures 7d and 7c). Labeling is still observed in the cytosol and central vacuole
of cells along with the cell wall while no labeling was observed along the pericycle and
epidermis (Figure 7f). The four day cold-stress root tips appear to demonstrate a similar
signaling pattern to the four day control where signals are localized to the cytosol and cell
walls (Figures 7g, 7h, and 7i).
Five day control root tips exhibit a signal, but the spread of the label covers the cortex
and appears closer to the epidermis. Distribution of the signal patterns appears less
homogenous than the 3 day control. Specific “hot spots” are observed with intense
labeling of the cytosolic areas closest to the cell walls. Signal is also apparent in the pith,
although not with the same intensity as the 3 day control (Figures 8a, 8b, and 8c). The
five day cold-stress, root tips were subjected to two days of cold stress and distribution of
β-glucosidase appears similar to the five day control. Again the signal occurs in the
cortex and the pith while the epidermis, quiescent center and pericycle remain free of
label. Signal is observed in the cytoplasm and cell wall with less signal in the apical
meristem when compared to the five day control (Figures 8d, 8e, 8f).
The distribution of signal in the six day control shows overall distribution similar to
previous control roots. However, the localization is limited to the cortex flanking the
pericycle. Very little signal is observed in the pith and some labeling of the epidermis is
also observed. Most of the β-glucosidase signal appears in the cytosol with limited
labeling of the cell walls (Figures 9a, 9b, 9c). The six day cold-stress shows almost no
signaling at the apical meristem and the β-glucosidase signal appears evenly localized in
22
FIGURE 8:
FIGURE 8: Immunolocalizations of 5C and 5CS
Control seedlings (C) were kept at 25°C for five days (5C). 5CS Treated seedlings were
grown at 25°C for 3 days and placed in 4°C for two days. Samples were incubated in 1°
rabbit anti β-glucosidase antibody and 2° goat anti-rabbit. Samples were labeled using
Vector Red Substrate kit. Β-glucosidase is labeled in red. Green color is
autofluorescence. Sections were visualized at 10X, 20X and 40X magnification using the
Alexa Red filter at 650nm on Zeiss AxioObserver.Z1 inverted microscope and the Zen
Imaging software.
AM= Apical Meristem
PC= Pith
C= Cortex
RC= Root Cap
E= Epidermis
QC= Quiescent Center
P= Pericycle
23
FIGURE 9:
FIGURE 9: Immunolocalizations of 6C and 6CS
Control seedlings (C) were kept at 25°C for six days (6C). 6CS Treated seedlings were
grown at 25°C for 3 days and placed in 4°C for three days. Samples were incubated in 1°
rabbit anti β-glucosidase antibody and 2° goat anti-rabbit. Samples were labeled using
Vector Red Substrate kit. Β-glucosidase is labeled in red. Green color is
autofluorescence. Sections were visualized at 10X, 20X and 40X magnification using the
Alexa Red filter at 650nm on Zeiss AxioObserver.Z1 inverted microscope and the Zen
Imaging software.
AM= Apical Meristem
PC= Pith
C= Cortex
RC= Root Cap
E= Epidermis
QC= Quiescent Center
P= Pericycle
24
the cortex between the epidermis and the pericycle. There is some label observed in the
pith. Cortex cells under all three conditions appear to have signals localized primarily to
the cytoplasm with some labeling of cell walls (Figures 9d, 9e, and 9f).
Seven day control root tips appear to have limited signal. Some labeling is observed in
the root cap, cortex and pith. The labeling is concentrated to the cytoplasm with some
signal observed in the cell walls (Figures 10a, 10b, 10c). Seven day cold-stress root tips
appear to have a slightly wider distribution with signal occurring in the cortex between
the epidermis and pericycle. Increased signal is also observed at the root cap. βglucosidase labeling can be observed primarily in the cytosols of cells located in the root
tip, while greater levels are observed in cell walls of the root cap (Figures 10d, 10e, 10f).
