Plant Cell Physiol. 47(6): 736–742 (2006)
doi:10.1093/pcp/pcj045, available online at www.pcp.oupjournals.org
JSPP © 2006
Increase of Homologous Recombination Frequency in Vascular Tissue of
Arabidopsis Plants Exposed to Salt Stress
Alex Boyko, Darryl Hudson 1, Prasanna Bhomkar, Palak Kathiria and Igor Kovalchuk *
Department of Biological Sciences, University of Lethbridge, Lethbridge, AB, T1K 3M4, Canada
;
al. 2005, Boyko et al. 2006). Another level of complexity is
introduced by the fact that different genome areas apparently
use the HR to different degrees (Puchta et al. 1995, Filkowski
et al. 2004a).
Estimation of a particular compound’s mutagenicity, being
either physical or chemical in nature, depends on the ability to
estimate the amount of DNA damage in the control and
exposed population of organisms. Of particular interest is the
analysis of DSBs and the activity of the repair machinery that
takes care of DSBs. Strand breaks are the most dangerous type
of DNA damage which, if left unrepaired, prevent cell cycle
progression and can result in cell death (Karanjawala et al.
2002, Sancar et al. 2004).
Most of the recent studies directed towards estimating the
frequency of HR are based on the visual detection of reporter
gene reactivation (Swoboda et al. 1994, Puchta et al. 1995,
Gorbunova et al. 2000, Kovalchuk et al. 2003, Filkowski et al.
2004a, Filkowski et al. 2004b, Filkowski et al. 2004c, Li et al.
2004, Abe et al. 2005, Boyko et al. 2005). Reactivation of the
inactive β-glucuronidase (GUS; uidA) transgene, the most
commonly used reporter gene, can be visualized through histochemical staining that requires the penetration of the cleavage
substrate [5-bromo-4-chloro-3-indolyl glucuronide (X-glu)]
into the cells. The detection of the reactivated gene is based on
the ability to observe the recombination events either with the
naked eye, or with the help of a dissecting microscope (with
minimal magnification). This allows recombination events in a
group of 200–400 plants to be counted in a relatively fast manner, thereby enabling processing of the results of an entire
experiment with 3–4 doses of mutagen in a couple of days. It is
of course possible to observe the recombination events at
higher magnifications; this, however, slows down the process
of counting recombination events tremendously, making the
repetitions of experiments with multiple doses of mutagens virtually impossible. In addition, this method cannot detect all of
the recombination events that have taken place, especially
those occurring in subepidermal cells. It is therefore possible
that recombination is occurring at different rates in control and
mutagen-treated plants, but is undetectable using conventional
methodology.
Here we analyzed the influence of salt stress on the frequency and distribution of recombination events by performing dissections of paraffin-embedded histochemically stained
Here we analyzed the influence of salt stress on plant
genome stability. Homologous recombination events were
detected in transgenic Arabidopsis plants that carried in
their genome a β-glucuronidase recombination marker.
Recombination events were scored as blue sectors using a
stereo microscope. Exposure to 50 mM salt resulted in a
3.0-fold increase in recombination frequency. To analyze
the organ and tissue specificity of recombination events, we
examined cross-sections of leaves, stems and roots. We
found that nearly 30% of recombination events in plants
grown under normal conditions and nearly 50% of events
in plants grown on salt were undetected by the conventional
method. Most of the recombination events represented a
cluster/group of cells (12 on average), although events with
single cells were also detected. Recombination events were
very frequent in leaf mesophyll cells. On average, individual recombination events located on leaves contained more
cells than events located on roots or stems. Analysis of
recombination events in cross-sectioned tissue of salttreated plants revealed a shift in the distribution of recombination events towards the vascular tissue. We discuss
the significance of the finding for plant stress physiology.
Keywords: Arabidopsis thaliana — Homologous recombination — Salt stress — Tissue specific — Vascular tissue.
Abbreviations: DSB, double strand break; GUS, β-glucuronidase; HR, homologous recombination; MS, Murashige and Skoog;
PBS, phosphate-buffered saline; X-glu, 5-bromo-4-chloro-3-indolyl
glucuronide.
