2. ABSTRACT
BLADE-ON-PETIOLE1 (BOP1) and BOP2 genes in Arabidpsis thaliana have been long associated with
the control growth asymmetry- aspect of patterning of leaves and flowers. BOP1 and BOP2 proteins are part of
the NPR1 (NONEXPRESSOR OF PR GENES1) protein family. NPR1 is a positive regulator of the
systematic acquired resistance, and other members of this family have been assumed to participate in the
modulation of plant defense responses. BOP1 and BOP2 function redundantly using an NPR-1 like
mechanism – formation of oligomers, nucleus trans-localization; preferential interaction with some of
the TGA transcription factors. Recent experiments suggest that bop1bop2 mutants are defective in plant
defense responses, and may function in down-regulation of disease resistance pathways in the plant.
Upon inoculation with P. syringae DC3000 strains, bop1bop2 mutants showed elevated resistance and
decreased pathogen growth .It has also been reported that in short days photoperiods bop1 bop2 double
mutants fail to senesce at normal rate, due to a reduced rate of leaf initiation. Here bop1bop2 double
mutants were tested for alternations in hypersensitive response, induced by infection with Pseudomanas
syrinagae DC3000 (avrRpm1). Runaway cell death phenotype was observed in the leaves of these
mutants, however the results were highly variable. Rate of senescence analysis were performed through
measurement of key developmental parameters in plant longevity. Columbia WT and bop1bop2
mutants were grown in parallel trials under short and long day photoperiods. The results indicated a
delay in the bop1bop2 production of photosynthetic and reproductive organs in short day photoperiods.
There was also a noticeable delay in leaf initiation in long day photoperiods.
ii
3. ACKNOWLEDGEMENTS
I would like to thank my supervisor Dr. Shelley Hepworth for her continuous guidance, support and
patience throughout my work on this project. I would also like to thank Michael Bush for his valuable
help and advice, as well the rest of the members of Dr. Hepworth lab and Dr. Owen Rowland's lab for
sharing their experience and knowledge with me.
I am deeply grateful to my wonderful family and friends, especially to Lisa Watson, for always
believing in me and my abilities, and for inspiring me to be a better person.
iii
4. TABLES OF CONTENTS
ABSTRACT……………………………………………………………………………………………………………………ii
ACKNOWLEDGMENTS…………………………………………………………………………………………………….iii
LIST OF CONTENT…………………………………………………………………………………………………………..iv
LIST OF TABLES……………………………………………………………………………………………………………...v
LIST OF FIGURES……………………………………………………………………………………………………………vi
LIST OF ABBREVIATIONS ………………………………………………………………………………………………..vii
INTRODUCTION……………………………………………………………………………………………………………...1
BLADE -ON-PETIOLE (BOP) 1 and 2 proteins …………………………………………………………………..1
The Hypersensitive Response (HR) …………………………………………………………………………………2
Plant Longevity and Senescence …………………………………………………………………………………….4
Genetic Control of Plant Senescence ……………………………………………………………………………….5
Potential Role of bop1bop2 Mutants in Regulating Rate of Plant Senescence……………………………………6
MATERIALS AND METHODS………………………………………………………………………………………………6
Plant Materials and Cultivation……………………………………………………………………………………..6
Induction of Hypersensitive Response in bop1bop2 double mutant……………………………………………….7
Trypan Blue Staining…………………………………………………………………………………………………7
Measurements of Development Parameters in Plant Longevity…………………………………………………...8
RESULTS……………………………………………………………………………………………………………………….8
bop1 bop2 Mutants Exhibited an Altered Hypersensitive Response …………………………………………………...8
bop1bop2 Mutants Grown Under Long Day Photoperiods Displayed a Delay in Leaf Initiation…………………..12
Delay in Production of Photosynthetic and Reproductive Organs in Short Day Photoperiods………………..14
DISCUSSION…………………………………………………………………………………………………………………16
Evidence of Runaway Cell Death HR Phenotype………………………………………………………………..16
Altered Rate of Senescence in bop1bop2 Mutants………………………………………………………………..17
FUTURE DIRECTIONS……………………………………………………………………………………………………18
REFERENCES………………………………………………………………………………………………………………19
APPENDIX OF TABLES……………………………………………………………………………………………………20
iv
5. LIST OF TABLES
Table 1. Correlative analysis of longevity markers in Col-0 and bop1bop2 mutants development in long day
photoperiods. Observations were collected for 80 days after planting.
Table 2. Correlative analysis of longevity markers in Col-0 and bop1bop2 mutants development in short day
photoperiods. Observations were collected for 81 days after planting.