The eight day control root tips demonstrate consistent signal throughout the cortex,
epidermis, pith and root cap. No labeling is observed in the quiescent center, the
pericycle and limited signal is observed in the pith and apical meristem. Individual cells
show concentration of β-glucosidase signal in the cytoplasm near the edges of the cells
and the cell walls. This was observed for both the root cap and root tip (Figures 11a, 11b,
11c). In the eight day cold-stress a significant shift was observed in the signal
distribution. Localization was confined to the pith, pericycle and cortex flanking the
pericycle. Some signal in the epidermis was also observed. In the cortical cells, a
significant increase of β-glucosidase label is concentrated specifically in the cell walls
with some signal of the cytoplasm (Figures 11d, 11e and 11f). This distribution differs
significantly when compared to the eight day control or three day control.
25
FIGURE 10:
FIGURE 10: Immunolocalizations of 7C and 7CS
Control seedlings (C) were kept at 25°C for seven days (7C). 7CS Treated seedlings
were grown at 25°C for 3 days and placed in 4°C for four days. Samples were incubated
in 1° rabbit anti β-glucosidase antibody and 2° goat anti-rabbit. Samples were labeled
using Vector Red Substrate kit. Β-glucosidase is labeled in red. Green color is
autofluorescence. Sections were visualized at 10X, 20X and 40X magnification using the
Alexa Red filter at 650nm on Zeiss AxioObserver.Z1 inverted microscope and the Zen
Imaging software.
AM= Apical Meristem
PC= Pith
C= Cortex
RC= Root Cap
E= Epidermis
QC= Quiescent Center
P= Pericycle
26
FIGURE 11:
FIGURE 11: Immunolocalizations of 8C, 8CS and 8CSR
Control seedlings (C) were kept at 25°C for eight days (8C). 8CS Treated seedlings were
grown at 25°C for 3 days and placed in 4°C for five days.8CSR treated seedlings were
grown at 25°C for 3 days, placed in 4°C for four days and recovered at25°C for one day.
Samples were incubated in 1° rabbit anti β-glucosidase antibody and 2° goat anti-rabbit.
Samples were labeled using Vector Red Substrate kit. Β-glucosidase is labeled in red.
Green color is autofluorescence. Sections were visualized at 10X, 20X and 40X
magnification using the Alexa Red filter at 650nm on Zeiss AxioObserver.Z1 inverted
microscope and the Zen Imaging software.
AM= Apical Meristem
PC= Pith
C= Cortex
RC= Root Cap
E= Epidermis
QC= Quiescent Center
P= Pericycle
27
ALTERED β-GLUCOSIDASE LOCALIZATION MAY EXPLAIN THE DIFFERENCE
IN ACTIVTY IN RESPONSE TO COLD STRESS RECOVERY
On the first day of recovery (eight day cold-stress-recovery) signaling peaks and exceeds
the signal output of all other treatments. The eight day cold-stress recovery signal
patterns are localized to the cortex, pith and root cap. No label was observed in the
quiescent center or pericycle. Additionally, cortical cells appear to contain the highest
concentration of β-glucosidase in the cell walls, while minimal β-glucosidase is observed
in the cytosol of the cells. This pattern remains consistent between the root tip and root
cap (Figures 11g, 11h, and 11i).
Nine day control root tips demonstrate a significant decrease in label when compared to
the eight day cold-stress-recovery and eight day cold-stress conditions. In nine day
control root tips labeled areas include the root cap with some labeling of the cortex,
although signal is weak. Some labeling is also observed in the pith with some
fluorescence detected in the epidermis and apical meristem. Cells appear to fluoresce
primarily in the cytoplasm of the root tip while strong signal remains in the root cap.
Little signal is detected in nine day cold-stress with stronger labeling along the cells in
the root cap region. Signal appears greatly reduced and localized to the cytoplasm of cells
located primarily in the cortex, root cap and pith (Figures 12d, 12e and 12f). The nine day
cold-stress recovery shows very little observable fluorescence being localized in the
cortex cells flanking the pericycle and with little labeling in the root cap. Most labeling
appears diminished and cells that show labeled β-glucosidase have the protein distributed
throughout the cytoplasm (Figures 12g, 12h, and 12i).