Introduction
Homologous recombination (HR) is one of two major
double strand break (DSB) repair mechanisms (Bleuyard et al.
2005, Schuermann et al. 2005). Its contribution to repair of
breaks varies between organisms belonging to different kingdoms, families and even different species (Cromie et al. 2001).
Moreover, the rate at which cells repair the breaks using HR
depends on factors such as the type of organ the cell is in, the
plant developmental stage and the growth conditions (Boyko et
1
*
Present address: Department of Molecular and Cellular Biology (MCB), University of Guelph, Axlerod building, Guelph, Ontario, N1G 2W1,
Canada
Corresponding author: E-mail, [email protected]; Fax, +1-403 329 2242.
736
Homologous recombination in Arabidopsis tissue
737
Fig. 1 Detection of homologous recombination events. (A) The
homologous recombination substrate consisted of the 5′ end (GU) and 3′
end (US) of the uidA transgene cloned in a direct orientation (Swoboda
et al. 1994). A recombination event between two regions of homology
(‘U’) restores the active GUS gene. (B) Restoration of transgene activity is visualized upon histochemical staining as blue sectors.
Arabidopsis plants. We show that exposure to salt results in a
3-fold increase in the number of recombination events primarily due to the increase in number of events in vascular tissue.
Results and Discussion
Recombination lines used in the experiment
The repair of DSBs was analyzed using Arabidopsis
thaliana line #11 plants transgenic for a uidA (cv C24) HR substrate (Puchta et al. 1995). The substrate for repair consisted of
two disrupted, non-functional, overlapping copies of the
marker gene cloned in direct orientation (Fig. 1a). A single
DSB generated in the region of homology, ‘U’, can potentially
be repaired via HR, restoring the functional gene. Histochemical staining reveals the cells and their progeny where recombination took place as blue sectors (Fig. 1b).
Exposure to 50 mM NaCl resulted in increase of HR frequency
To analyze the influence of salt on genome stability, plants
of line 11 were germinated on Murashige and Skoog (MS)
medium supplemented with 50 mM NaCl. Control plants were
grown without NaCl. Three independent groups, consisting of
50 plants each, were used for control and salt treatment. Plants
germinated on the medium supplemented with salt were phenotypically similar to those grown on normal medium. The average recombination frequency fluctuated in controls from 1.64
to 1.80 events per plant (as calculated by relating the number of
events to the number of plants). Exposure to salt resulted in a
2.90- to 3.03-fold increase in the number of recombination
events, bringing it to 4.76–5.46 events per plant. The difference in recombination frequency was statistically significant in
all three cases (P = 0.002, P = 0.002 and P = 0.009). An interesting phenomenon that was observed was the shift in distribution of recombination events under the influence of salt,
changing it from a nearly Poisson distribution to a non-Poisson
distribution (Fig. 2). The increase in HR frequency was primarily due to the appearance of plants with a very high number of
Fig. 2 Distribution of recombination events in control and saltexposed plants. Three independent experiments were performed. The
distribution of recombination events in a group of 50 control and saltexposed plants is presented. The x-axis shows the number of spots per
plant, whereas the y-axis shows the number of plants with this number
of events.
events, 32, 43 and 52, etc. This phenomenon was described
previously by Swoboda et al. (1994).
To understand the mechanism of the NaCl-induced
increase in HR frequency, we decided to analyze in what tissue/
cells the recombination was most frequent.
Cross-sectioning of control plants showed that >30% of recombination events remained undetected
To analyze the number of recombination events in different tissues of control plants, we used 50 plants from the first
738
Homologous recombination in Arabidopsis tissue
Fig. 3 Homologous recombination events
in leaves. (A) Schematic representation of
the leaf cross-section; epidermal, mesophylic and vascular tissues are shown. (B)
Schematic representation of a recombination event (in blue) located in the spongy
parenchyma (mesophylic tissue). (C)
Schematic representation of a recombination event (in blue) located in mesophylic
and vascular tissue. (D) A recombination
event (with a close up) comprised of several mesophylic and vascular cells
(stained blue). The size bar represents
100 µM. (E) A recombination event (with
a close up) comprised of spongy parenchyma cells (stained blue) located around
the vascular tissue (stained in red). The
size bar represents 80 µM. (F) A recombination event comprised of several mesophylic and vascular cells; vascular cells
with no GUS activity are stained in red.