Table 1A. Columbia plants grown under long day conditions ( 16 hours of light, 8 hours of dark) for 80 days after
planting
Table2A. bop1bop2 mutants grown under long day conditions for 80 days
Table 3A. Columbia plants grown under short day conditions for 81 days after planting
Table 4A. bop1bop2 mutant plants grown under short day conditions for 81 days
Table 5A. Longevity of Columbia plant leaves grown under long day conditions
Table 6A. Longevity of bop1bop2 mutant leaves grown under long day conditions
Table 7A. Longevity of Columbia leaves grown under short day conditions
Table 8A. Longevity of bop1bop2 leaves grown under short day conditions
v
6. LIST OF FIGURES
Figure1. Gene-for-gene resistance and the hypersensitive response
Figure 2. Hypersensitive response signaling during pathogen infection
Figure3. Progress of hypersensitive response cell death 3 days after inoculation.
Figure 4. Runway cell death phenotype in bop1bop2 leaves.
Figure 5. Quantification of local cell death 3 days after bacterial infection
Figure 6. Quantification of local cell death 5 days after bacterial infection
Figure 7. Effects of bop1bop2 on development and longevity in Arabidopsis thaliana in long day photoperiods
Figure 8. Effects of bop1bop2 on development and longevity in Arabidopsis thaliana in short day photoperiods
vi
7. LIST OF ABBREVIATIONS
AVR avirulence
BOP BLADE-ON-PETIOLE
DAP days after planting
CPR1 constitutive expression of pathogen related genes
NPR1 NONEPRESSOR OF PR GENES1
HR hypersensitive response
LD long day photoperiods
LSD lesion simulating disease
NO Nitric oxide
PCD programmed cell death
PR pathogen related genes
R resistance
ROI reactive oxygen intermediates
SA salicylic acid
SAR systematic acquired resistance
SD short day photoperiods
TB trypan blue
WT Wild type
vi
i
8. INTRODUCTION
BLADE -ON-PETIOLE (BOP) 1 and 2 proteins
BLADE-ON-PETIOLE (BOP) 1 and 2 are part of the NPR1 (NONEXPRESSOR OF PR
GENES1) protein family found in Arabidopsis thaliana. NPR1 is an essential regulator of plant defense
responses during systematic acquired resistance, and it does so by interactions with TGA transcription
factors, which activate PR defense genes, (Hepworth et al, 2005). BOP1 and BOP2 function
redundantly using an NPR-1 like mechanism- formation of oligomers, nucleus trans-localization;
preferential interaction with some of the TGA transcription factors, (Hepworth et al, 2005). These
proteins have two conserved domains characteristic for all the members of the NPR1 protein family -
BTB/POZ motif and ankyrin repeats, (Despres et al, 2003, Hepworth et al, 2005). BTB/POZ domain in
NPR1 is required for PR-1 activation by SA, and mutations in this regions lead to loss of function,
(Rochon et al, 2006). The ankyrin in NPR1 repeats are involved in the interactions with members of the
TGA family of transcription factors. BOP1/2 proteins have two conserved ankyrin repeats which are
likely used in their interactions with specific TGA transcription factors such the once encoded by
PARIANTHIA, (Hepworth et al, 2005). They also have conserved Cys residues, which are required for
the redox control of NPR1 and its oligomerization state. Mutations in the bop genes have been always
associated with defects in growth and development of lateral organs, and loss of floral abscission.
Recent experiments suggest that bop1bop2 mutants are defective in plant defense responses, and
may function in down-regulation of disease resistance pathways in the plant. Upon inoculation with P.
syringae DC3000 strains, bop1bop2 mutants showed elevated resistance and decreased pathogen
growth. It has also been reported that in short days photoperiods bop1 bop2 double mutants fail to
senesce at normal rate, due to a reduced rate of leaf initiation, (Norberg et al, 2005). The experiments
described in this paper, were designed to test whether bop1bop2 mutants show alternation in
hypersenasative response or runway cell death similar to other plant defense mutants with increased
resistance to pathogens such as the lsd mutants group,(lesion simulating disease),or the cpr mutants
(constitutive expressor of PR genes) group. It was also investigated through quantitative analysis of
longevity markers whether bop1bop2 mutants have a reduced rate of senescence when they are grown in
short day, or long day photoperiods.
1
9. The Hypersensitive Response (HR)
HR is a form of programmed cell death associated with the restriction of pathogen growth. It can
be recognized by the presence of brown, dead cells at the infection site. Hypersensitive cell death is
distinct from necrosis, which is caused by metabolic toxins or severe trauma.HR requires active host cell
metabolism, (Heath, 2000). HR elicitors could be classified as non-specific and specific elicitors. Plant
defense responses could be preformed or induced by pathogen ingress, and they all relay on multiple
pathways that lead to inhibition of the pathogen growth. Activation of inducible resistance response is
achieved through the gene –for-gene mode where complex array of R (resistance) genes in the plant
trigger resistance: the R-gene product recognizes directly or indirectly a corresponding pathogen
avirulent gene (avr gene) (Figure 1). This recognition often but not aways is associated with the rapid
cell death or HR that is well defined within the attempted infection site (Devadas and Raina, 2002,
Heath, 2000).