28
FIGURE 12:
FIGURE 12: Immunolocalizations of 9C, 9CS and 9CSR
Control seedlings (C) were kept at 25°C for nine days (9C). 9CS Treated seedlings were
grown at 25°C for 3 days and placed in 4°C for six days.9CSR treated seedlings were
grown at 25°C for 3 days, placed in 4°C for four days and recovered at25°C for two days.
Samples were incubated in 1° rabbit anti β-glucosidase antibody and 2° goat anti-rabbit.
Samples were labeled using Vector Red Substrate kit. Β-glucosidase is labeled in red.
Green color is autofluorescence. Sections were visualized at 10X, 20X and 40X
magnification using the Alexa Red filter at 650nm on Zeiss AxioObserver.Z1 inverted
microscope and the Zen Imaging software.
AM= Apical Meristem
PC= Pith
C= Cortex
RC= Root Cap
E= Epidermis
QC= Quiescent Center
P= Pericycle
29
FIGURE 13:
FIGURE 13: Immunolocalizations of 10C, 10CS and 10CSR
Control seedlings (C) were kept at 25°C for ten days (10CS). 10CS Treated seedlings
were grown at 25°C for 3 days and placed in 4°C for seven days.10CSR treated seedlings
were grown at 25°C for 3 days, placed in 4°C for four days and recovered at25°C for
three days. Samples were incubated in 1° rabbit anti β-glucosidase antibody and 2° goat
anti-rabbit. Samples were labeled using Vector Red Substrate kit. Β-glucosidase is
labeled in red. Green color is autofluorescence. Sections were visualized at 10X, 20X and
40X magnification using the Alexa Red filter at 650nm on Zeiss AxioObserver.Z1
inverted microscope and the Zen Imaging software.
AM= Apical Meristem
PC= Pith
C= Cortex
RC= Root Cap
E= Epidermis
QC= Quiescent Center
P= Pericycle
30
The ten day control root tips demonstrate little fluorescence and detectible signals appear
localized to the cortex with limited labeling in the root cap. Cells that exhibit any
signaling have the β-glucosidase localized to the cytoplasm (Figures 13a, 13b and 13c).
Similarly, ten day cold-stress root tips demonstrate decreased levels of fluorescence
displaying similar patterns of β-glucosidase distribution appear greatly reduced with
signals scattered throughout the cortex and some of the pith. More fluorescence is
observed in the root cap and individual cells appear to primarily localize the βglucosidase signal to the cytoplasm in the root tip while some signaling in the root cap is
localized to the cell walls (figures 13d, 13 e and 13f). The ten day cold-stress recovery
exhibits signal patters along the cortex away from the apical meristem and root cap with
some signal in the pith. No labeling is observed in the pericycle and diminished signal is
observed at the apical meristem. The cells show localization in the cell wall along with
the cytoplasm of both the root tip and root cap (Figures 13g, 13h and 13i).
Figure 14 summarizes the subcellular relocalization of β-glucosidase in the three day
control, four day cold stress and eight day cold stress recovery. The samples were
magnified to 40X and illustrate regions of the cortex flanking the pericycle. The spread of
β-glucosidase in the 3 day control localizes throughout the cytoplasm and plasma
membrane whereas a concentration of label toward the plasma membrane with cytosolic
labeling is observed in the 4 day cold stress. The eight day cold stress recovery shows
strong labeling directed toward the cell wall and plasma membrane.
31
Table 2 summarizes the immunolocalization results between the 3C, 4C, 4CS, 8C, 8CS,
8CSR and 10C, 10CS and 10CSR and sites of signal within the root tip and at subcellular
levels.