The size bar represents 10 µM.
Fig. 4 Homologous recombination events
in roots. (A) Schematic presentation of the
root cross-section; epidermal, cortex and
vascular tissues are shown. (B) Schematic
presentation of a recombination event
located (from left to right) in vasculature,
cortex or epidermis (in blue). (C) A singlecell recombination event located in the
cortex; the size bar represents 20 µM. (D)
A two-cell recombination event in the epidermis; the size bar represents 30 µM. (E)
A recombination event comprised of multiple cortex and vascular tissue cells; the
size bar represents 10 µM. (F) A recombination event consisting of multiple epidermal cells; the size bar represents 200 µM
Homologous recombination in Arabidopsis tissue
739
Fig. 5 Homologous recombination events in the stem. (A) Schematic presentation of the stem cross-section; epidermal, cortex and vascular tissue are shown. (B) A recombination event located in pith between two conductive vessels; the size bar represents 50 µM.
experimental control group. Whole histochemically stained
plants were embedded in paraffin and sectioned. Slides that
were prepared from 50 stained plants were used for screening
for recombination events using a high resolution microscope
(Fig. 3–5). A total of 107 spots were recorded, thus resulting in
an HR frequency under high magnification of 2.14 per plant on
average (Table 1). In contrast, observation of plants using the
dissecting microscope could detect 82 spots in 50 plants,
thereby resulting in an HR frequency of 1.64 per plant. Thus,
the HR frequency calculated using high magnification was
30% (107 vs. 82) higher than that which was calculated using
the dissecting microscope. This change was primarily due to a
significant increase in the number of blue spots observed in the
roots (25 vs. 9). In contrast, there was only a marginal (10%)
increase in the number of recombination events recorded in
cross-sectioned leaves (80 vs. 73 observed under a binocular
microscope) (Table 1). Interestingly, of the 107 spots detected
Table 1 Homologous recombination frequency in control and
salt-treated plants, as observed under the binocular microscope
and upon cross-sectioning
Control
Binocular Cross-section
Leaves
Stems
Roots
Total
HRF
Salt
Binocular Cross-section
73
0
9
82
80
2
25
107
215
0
23
238
233
9
107
349
1.64
2.14
4.76
6.98
Recombination events were counted in each of 50 plants for both the
control and salt-treated groups. This was done either using a binocular
microscope or after cross-sectioning. The table shows the number of
total of recombination events. HRF, homologous recombination
frequency observed under the binocular microscope or after crosssectioning.
after cross-sectioning, only two were present in the stem. This
suggests that recombination events are a rare phenomenon in
stem cells.
This experiment showed that the conventional method
used for scoring recombination events does not allow the
detection of all events; nearly 30% of the events remained
undetected. This was primarily due to the events in roots; out
of 25 (107 vs. 82) undetected events, 16 (25 vs. 9) were found
in roots.
Next, in an effort to understand why the recombination
events were so significantly underestimated when using dissecting microscopes, we counted the total number of cells in all
the blue sectors and related it to the total number of sectors
observed in each plant organ. We found that leaves on average
had the highest number of cells per spot, 31.5 ± 5.9, whereas
roots had only 18.1 ± 3.7 and stems as few as 5.0 ± 3.5. Fig. 6
shows the distribution pattern for the cell number in each individual recombination event. There was a substantial variation
observed in the number of cells in each recombination spot.
Whereas most recombination events contained between two
and 30 cells, events with up to 110 cells per 15 µm section
were also found (Fig. 3–6). The difference between leaves,
roots and stems in the average number of cells per spot could
reflect either that the recombination events were happening
earlier in leaf development than in roots, or that leaf cells
divide more frequently than root cells. It has been shown previously by our laboratory that roots contain approximately 10fold fewer genomes as compared with leaves (Boyko et al.