(Stuvier and Clusters, 2001)
Figure1. Gene-for-gene resistance and the hypersensitive response
2
1
10. The initiation of HR is marked by the on fluxes across plasma membrane of infected cells, which
is followed by oxidative burst in the infected area and surrounding cells. This is accompanied with the
production of reactive oxygen intermediates ROI such as superoxide, and hydrogen peroxide, and Nitric
oxide (NO), which is a signaling molecule in plant defense, (McDowell and Dangl, 2000; Shirasu and
Schulze-Lefert,2000). There is also significant tissue reinforcement production of antimicrobial
metabolites and induction of R genes expression at the site of infection. This local response is followed
by a secondary resistance response established throughout the plant, and known as a systemic acquired
resistance, (SAR), which is long lasting resistance effective against broad range of pathogens
(McDowell and Dangl, 2000; Shirasu and Schulze-Lefert,2000).
(McDowell and Dangl, 2000)
Figure 2. Hypersensitive response signaling during pathogen infection
The control of HR is established through temporal and stoichiometric balance between ROI, NO
and salicylic acid (SA), a singling molecule and HR and SAR. Their action appears to be synergistic
rather than additive. There is a positive feedback loop of ROI-NO production and SA accumulation
which amplifies the initial signal, (Figure 2). ROI-SA-cell death cycle in HR ends with release of ROI ,
NO and SA in intracellular spaces to inhibit pathogen growth and to worn surrounding cells,( McDowell
3
11. and Dangl, 2000). It was proposed that ROI-SA death cycle was repeated at lower amplitudes in
uninfected tissues in Arabidopsis leaves two hours after inoculation with avirulent strain of
Pseudomonas syringae, (Alvarez et al, 1998). HR micro-oxidative burst at low level amplitudes were
detected throughout the plant, which contributed for the induction of SAR in secondary tissues.
Some of the genes acting upstream of HR signal are lsd, cpr, constitutive immunity (cim3), and
defense, no death (dnd) . They function in the production of ROS and synthesis of SA during the first
stages of SAR induction, (Mauch-Mani and Metraux, 1998). Mutations in these genes leads to increased
pathogen resistance and alternations in HR phenotype such as spontaneous lesions or early HR (cpr
mutant group), or runway cell death which spread beyond the infected site, (lsd mutant group), (Clarke
et al, 2001; Torres et al, 2005)
Plant Longevity and Senescence
Plant ageing in Arabidopsis thaliana is influenced by its reproductive strategy. The ability of the
plant to generate photosynthetic organs such as leaves and stems, and loss of meristem activity appears
to play a central role in plant senescence, (Nooden and Penney, 2001). Senescence is associated with
programmed cell death, which also occurs during HR. Whole plant or monocarpic senescence is
associated with senescence of photosynthetic tissues and suppression of meristematic development. It
was suggested that monocarpic senescence has a polyphyletic origins due to its diversity of forms,
(Bleecker and Patterson, 1997). Development of vegetative organs in plants is indeterminate and
modular; the continuous generation of new organs by the meristem activities throughout the plant is
opposed by the programmed senescence and abscission of existing organs. In other words the plant
development and longevity is affected by the suppression of apical dominance and loss of meristem
activity in the development of flowers, (Bleecker and Patterson, 1997). In monocarpic plants senescence
is governed by the reproductive development, during which old vegetative organs are not replaced with
new once, and the remaining organs are signalled to die. Factors that delay reproductive development
can also affect the whole plant senescence, (Nooden and Penney, 2001). Senescence of individual leaves
in Arabidospis unlike in other monocarpic plants is not government by the reproductive development,
and the leaf longevity is not affected by the presence or the absence of reproductive organs. However the
rate of the whole plant senescence is altered, when the plant is able to regenerate more vegetative organs
and there is significant delay in reproductive organ generation. Senescence in Arabodpsis can be taken
as a result of favoring reproduction at expense of somatic tissues. It can be assumed that plant is set on
4
12. its path to senescence with the onset of the early stages of to reproductive development. During this
development stage the apical meristem is enlarged, and there is initiation of flower meristerms instead of
leaf primordia on the flanks of the apical meristem, (Bleecker and Patterson, 1997). Primary
inflorescence meristerm can produce only certain number of flowers before its arrest, and by this point
all shoot meristems in the plant have become inflorescence meristems. When these meristems arrest, the
plant stops generating new photosynthetic organs, which speeds the rate of senescence. The arrest of all
inflorescence meristems happens within 24 hours and it directly correlates with seed production,
(Bleecker and Patterson, 1997). The programmed nature of vegetative senescence ensures that enough
energy and nutrients are redirected from the dying tissues to sites of seed development. This depletes the
plant of renewable resources and leaves it with no generation capacity for new photosynthetic organs,
which could explain why Arabidopsis is a relatively short-lived plant. It has been observed that
Columbia ecotype plants tend to live longer than Landsberg plants, probably due to a higher number of
photosynthetic organs produced, (Nooden and Penney, 2001).
Genetic Control of Plant Senescence
The timing and location of senescence requires new gene transcription, and it is a process
undergoing significant post-transcriptional and post-regulational modifications, (Thomas et al, 2003).