32
FIGURE 14:
FIGURE 14: Immunolocalization of 3C, 4CS and 8CSR
33
TABLE 2:
Sample
Root Cap
3C
-Strong in the
cytosol and
cell wall, cell
membrane
Strong label
close to cell
membrane/
cell wall and
some in
cytosol
4C
Apical
Meristem
cytosol with
some label in
cell wall, cell
membrane
-Some
throughout
-Strong label
close to cell
membrane/
cell wall some
in cytosol
-Some
-Some label
close to cell
membrane/
cell
wall/cytosol
Cortex
Pericycle
Pith
Epidermis
cytosol and cell
wall, cell
membrane
none
Label in the
cytosol
none
Label close to
pericycle. Cells
labeled in
cellmembrane/
cell wall
Some in
cytoplasm
Light label in cell
membrane/ cell
wall/cytoplasm
none
Some along
cell
membranes/
cell walls
none
none
Light label in
cytoplasm
none
4CS
-Strong in
center of root
cap
-label mostly
in cell
wall/cytosol
8C
Moderate
label along
cell wall/ cell
membrane
Strong with
concentration
on cell walls/
some in
cytoplasm
Strong in cell
walls/cell
membrane
-some in
cytoplasm
Weak with
some signal in
cytoplasm
Some
-cells labeled in
cell wall
Strong label in
cell walls
none
Label in cell
walls
Some in
cytoplasm
Strong with
concentration
on cell walls/
some in
cytoplasm
Strong in cell
walls/cell
membrane
-some in
cytoplasm
Weak to no
label in
cytoplasm
-Label closer to
pericycle
Strong in cell
walls/ some in
cytoplasm
Strong in cell
walls/ cell
membrane
-some in
cytoplasm
Weak with
“clumping” in the
cytoplasm
Strong in
cell walls
-some in
cytoplam
Strong in cell
walls
-some in
cytoplasm
none
none
Some in
cell walls
Weak with
some signal in
cytoplasm
Moderate
label mostly in
cell walls
Weak to no
label in
cytoplasm
Weak to none
Weak with
“clumping” in the
cytoplasm
Moderate with
concentration on
cell membrane
and clumping in
the cytoplasm
none
Strong in cell
walls/ cell
membrane
-some in
cytoplasm
Weak with
label
concentrated
in cytoplasm
Weak with
“clumping” in
the cytoplasm
Weak signal in
the cytoplasm
8CS
8CSR
10C
10CS
10CSR
none
none
TABLE 2: Summary of localization of β-glucosdiase in 3C, 4C, 4CS, 8C, 8CS,
8CSR, 10C, 10CS, and 10CSR groups
34
Some in
cytoplasm
none
Strong in
the cell
walls
We hypothesized that an increased accumulation of β-glucosidase would parallel an
increased activity of the enzyme in cold stress recovery root tips. In order to determine if
protein concentrations correlated with the constant transcript concentrations visualized in
the northern blot in Figure 5, we conducted a western blot analysis to quantify protein
accumulation (Figure 14).
EFFECT OF COLD STRESS and COLD STRESS RECOVERY on β-GLUCOSIDASE
ACCUMULATION
Samples were collected from approximately 60 to 100 excised Zea mays root tips. A ratio
of 1:1 of dry weight to extraction buffer was used to extract protein from the root tips. A
purified almond β-glucosidase was used as a control to compare protein samples. There
were two bands observed, one at 72kDa and the other at 65kDa (Figure 15). The two
bands may indicate 2 different isozymes that have been reported in previous research
(Allen 2002).
The heavier band at approximately 72kD showed strongly at the 3 day control root tips.
Concentration dropped in the 4C samples then remained constant across different
treatments up to 7 day control group. An increase in the 72kDa band is observed at the
first day of cold stress at 4CS when compared to the other groups; it drops for the 5CS,
6CS and 7CS groups. All 8 day treatments: 8 day control (C), 8 day cold stress (CS), and
8 day cold stress recovery (CSR) this peptide decreases significantly. In contrast, levels
increase and are similar for 9C, 9CSR, 10C, 10CS, and 10CSR groups. A slight increased
accumulation is detected at 9 day cold stress before decreasing at 10 day cold stress.
35
Conversely, the lighter 65kDa peptide levels remain constant for all control samples from
3 to 7 days. Similarly, concentrations remain constant for cold-stress samples from 4CS
to 7CS. At the 8C, 8CS and 8CSR samples a slight increase of the 65kDa band is
observed. The β-glucosidase decreases slightly at 9 and 10 control, cold stress and cold
stress recovery groups.