2006). This could be due either to the fact that there were more
cells in general in all leaves of one plant or simply because of
higher levels of endoreduplication in leaf tissue. If the ploidy
level is similar in these two organs, then it means that leaves
contain 10-fold more cells than roots. In this case, if recombination events were happening more or less at the same time in
both organs, leaves would have more cells resulting from each
740
Homologous recombination in Arabidopsis tissue
Fig. 6 Distribution of recombination events in
leaves, roots and stems presented as the number
of cells per spot. The number of cells in each
recombination event observed upon crosssectioning was calculated. The y-axis shows the
number of recombination events, whereas the xaxis shows the number of cells in each recombination event. The close up shows the recombination events with 0–20 cells per event.
recombination event, but would have the same average number
of cells per each event. The finding that an average recombination event in leaves had significantly more cells than in roots
suggests that recombination events in leaves are happening earlier than in roots.
When we analyzed the spot distributions among different
tissues of leaves (Fig. 3), root (Fig. 4) and stem organs (Fig. 5),
Fig. 7 Distribution of recombination events in epidermal, cortex and
vascular tissue of leaves, roots and stem of plants grown under normal
conditions or exposed to salt stress. The percentage of recombination
events (y-axis) in various tissue was calculated individually for each
plant organ of 50 plants per experimental group. Unshaded bars show
the data (with SE) for plants grown under normal conditions, whereas
dark-shaded bars represent the data (with SE) for plants grown at
50 mM NaCl. A single asterisk shows the 95% confidential interval,
whereas double asterisks show the 99% confidential interval.
we found that recombination events were most frequent in the
leaf mesophyll (in both the palisade and spongy parenchyma),
root and stem cortex, 71.4, 66.8 and 60%, respectively (Fig. 7).
This is not surprising as these tissues contain most of the viable cells in the aforementioned organs. For instance, it is possible that high photosynthetic activity in chlorenchyma cells
predispose them to higher free radical production that can
cause strand breaks. Indeed, it was previously observed that the
lateral parts of the leaves that had higher photosynthetic activity produced more radicals and had a higher HR rate (Boyko et
al. 2006).
Exposure to salt increases the frequency of recombination
events in vascular tissue
To analyze the differences in tissue distribution between
plants grown on normal medium and plants grown on soil, we
examined cross-sections of salt-treated plants. When HR was
monitored in cross-sectioned plants, we found that 50% of previously undetected events could be visualized and consequently the HR frequency increased from 4.76 (238 events in
50 plants) to 6.98 per plant (349 events in 50 plants) (Table 1).
This resulted in a statistically significant (P < 0.01) 4.26-fold
increase of recombination frequency in salt-treated plants using
high magnification as compared with a 2.9-fold increase calculated using the binocular microscope.
Also, using high magnification, we found that there was a
larger increase in the number of recombination events visible in
the roots of salt-exposed plants. The number of recombination
events in roots of the ‘control’ plants observed upon crosssectioning (25 events) was found to be 2.8-fold higher than the
Homologous recombination in Arabidopsis tissue
number of events observed using the binocular microscope
(nine events) (Table 1). In contrast, the number of events in
roots of salt-treated plants increased by 4.7-fold (107 events
upon cross-sectioning vs. 23 events with the binocular microscope) (Table 1).
We also noticed that there was a decrease in the average
number of cells resulting from the recombination events in saltexposed plants compared with plants grown under normal conditions. For instance, plants grown on salt contained 25.3, 11.7
and 4.1 cells on average per recombination events in their
leaves, roots and stems, respectively, while those grown under
normal conditions had 31.5, 18.1 and 5.0 cells per recombination event (data not shown). This suggests that the increase of
HR in plants exposed to salt was partially due to the events
occurring later in time. Another reason for the observed difference could be due to the different ploidy level in control and
exposed plants. We do not think that this difference was due to
the change in endoreduplication status in plants exposed to
stress since we found that there was a comparable number of
genomes in organs of plants grown under normal conditions
and of plants exposed to salt stress (data not shown).