Increasing number of genes have been associated with senescence, (more than 100 genes) based on
elevated levels of their mRNAs in senescing tissues, (Bleecker and Patterson, 1997, Jing et al, 2003).
Senescence associated genes (SAGs) may code for proteins with scavenger functions; based on their
deduced amino acid sequence these proteins are similar to proteaseases, nucleases and proteins involved
in nitrogen and lipid metabolism, (Bleecker an Patterson, 1997). During whole plant senescence the
levels of nitrogen and carbon decline in the dying tissues; there is implied transport of metabolites out of
the dying tissues, which become a source of energy and nutrients to the whole plant. It is quite possible
that SAGs play role in this transport, however there is no common mechanism controlling SAG
expression, and these genes have been found to act in other biological processes too. Senescence is a
considered a complex development stage regulated by diverse biochemical pathways, and it is
modulated by variants in a large number of genetic loci, (Jing et al, 2003).
5
13. Potential Role of bop1bop2 Mutants in Regulating Rate of Plant Senescence
Norberg et al, 2005, has discovered that short days bop1bop2 double mutants fail to senescence
at normal rate. Flowering in these double mutants was delayed compared to the wild type Col-0.
Apparently this delay was largely due to the slower leaf initiation rate. bop1bop2 mutants formed 15
more leaves until flowering compared to the wild type. The plants continued to grow for at least 10
more months, before they died, while it took only 4 months for wild type plants under short day
conditions. Since whole plant senescence has such a diverse genetic regulation, it is possible that
bop1bop2 mutants could participate in some of the signalling biochemical pathways controlling the
timing and rate of senescence. The experiments described here were designed to test through
quantitative analysis of longevity markers whether bop1bop2 mutants have a reduced rate of senescence
when they are grown in short day, or long day photoperiods.
MATERIALS AND METHODS
Plant Materials and Cultivation
Plant genotypes for HR analysis: Wild-type Arabidopsis thaliana plants were the Columbia-0 (Col-0)
ecotype; bop1bop2 double mutants, bop1 and bop2 single mutants, npr1 and cpr1. All seeds except cpr1
mutants were obtained from Dr. Hepworth’s seed collection, Carleton University, Ottawa. Seeds for
cpr1 mutants were obtained from Dr. Subramanian, Eastern Oil and Seeds Research Center, Agriculture
Canada, Ottawa.
Seeds were sterilized in a 50% bleach/0.5% SDS solution for 10 minutes and rinsed 5 times with
sterile water. All seeds were planted directly in 3.2 inches flower pods on fertilized (20-20-20 fertilizer)
soil (BMI Growing Mix peat moss, Berger Horticulture, Canada). Dormancy of the seeds was broken
by storing the seeds the dark at 4°C for 3 days. 10 days old seedlings grown under short day conditions
(SD) (light cycle: 8 hours day, 16 hours night; 22°C temperature; 4% relative humidity ) were replanted
in 1 inch flower pods. 18 plants per genotype were grown for 3.5 weeks before infection.
6
14. Plants genotypes for rate of senescence analysis: Wild-type Arabidopsis thaliana, Col-0 ecotype, and
bop1bop2 mutants. Seeds were obtained from Dr. Hepworth’s seed collection, Carleton University,
Ottawa.
Seeds were sterilized as described above, and they were sown onto Arabidopsis thaliana (AT) minimal
media, and transplanted into fertilized soil 10 days later. Dormancy of the seeds was broken by storing
them in dark at 4°C for 3 days. 12 plants per genotype were grown in 3.2 inches flower pods, one
flower per pod, in two parallel trials in short day (SD) and long days photoperiods (LD)(light cycle: 16
hours day, 8 hours night; 20°C temperature; 60% relative humidity). Plants were monitored for 3 months
for alternations of earlier stages of development that could alter rate of senescence.
Induction of Hypersensitive Response in bop1bop2 double mutant
Bacterial induction of HR in 3.5 weeks old plants of the genotypes described above was done by
infiltration of Pseudomonas syringae DC3000 (avrRpm1) suspension (OD=0.0002) into one side of the
leaf using needless syringe. Two plants per genotype were infected with distilled water as water
controls. 4 to 5 leaves were infected per plant of each genotype. Plants were inspected for disease
symptoms and/or altered HR phenotype over 6 days under the same conditions as described above.
Runway cell death phenotype was recorded when the HR cell death expanded beyond the infection site,
across the leaf midvain.
Trypan Blue Staining
Cell death induced by pathogen inoculation on leaf tissues was monitored by staining with
lactophenol-trypan blue (TB) and distaining in saturated chloral hydrate. 4 leaves were sampled from
different plants of each genotype 6 hours and 3 days post inoculation. One leaf was sampled from the
water control plants. Leaf tissues were placed in 12-well microtiter plat- 4 leaves per genotype per well.