Across the 8 day treatments, the difference in the 65kDa β glucosidase was more
pronounced when compared to all of the other samples. When compared to the control,
the 8 CS and 8 CSR exhibited comparable concentrations of 65kDa β-glucosidase, thus
indicating that accumulation does not depend on stress, but depends on development
instead. General trends were graphed using Excel (Figure 16) showing that the 65kDa
band decreases until the eighth day when there is a slight increase while the 72kDa band
decreases for all groups after the third day.
Two minor contaminating proteins of 36 and 38 kD (not shown) possibly binding to βglucosidase, also appeared on the Western blot gels. They may be due to unspecific
binding but also have been identified as a β-glucosidase-aggregating factor (BGAF)
(Esen 2000).
BIOINFORMATICS
In order to investigate putative glycosylation, the Gluc1 amino acid sequence was
submitted to NetNGlyc 1.0 Server. This prediction program found that there were four
likely putative sites at amino acid positions (Figure 17) at amino acid positions 27 (NESF
36
with a potential of 0.5248), 48 (NLSF with a potential of 0.7122), 400 (NYSP with a
potential of 0.1226) and 537 (NCTR with a potential of 0.5589) (Figure 18).
37
FIGURE 15:
FIGURE 15: Western blot analysis of β-Glucosidase in control (C), cold-stressed
(CS) and cold-stressed-recovery (CSR) conditions
Control seedlings (C) were kept at 25°C throughout the experimental period of ten days.
Treated seedlings were grown at 25°C for days 1-3 “normal” conditions, placed in 4°C
for days 4-7 for ‘cold-stress’ CS, days 8-10 are ‘cold stressed recovered’ CSR. A rainbow
ladder from GE Healthcare (RPN 800E) was used in the first lane. Almond β-glucosidase
was used as a control. The blot shown below is representative of four different western
blots.
38
FIGURE 16:
3.5
Control 65kDa band
Cold stress recovery 65kDa band
Cold stress 65kDa band
Control 72kDa band
Cold stress 72kDa band
Cold stress recovery 72kDa band
Relative Density
3
2.5
2
1.5
1
0.5
3
4
5
6
7
8
9
10
Time (Days)
FIGURE 16: Average Relative Densities of Beta-glucosidase quantities according to
time for Control, Cold Stress and Cold stress recovery Zea mays root tips for 65kDa
and 72kDa bands
Control seedlings (CC) were kept at 25°C throughout the experimental period of ten
days. Treated seedlings were grown at 25°C for days 1-3 “normal” conditions, placed in
4°C for days 4-7 for ‘cold-stress’ CS, days 8-10 are ‘cold stressed recovered’ CSR.
Western blot analysis shows that label peaks in the 3 day control and drops significantly
for 65kDa and 72kDa bands. 65kDa increases at the eighth day for controls and treated
samples. Representative of 3 Western blots.
39
FIGURE 17:
1 mapllaaamn haaahpglrs hlvgpnnesf srhhlpsssp qsskrrcnls fttrsarvgs
61 qngvqmlsps eipqrdwfps dftfgaatsa yqiegawned gkgesnwdhf chnhperild
121 gsnsdigans yhmyktdvrl lkemgmdayr fsiswprilp kgtkegginp dgikyyrnli
181 nlllengiep yvtifhwdvp qaleekyggf ldkshksive dytyfakvcf dnfgdkvknw
241 ltfnepqtft sfsygtgvfa pgrcspgldc ayptgnslve pytaghnill ahaeavdlyn
301 khykrddtri glafdvmgrv pygtsfldkq aeerswdinl gwflepvvrg dypfsmrsla
361 rerlpffkde qkeklagsyn mlglnyytsr fsknidispn yspvlntdda yasqevngpd
421 gkpigppmgn pwiymypegl kdllmimknk ygnppiyite ngigdvdtke tplpmedaln
481 dykrldyiqr hiatlkesid lgsnvqgyfa wslldnfewf agfterygiv yvdrnnnctr
541 ymkesakwlk qfnaakkpsk kiltpa
FIGURE 17: Putative N-glycosylation sites as predicted by NetNGlyc program.