The most significant change found was the profound
increase in the percentage of recombination events occurring in
vascular tissue of plants grown on salt. The number of recombination events observed in vascular tissue of roots and stems
of the plants grown on salt increased from 24.5 and 40% to 46
and 80%, respectively (Fig. 7; P < 0.01 in all cases). The effect,
however, was less pronounced but still statistically significant
in leaves, where a comparatively minor change (from 18.4 to
27.3%) was observed (Fig. 7; P < 0.05).
These results show that the majority of newly appearing
recombination spots occurring in roots appeared primarily in
vascular tissue. As expected, the enucleated vessel elements of
the xylem were never GUS positive, being incapable of protein expression, but the surrounding stellar parenchyma cells of
the vascular tissue were often intensely blue. This could be due
to the direct influence of salt on the conductive vessels as salt
transport from the area of high concentration to the area of low
concentration could result in direct diffusion of salt to neighboring cells, leading either to direct DNA damage or to stimulation of stress-related signal transduction cascades resulting in
increased HR. High salt concentrations are known to inhibit
cell cycle progression (Burssens et al. 2000) and, since the
activity of HR is negatively regulated by cyclin-dependent
kinases (Aylon et al. 2004, West et al. 2004), it is quite possible that high salt concentrations result in inhibition of cyclindependent kinases (such as CYCB1) with the ensuing release
of the inhibition of HR activity.
Alternatively, it is possible that different stresses could
lead to different changes in the distribution of recombination
events. It would thus be interesting in the future to analyze
tissue-specific changes in frequency of recombination events
741
upon application of a variety of stresses. Efforts in this direction are currently ongoing in our laboratory.
Materials and Methods
GUS transgenic lines used
The structure of the GUS-based substrate for detection of HR
events has been described previously (Swoboda et al. 1994). The
single-copy transgenic Arabidopsis thaliana line #11 (homologous
parts are in direct orientation) was used in the experiments (Fig. 1a).
Plant growth and sampling
Seeds were vernalized at 4°C for 48 h and then germinated and
grown on soil at 22°C with a 16 h/8 h day/night light regime with illumination at 100 µM m–2 s–1. Sampling for histochemical GUS staining
was done either at the full rosette stage (4 weeks) or at the flowering
stage (8 weeks).
To analyze the pattern of HR in plants exposed to stress, seedlings were grown on MS medium supplemented with 50 mM NaCl.
Seedlings grown on basic MS medium were maintained as controls.
Histochemical GUS staining procedure
Histochemical staining was performed as described by Jefferson
(1987). Fifty plants from the control and stress-exposed groups were
used for staining. Plants were vacuum infiltrated (2×10 min) in a sterile staining buffer containing 100 mg of X-glu substrate (Jersey Labs
Inc., USA) in 300 ml of 100 mM phosphate buffer (pH 7.0), 0.05%
NaN3, 0.05% Tween-80 and 1 ml of dimethylformamide. Afterwards,
plants were incubated at 37°C during a 48 h period and subsequently
bleached with periodic changes of 70% ethanol (Fig. 1b).
Plant sectioning
GUS-stained plants were removed from 70% ethanol (the final
step of histochemical staining, see above), rinsed with 1× phosphatebuffered saline (PBS) and fixed overnight in 4% paraformaldehyde.
These were then dehydrated using ethanol/Citrisolv (Diamed) incubations and embedded in paraffin for sectioning using methods similar to
those described (Jackson 1991). Each plant was sectioned into 15 µm
squares using a Leitz microtome and fixed to glass slides at 45°C. The
paraffin was removed with Citrisolv and the sections were rehydrated
through a graded series of ethanol incubation followed by 10 min incubation in PBS prior to applying very dilute Safranin O (0.01% in
water) for 1 min to differentiate GUS-producing cells more clearly.
The tissue was mounted in 10% glycerol for microscopy.