The leaves were covered with trypan blue-lactophenol solution, and the plate was heated in boiling
water bath for 1.5 min. The plate was allowed to sit at room temperature for 20 mins. The staining
solution was removed, and the tissue was covered in 7.5M choral hydrate solution. The leaves were
incubated at room temperature in the distaining solution with gentle shaking for 3 days. The chloral
hydrate solution was replaced 3 times during the incubation.
7
15. Measurements of Development Parameters in Plant Longevity
Several key development parameters in plant longevity were measured: the birth and longevity
of individual leaves, the date at which the initial stem with inflorescence had reached 1 cm, the opening
of the first flower with white petals showing spread apart, the production of the first full silique.
Marking of Leaf Birth
Leaves including the cotyledons were counted in order of appearance, and four leaf cohorts (leaf
#12, 14, 16, and 18) were marked near the tip with a drop of water-soluble correction fluid. The date of
birth was designated as the day when the new leaf reached 1 mm in length, (Nooden and Penny, 2001)
Leaf Death and Longevity
Leaves were scored as dead when they were flaccid or dried over more than half of their surface
(Nooden and Penny, 2001). The leaf death could be determined within 1 day. The longevity of the
individual marked leaves was taken to be the time span from the date of birth until the date of death.
RESULTS
bop1 bop2 Mutants Exhibited an Altered Hypersensitive Response Phenotype
Similar to Runway Cell Death.
Many of the mutants displaying increased resistance to pathogen infection or defective in
defense response pathways display altered hypersensitive response such as early onset or runway cell
death. Since there have been recent reports of increased resistance in bop1bop2 mutants to various
strains of the bacterial pathogen Pseudomanas syrinagae pv. tomato DC3000 , it was possible that these
mutants also have an altered hypersensitive response phonotype similar to other plant defense mutants
such as lsd mutants or the cpr mutant group. This was tested with infection of 3.5 weeks old plants
grown in short day photoperiods with Pseudomanas syrinagae DC3000 (avrRpm1). Induction of HR
was done in bop1bop2 double mutants, bop1 and bop2 single mutants. The negative control was the
npr1 mutant, which is insensitive to all SAR inducers and avirulent pathogens, and it is compromised in
its ability to express PR-1, PR-2, and PR-5, (PR- pathogen resistant genes), (Dong, 2004). It was
8
16. assumed that the HR response in npr1 mutants will be either not detectable or highly delayed compared
to the Col-0 WT plants, despite the fact that NPR1 acts downstream of HR gene signaling. The positive
control was cpr1 mutants, which is one of the early genes involved in HR signaling. It displays an
increased resistance to various pathogens such as Pseudomonas syringae ES4326 and the oomycete
pathogen Peronospora parasitica Noco2, (Clarke et al, 2000). However it was discovered later in the
experiment that this member of the cpr mutant group does not exhibit spontaneous lesions or early HR
phenotype like cpr5 for example. The progress of the HR cell death was monitored for 5 days after
infection. There was little or no visible HR in any of the mutant combinations infected 6 h after
inoculation. By 3 days after inoculations spontaneous lesions and expanding cell death beyond the
infection site was observed in leaves of bop1bop2 double mutants, bop1 and bop2 single mutants,
(Figure 3.). Col-0 WT leaves displayed only local cell death restricted to the site of infection. Local cell
death or no HR symptoms were detected in npr1 and cpr1 mutants. These observations were confirmed
with trypan blue staining, (Figure 4.). Sampled leaves from all bop1/2 double and single mutants
revealed spread of cell death across the leaf midvain into uninfected tissues. This HR phenotype was
consistent with runway cell death displayed by the lsd mutant group in similar experiments, (Torres et
al, 2005). Local cell death or micro-HR bursts were only visible in the infected site of TB stained leaves
of WT plants (Figure 4). The cpr1 mutant also displayed small micro-HR burst throughout stained
leaves, which were otherwise not detectable. The npr1 mutant displayed micro-HR burst only in the
inoculated site, however their number and area of range was smaller compared to WT.
Quantification of cell death was done based on 30 leaves count per genotype, (Figure 5. and 6).
No runway cell death was observed in Col-0 WT during the 5 day trial. Col-0 WT leaves exhibited 22
leaves with local cell death 3 days after infection; the number increased to 25 leaves 5 days after
infection,(Figure 6.). bop1bop2 mutants displayed 22 leaves with local cell death, and 9 leaves with
runaway cell death phenotype 3 days after infection; 5 days after infection 11 leaves displayed this HR
phenotype. bop1 and bop2 single mutants produced low number of leaves with runway cell phenotype –
3 leaves at day 3, and 8 leaves at day 5. The positive control cpr1 mutant exhibited only 14 leaves with
local cell death at day 5; the negative control npr1 mutant displayed even higher numbers than the
positive control – 17 leaves in total with local cell death symptoms, (Figure 6.). Early HR phenotype in
cpr1 mutants could be only detected after TB staining, which revealed small HR burst of cell death
spread across the entire leaf tissue (Figure 4.)