Sequences highlighted in yellow indicate possible N-glycosylation sites. The underlined
red amino acids are the catalytic sites.
40
FIGURE 18:
FIGURE 18: Putative glycosylation sites and proposed potentials as predicted by
NetNGlyc 1.0 program.
41
DISCUSSION
We predicted relocalization patterns of β-glucosidase in the Zea mays root tip during cold
stress recovery. β -glucosidases can release active cytokinins from zeatin-o-glucoside
(ZOG) and are restricted to zones of active cell division in the young root tip with almost
no labeling in the quiescent center. In previous studies, it has been shown that there is an
inverse relationship between the active form, zeatin, and the storage form, ZOG
(Brandon, et al. 1992). β-glucosidase has been implicated in the deglycosylation of ZOG
that activates zeatin, a cytokinin, that regulates primary root growth at the apical
meristem in actively growing roots and cold stressed roots. Conversely, zeatin-Oglycotransferase has been implicated in the glycosylation of zeatin into its storage form
under cold-stress conditions (Li et al. 2000).
Therefore, β-glucosidases can regulate the liberation of active cytokinins and ultimately
alter primary root development. Studies have shown that the ectopic over-expression of
the β-glucosidase perturbs phytohormone concentrations in leaves, but no discernible
difference in the accumulation of zeatin was observed (Kiran et al. 2005). Environmental
cues ultimately influence the regulation or accumulation of β-glucosidases. Cold stress
induces the glycosylation of zeatin to form ZOG and is accompanied with an increase in
concentration of zeatin-O-glucotransferase. We tested the hypothesis that cold-stress of
3-10 day roots followed by a recovery period would result in increased levels of βglucosidase.
42
Twenty-six β-glucosidase isozymes derived from 2 β-glucosidase genes (Glu1, Glu2)
have been identified (Anduro et al 2010). Singular genes can code for a multitude of
similar proteins base on post-transcriptional and post-translational modifications. In
antibodies, for instance, post-translational modifications alter substrate specificity (Mann
and Jenson 2003). There are also examples of β-glucosidase aggregation and βglucosidase binding proteins in various plants. Two myrosinase (β-thioglucosidase)binding proteins (50 and 52 kDa) were reported from rapeseed (Falk and Rask 1995). A
specific β-glucosidase aggregating factor (BGAF 35 kDa) was reported to limit βglucosidase activity (Esen and Blanchard 2000) suggesting that post-translational
modifications that occur in stressed recovered root tips could lead to different activities.
Previous research reported significant increase in β-glucosidase activity within the first
24 hours of recovery (Figure 4 p7) at the 8 day cold stress recovery. This behavior is
parallel to zeatin levels which increase under the same conditions. β-glucosidase activity
levels remained unchanged while the activation of zeatin-O-glucotransferase was
observed under cold-stress (Li, Sosa, Zavala, 2000). Activity of β-glucosidase declines
after 9 day cold stress recovery and 10 day cold stress recovery respectively (Allen
2002). Previous research also reported steady levels of transcriptional activity across all
treatments (Figure 5 p9). Constant mRNA levels across all treatments ranging from 3 day
control root tips through 10 day control root tips, cold stress 4 day through 10 day cold
stress root tips and 9 day cold stress recovery through 10 day cold stress recovery root
tips. Interestingly, there was a decline in transcription during the 8CSR (Silva 2004
43
Figure 5 p9). This suggests little relationship between transcript accumulation and protein
translation.
IMMUNOLOCALIZATION
The eight day cold-stress conditions exhibits high levels of β-glucosidase labeling, but in
a pattern not different from the eight day cold stress recovery. Signal is not localized to
the pith and cortex. There is also visible labeling of the pericycle with only the epidermis
left unlabeled. The increased labeling of these areas accompany increased growth in the
diameter of root tips under cold stress conditions. After five days of cold stress, this may
be considered a critical point in the root which results in damage to the root tip and
growth responses that prevent roots from further damage.