9
17. Figure3. Progress of hypersensitive response cell death 3 days after inoculation. Arrows indicate points of
expanding cell death beyond the infection site in the leaves of bop1bop2 double mutants, bop1 and bop2 single
mutants.
Figure 4. Runway cell death phenotype in bop1bop2 leaves. Spread of runway cell death 3 days after bacterial
infection beyond the midvain of the leaf is only visible in bop1bop2 double mutants, bop 1 and 2 single mutants.
Col-0 bop1bop2 bop1 bop2 npr1 cpr1
10
18. -5
0
5
10
15
20
25
30
1 2 3 4 5 6
#OFLEAVES
SYMPTOMS DISPLAYED 3 DAYS AFTER INFECTION
No symptoms Local cell death Runway cell death
Columbia bop1bop2 bop1 bop2 npr1 cpr1
Figure 5. Quantification of local cell death 3 days after bacterial infection. Results are based on 30 leaves
inoculated per genotype. 9 leaves displayed runway cell death for bop1bop2 double mutants; bop1 and bop2
single mutants displayed only 3 leaves with this HR phenotype.
-5
0
5
10
15
20
25
30
1 2 3 4 5 6
#OFLEAVES
SYMPTOMS DISPLAYED 5 DAYS AFTER INFECTION
No Symptoms Local cell death Runway cell death
Columbia bop1bop2 bop1 bop2 npr1 cpr1
Figure 6. Quantification of local cell death 5 days after bacterial infection. 30 inoculated leaves per genotype
were examined for altered HR phenotype. 11 leaves displayed runway cell death for bop1bop2 double mutants;
bop1 and bop2 single mutants displayed.
11
19. bop1bop2 Mutants Grown Under Long Day Photoperiods Displayed a Delay in Leaf
Initiation.
Analysis of key developmental parameters in plant longevity in long day photoperiods revealed
altered rate of leaf initiation in bop1bop2 double mutants (Figure 7, Table 1). 12 plants per genotype
(Col-0 and bop1bop2 mutants) were grown for 80 days in the conditions described above.
Figure 7. Effects of bop1bop2 on development and longevity in Arabidopsis thaliana in long day photoperiods.
Delay in leaf initiation was observed in bop1bop2 double mutants. Production of shoots, flowers and siliques
followed closely Col WT time points.
Col-0 bop1bop2
18
DAP
26
DAP
30
DAP
31
DAP
12
20. Measurements of development markers that could indicate altered rate of senescence included:
birth and longevity of individual leaves, production of shoots, flowers and siliques, (Table 1.). The
bop1bop2 mutants had a retard growth in the first part of the trail, and reached normal size at 46 days
after planting (DAP). 18 DAP all WT plants displayed leaf# 12 and 14, while bop1bop2 mutants
displayed only leaf #9 or #10, (Figure 7). 26 DAP, WT displayed leaf #16 and 18; bop1bop2 mutants
developed leaf #12. 33 DAP, all WT plants had leaf #18, (Table 1A.), and 36 DAP they have all
developed shoots, flowers and siliques. At 33 DAP some bop1bop2 mutants displayed leaf #14 without
shoots. The majority of the mutant plants produced shoots, flowers and siliques while having 11 or 12
leaves in total.
Table 1. Correlative analysis of longevity markers in Col-0 and bop1bop2 mutants development in long day
photoperiods. Observations were collected for 80 days after planting.
Development parameters Col (wt)
Days since planting ± s.e.m.
N=12
bop1bop2
Days since planting ± s.e.m.
N=12
Leaf 12 birth 17.83 (0.11) 30.17 (1.53)
Leaf 14 birth 20.33 (0.43) 37.83 (1.60)
Leaf 16 birth 25.00 (0.92) 41.83 (1.50)
Leaf 18 birth 30.41 (0.90) 45.42 (0.90)
Bolting 27.25 (1.03) 32.33 (1.63)
1st
Flower open 29.25 (1.10) 34.33 (1.70)
1st
full silique 32.00 (0.78) 36.33 (1.70)
Longevity (d) Longevity (d)
Leaf 12 longevity 32.83 (0.32) 36.08 (0.31)
Leaf 14 longevity 34.75 (0.28) 36.25 (0.33)
Leaf 16 longevity 35.66 (0.38) 36.17 ( 0.27)
Leaf 18 longevity 33.17 (0.30) 35.83 (0.53)
36 DAP only two bop1bop2 mutants displayed leaf #16 and 18. Eight plants had only leaf# 12, and
seven of these plants were bolting and flowering (Table 2A.). Ten plants in total were bolting, nine
13
21. plants had flowers, and seven plants had full siliques. 46 DAP all bop1bop2 mutants had shoots, 1st
open
flowers, and 1st
fully extended siliques. 47 DAP. All bop1bop2 mutants had leaf # 18, which was
fourteen days later after all Columbia plants finally displayed their leaves #18, (Table 1A and 2A.). The
leaf longevity in bop1bop2 plants did not differ significantly from WT. (Table 1). The life span of the
individual marked leaves (#12, 14, 16, and 18) in the mutants was prolonged with only 2 days compared
to WT leaves. Refer to supplemented material in the Appendix of Tables for recorded leaf longevity
observations for each mutant plant in the trail.
bop1bop2 Mutants Showed a Delay in Production of Photosynthetic and
Reproductive Organs in Short Day Photoperiods.
Figure 8. Effects of bop1bop2 on development and longevity in Arabidopsis thaliana in short day photoperiods.
bop1bop2 mutants showed significant delay in production of photosynthetic and reproductive organs compared to
WT.
Col-0 bop1bop2
24
DAP
28
DAP
42
DAP
77
DAP
14
22. The same measurements of development parameters were done in short day periods with 12 plants per
genotype. The plants were monitored for 81 days. bop1bop2 plants had slower rate of production of
photosynthetic and reproductive organs compared to WT. 24 Days after planting, WT plants displayed
leaf #12 and #14; bop1bop2 mutants had only leaf # 9 or #10, (Figure 8, Table 2). 28 DAP most of the
WT plants displayed leaf #18,( Table 3A); bop1bop2 mutant have just developed leaf #12 and only two
plants had leaf #14, (Table 4A).
Table 2. Correlative analysis of longevity markers in Col-0 and bop1bop2 mutants development in short day
photoperiods. Observations were collected for 81 days after planting.
Development parameters Col (wt)
Days since planting ± s.e N=12
bop1bop2
Days since planting ± s.e N=12
Leaf 12 birth 19.75 (0.25) 27.92 (0.74)
Leaf 14 birth 23.75 (0.41) 32.58 (0.94)
Leaf 16 birth 26.17 (0.30) 36.58 (0.56)
Leaf 18 birth 28.33 (0.31) 40.83 ( 0.21)
Bolting 63.90 (0.43) 79.2 (1.2)
1st
Flower open 65.8 (0.51) ND
1st
full silique 68.3 (61.5) ND
Longevity (d) Longevity (d)
Leaf 12 longevity 41.92 (0.20) 43.33 (0.26)
Leaf 14 longevity 42.33 (0.27) 44.58 ( 0.26)
Leaf 16 longevity 42.08 (0.23) 42.94 (0.23)
Leaf 18 longevity 41.83 (0.21) 41.83 (0.21)
By 44 DAP, WT plants were producing shoots, flowers and siliques, while bop1bop2 mutants had only
produced leaf #18. For 30 more days bop1bop2 plants were producing more leaves, without bolting. 81
DAP, half of the bop1bop plants produced shoots, but no flowers or siliques, (Table 2, Table 4A). The
bop1bop2 individual leaves lived 2 days longer than WT leaves; leaves #18 had the same longevity for
15
23. both genotypes – 41.83 days, and the life span was shorter compared to that of leaves #12,14, and 16 in
the same genotypes, (Table 2.).
DISCUSSION
Evidence of Runaway Cell Death HR Phenotype
Alternation in HR phenotype in bop1bop2 mutants were established 3 days after infection with
the bacterial pathogen Pseudomanas syrinagae DC3000 (avrRpm1). Expanding cell death beyond the
inoculated side was visible only in the leaves of bop1bop2 double and single mutants, (Figure 3). The
HR phenotype was similar to the runaway cell death in lsd mutants, (Aviv et al, 2002; Torres et al,
2005). TB staining of sampled leaves indicated HR cell death was spreading across the leaf midvain into
uninfected areas, (Figure 4). However quantification of leaves expressing runway cell death like
phenotype revealed very low numbers for bop1bop2 double mutant – only 9 out of 30 leaves, (Figure 5).
The number did not increased significantly at day 5 of the experiment; only 11 leaves were engulfed in
cell death, (Figure 6). bop1 and bop2 mutants produced even lower numbers of leaves with altered HR
phenotype – three leaves were counted for day 3, and between 7 and 8 leaves for day 5. The results were
highly variable based on the size and the age of the leaves. Smaller leaves more were likely to express
runway cell death HR phenotype than larger leaves. Conclusions can be based solely on the results
produced by one trail. Further experiments would be required to establish whether bop1bop2 mutation is
indeed defected in HR , and what is the place of BOP1 and 2 in the HR signaling pathway. The
possibility of developmental genes being involved in plant defence has been explored previously. It is
reported that another development gene ASYMMETRIC LEAVES 1 (AS1) could act as a repressor in the
inducible defense response against a relatively broad range of necrotrophic fungi, (Nurmberg et al,
2007). AS1 is known to act together with BOP1/2 in the suppression of the class I knox genes in the
shoot apical meristem, and thus promoting the normal leaf development, (Norberg et al, 2005).