Beta-glucosidase signal diminishes significantly in the nine day and ten day groups.
Overall labeling declines yet western blot analysis shows that the concentrations remain
consistent. This suggests that increased levels of β-glucosidase distribution along the
zones of elongation and differentiation may have in higher levels of β-glucosidase overall
while in root tips it remains largely absent.
It appears that β-glucosidase is either translocated to the sites of active cell division
primarily during the recovery from cold stress or is rapidly degraded in some root cells
and rapidly synthesized in others and results reflect how different cell types react
44
differentially to environmental stresses. This research shows that β-glucosidase relocates
to the perimeter of parenchyma cells, in the plasma membrane and cell wall.
WESTERN BLOT
Western blot analysis was used to assess whether protein levels remained constant
throughout cold-stress and control groups or if protein levels varied independently of
transcriptional activity. Western blot analysis with the anti-β-glucosidase yielded results
that demonstrate consistently low levels of the 65kDa β-glucosidase up to 8 days and
increased upon the eighth day (Figure 16 p39). Unlike the mRNA results, protein levels
increased upon the initial onset of cold stress for the 4 day treatment and according to day
during the eight day treatments. This suggests that β glucosidase abundance accumulates
independent of transcriptional activity. Two primary bands at 72kDa and 65kDa were
detected at each treatment and are presumed to be isozymes. A similar pattern was
observed in the expression of two β-glucosidases, Dhr1 and Dhr2, made up of 57- and 62
kDa monomers in Sorghum bicolor (Cicek and Esen 1998). These β-glucosidases
hydrolyze the substrate dhurrin to produce p-hydroxymandelonnitrile which subsequently
disassociates to free HCN and p-hydroxybenzaldehyde. Cicek and Essen also reported
that the lighter of the two β-glucosidases (Dhr1) was localized in the root.
Western analysis indicates that both isoforms are present in varying amounts depending
largely on the age of the root tips. The lighter-weight β-glucosidase appears strongly in
the three-day control then lessens until the 8 day control. Conversely, the quantities of the
45
heavier isoform appear equal to that of the lighter isoform in the three day control and
remain higher in the treatments until the eight day control. This isoform is barely detected
in the eight day control, cold-stress and cold-stress recovery, but appears again in the nine
through ten-day treatments (Figures 15a and15 b). The accumulation of the lighter
isoform corresponds to β-glucosidase activity levels (Figure 4 p7) up to the eighth day,
but on the eighth day, no observable difference in accumulation is detected between the
control, cold-stress and cold stress-recovery eight day groups. There is also little decrease
in this isoform after the eighth day. It is possible that the heavier isoform may indicate
differential glycosylation of the β-glucosidase. Previous studies suggest that a
glycosylated form of β-glucosidase is observed at 64 kDa in Brassica napus while the
deglycosylation of the enzyme with peptide-N-glycosidase reduced the apparent size of
the enzyme to approximately 60 kDa (Falk and Rask 1995).
The consistent quantities of the lighter isoform in the eight day control, eight day control
stress and eight day control stress recovery may suggest that activation requires
deglycosylation, but also that deglycosylation is not synonymous to activity.
Nikus and Jonsson reported that a partly purified cell wall β-glucosidase was analyzed
and found to lack glycosylation in rye (Nikus and Jonsson 2003). The program
NetNGlyc predicted three possible N-glycosylation sites for the primary amino acid
sequence. This includes the sites at amino acids 27, 48 and 537. (Fig 17) The probability
of the putative site at 537 is assigned a potential of 0.5589. The proximity of this putative
site to the catalytic site in position 457 may suggest a regulatory effect on the β-
46
glucosidase catalytic site and therefore the enzyme’s ability to deglycosylate ZOG into
zeatin as it relocates toward cell-wall sites.
Glycosylation of proteins occurs primarily in the rough ER as a form of protein
modification. Therefore, immunolocalization data supports the possibility that this type of
posttranslational modification takes place in controls and cold stress conditions ranging
from the third through seventh days. Results indicate localization of a β-glucosidase in
the cytoplasm of individual cells and these concentrations vary according to the age of
the root tip. High levels of cytoplasmic β-glucosidase are observed in the three day
control and drop off considerably in the four day control and four day cold-stress
conditions. Immunolocalization signal peaks at the three day control and diminishes
according to day up until the eighth day. The distribution of the signal concentrates in the
apical meristem in control groups, whereas cold-stress exhibit more labeling in the
cortex. This may indicate that β-glucosidases that are active along the cortex are more
primed to activate cytokinins in actively dividing or elongating cortical cells that add
width or length to the root tip. Other research reported that increase in the thickening of
the root diameter is common in response to stress (Degenhardt and Gimmler 2000).
Degnenhardt and Gimmler reported a 50% reduction in root length, but a 14% increase in
root diameter. It is possible that the liberation of zeatin from ZOG by β-glucosidase under
cold stress conditions promotes this increase of root diameter either through growth or
elongation in order to maximize the absorption of ions.
Figure 16 shows the decrease of both isoforms as a root tip ages within the first ten days.
However, the type of isoform varies in the 8 day root tips where the 65kDa band
47
intensifies while the 72kDa drops to intensity levels similar to the background. This again
suggests that the 65kDa isoform increases in quantity during the eighth day specifically,
but not as much as in the third day (Figure 16 p39). Studies show that β-glucosidases
exist as intermediates, and some vary in specificity, which may account for a variety of
intensities according to location and root tip development stage (Seshadri et al. 2009).
Interestingly, increased signal of β-glucosidase in the cell walls accompanied increases in
the possible lower molecular weight peptide observed in western blot with the strongest
labeling peaking at the eight day cold-stress recovery. This is supported by previous
research that reported β-glucosidase activity also peaking at the eight-day cold-stress
recovery (Figure 4 p7). Distribution of signal is localized along the apical meristem,
cortex and pith, therefore suggesting that distribution of β-glucosidase influences the
activity of cytokinins like zeatin which facilitate growth of the root tip and the cell wall.
A putative model of β-glucosidase and its relationship with ZOG and zeatin is illustrated
in Figure 19. Parenchyma cells located in the cortex are divided into the cell wall, plasma
membrane, cytoplasm, nucleus, and vacuole. A root tip growing under permissive
conditions shows that DNA (Gluc1) transcribes RNA which translates β-glucosidase
enzyme in both active and inactive form in the clytoplasm. Once exposed to cold stress,
zeatin is converted into ZOG, growing ceases and inactive β-glucosidase is localized near
the plasma membrane. Upon return to room temperature, β-glucosidase relocalizes to the
parameters of the cell in the plasma membrane and cell wall and de-glycosylates ZOG to
liberate the active zeatin form which induces growth of the root tip and cell wall.
48
FIGURE 19: Model of relocalization of active β-glucosidase according to activity
This is a putative model of how relocation of β-glucosidase to the plasma membrane/ cell
wall region may possibly activate zeatin from ZOG and induce root growth. The initial
location of cytoplasmic β-glucosidase in the 3 day control, moves toward the plasma
membrane during cold stress and becomes inactive. When returned to 25°C, βglucosidase is relocated near and within the cell wall to liberate zeatin from ZOG and
resume growth.
49
FIGURE 19: Model of relocalization of active β-glucosidase according to activity
50
FUTURE STUDY
If glycosylation plays a part in activity of β-glucosidase, proteins can be purified from
specific parts of the cell such as the cell wall and cell membrane. Based on bioinformatics
data, putative glycosylation sites exist. Determination of the type of modification in
relation to the catalytic sites will provide invaluable understanding of how glycosylation
of β-glucosidases can affect specificity and activity with certain substrates.
Also, location of where ZOG is modified in relation to zeatin would lead to a greater
understanding of the relationships between this hormone and β-glucosidase. Although it
has been reported that ZOG is stored in the vacuole (Kiran et al. 2012) due to the location
of β-glucosidase in the plasma membrane and cell wall when activity is highest, it is
probable that ZOG is de-glycosylated outside of the vacuole.
51
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