The positive control cpr1 displayed local cell death signs, although the areas of visible cell death
in the form of lesions was very small. Only after TB staining small-HR microburst were detected
throughout the entire leaf indicating the presence of early HR, (Figure 4) , which phenotype is
associated with the cpr mutation, (Clarke et al, 2001, Jirage et al, 2001). The negative control npr1 did
16
24. show signs of local cell death, and the number of leaves were unexpectedly high, and very close to
these of the positive control, and WT (Figure 5 and 6). This could be attributed to the fact that npr1
mutation affects defense response signaling downstream of HR and SA accumulation, (Dong, 2004). It
is quite possible that HR response is not affected at all in npr1 mutation, or at least not altered
significantly.
Altered Rate of Senescence in bop1bop2 Mutants
Analysis of senescence rate revealed delay in the bop1bop2 production of photosynthetic in short
day photoperiods. There was also a noticeable delay in leaf initiation in long day photoperiods.
Arabidopsis is monocarp plant that normally reproduces and senesces under LDs. However it is not an
obligate LD plant and it can produce reproductive organs and die in SD, (Nooden et al, 1996). The rate
of senescence is usually slower in SD photoperiods, (Nooden et al, 1996). Effect of bop1bop2 mutation
in plant development under LD conditions did not reveal any delay in generation of productive organs,
which influence the whole plant senescing rate. Mutant plants were developing shoots, flowers and
siliques relatively at the same time as WT (Figure 3, Table 1). The rate of leaf initiation was slower, and
the mutants had produced less leaves than WT when they started bolting. Environment factors such as
photoperiods and intensity of light could speed up rate of senescence in the sense that development rate
of the reproductive structures is accelerated. There were no significant changes in individual leaf
longevity in bo1bop2 mutants under LD. Although the leaves were initiated slower than WT, their life
span was not prolonged, (Table 1.). It was established previously that delayed flowering mutations and
inflorescence- removal treatments did not exert correlative controls on leaf senescence, (Nooden et al,
1996). This was observed again here in SD photoperiods, where the delay in bop1bop2 inflorescence
generation did not prolonged leaf longevity. The observations were consistent with previous reports by
Norberg et al, in that bop1bop2 plants produced more leaves than WT before developing flowers and
fruits.
It can be assumed that the late formation of reproductive structures is the factor which slows than
the rate of senescence in bop1bop2 mutants. Previous experiments have established that some growth
mutations in Arabidopsis prolonged plant life through the production of more leaves and bolts, without
affecting individual leaf longevity, (Nooden and Penny, 2001). Among these mutations are clavata,
17
25. vam1 enh (rev-1), dark-green and det3 mutants. Clavata and vam1 enh (rev-1) mutations the growth and
function of shoot meristem. The loss of meristem activity plays significant role in the delay of whole
plant senescence, (Bleecker and Patterson, 1997). BOP1 and BOP2 are known to regulate the
meristematic activity at discrete locations in developing lateral organs, (Ha et al, 2004). They are
involved in the leaf formation by suppressing the development of ectopic meristematic activity that leads
to the formation of new organs, (Norberg et al, 2005). The role of BOP1/2 proteins in modulating the
identity of the shoot apical meristem could be connected with a role in the regulation of the whole plant
senescence. It is also possible that BOP1/2 act in entirely different biochemical pathway controlling the
rate of senescence.
FUTURE EXPERIMENTS
Further tests with other P. syringae pt. DC3000 strains could be performed to confirm wheather the
bop1bop2 mutation is constitutively associated with a runway cell death HR phenotype. The plants
should be inoculated 4 or 5 weeks after planting to insure the size of the leaves would not introduce any
variation in the results. The leaves chosen for infection should be of the same shape, age and the same
order of appearance in the leaf cohorts.
The rate of senescence analysis will be continued with the plants growing in short day photoperiods.
The plants should be monitored for at least 6 more months, and other longevity parameters will be
recorded such as the death of the last caulin and rosette leaf which correlates with the death of the whole
plant, (Nooden and Penny, 2001). Based on these results conclusions could be made whether the slow
leaf imitation rate and the delay in reproductive organ generation observed in bop1bop2 are contributing
factors in an altered rate of senescence.
18
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20
28. APPENDIX OF TABLES
Table 1A. Columbia plants grown under long day conditions ( 16 hours of light, 8 hours of dark) for 80
days after planting.
Plant # Leaf# 12
birth ( days
after
planting)
Leaf# 14
birth
(days
after
planting)
Leaf#16
birth
(days
after
planting)
Leaf#18
birth
(days
after
planting)
Bolting
(days
after
planting)
1st
flower
open
( days
after
planting)
1st
Silique
(days
after
planting)
1 18 22 25 28 36 38 39
2 17 18 19 26 29 30 32
3 18 20 25 35 27 31 31
4 18 22 29 33 27 29 31
5 18 20 25 28 29 31 33
6 18 22 25 28 29 31 33
7 17 18 21 26 27 29 31
8 18 19 23 34 23 25 29
9 18 20 23 33 23 24 31
10 18 20 27 32 23 25 30
11 18 22 29 31 27 29 32
12 18 21 29 31 27 29 32
21