Calculation of homologous recombination frequency
The HR frequency was calculated by counting the number of
recombination events observed, either with the help of a dissecting
microscope or upon greater magnification after cross-sectioning, and
relating the obtained numbers either to the total number of plants (typically 50 for each experimental group) or to plant organs.
Statistical treatment of the data
The statistical significance of the experiments was confirmed by
performing either Students t-test (two-tailed paired or non-paired) or
single-factor analysis of variance (ANOVA). Statistical analyses were
performed using MS Excel software and Microcal Origin 6.0.
Acknowledgments
The authors wish to thank Michael Greer for critical reading of
the manuscript, and NSERC for funding the research.
742
Homologous recombination in Arabidopsis tissue
References
Abe, K., Osakabe, K., Nakayama, S., Endo, M., Tagiri, A., Todoriki, S.,
Ichikawa, H. and Toki, S. (2005) Arabidopsis RAD51C gene is important for
homologous recombination in meiosis and mitosis. Plant Physiol. 139: 896–
908.
Aylon, Y., Liefshitz, B. and Kupiec, M. (2004) The CDK regulates repair of
double-strand breaks by homologous recombination during the cell cycle.
EMBO J. 23: 4868–4875.
Bleuyard, J.Y., Gallego, M.E. and White, C.I. (2005) Recent advances in understanding of the DNA double-strand break repair machinery of plants. DNA
Repair 5: 1–12.
Boyko, A., Filkowski, J. and Kovalchuk, I. (2005) Homologous recombination
in plants is temperature and day length dependent. Mutat. Res. 572: 73–83.
Boyko, A., Filkowski, J., Hudson, D. and Kovalchuk, I. (2006) Homologous
recombination in plants is organ specific. Mutat. Res. 141(2): in press.
Burssens, S., Himanen, K., van de Cotte, B., Beeckman, T., Van Montagu, M.,
Inze, D. and Verbruggen, N. (2000) Expression of cell cycle regulatory genes
and morphological alterations in response to salt stress in Arabidopsis
thaliana. Planta 211: 632–640.
Cromie, G.A., Connelly, J.C. and Leach, D.R. (2001) Recombination at doublestrand breaks and DNA ends: conserved mechanisms from phage to humans.
Mol. Cell 8: 1163–1174.
Filkowski, J., Kovalchuk, O. and Kovalchuk, I. (2004a) Dissimilar mutation and
recombination rates in Arabidopsis and tobacco. Plant Sci. 166: 265–272.
Filkowski, J., Kovalchuk, O. and Kovalchuk, I. (2004b) Genome stability of
vtc1, tt4 and tt5 Arabidopsis thaliana mutants impaired in protection against
oxidative stress. Plant J. 38: 60–69.
Filkowski, J., Yeoman, A., Kovalchuk, O. and Kovalchuk, I. (2004c) Systemic
plant signal triggers genome instability. Plant J. 38: 1–11.
Gorbunova, V., Avivi-Ragolski, N., Shalev, G., Kovalchuk, I., Abbo, S., Hohn,
B. and Levy, A. (2000) A new hyperrecombinagenic mutant of Nicotiana
tabacum. Plant J. 24: 601–611.
Jackson, D. (1991) In situ hybridisation in plants. In Molecular Plant Pathology,
A Practical Approach. Edited by Bowles, D.J., Gurr, S.J. and McPhereson,
M. Oxford University Press, UK. pp. 17–25.
Jefferson, R. (1987) Assaying chimeric genes in plants: the GUS gene fusion
system. Plant Mol. Biol. Reporter 5: 387–405.
Karanjawala, Z.E., Murphy, N., Hinton, D.R., Hsieh, C.L. and Lieber, M.R.
(2002) Oxygen metabolism causes chromosome breaks and is associated with
the neuronal apoptosis observed in DNA double-strand break repair mutants.
Curr. Biol. 12: 397–402.
Kovalchuk, I., Bojko, V., Kovalchuk, O., Gloeckler, V., Filkowski, J., Heinlein,
M. and Hohn, B. (2003) Pathogen induced systemic plant signal triggers
genome instability. Nature 423: 760–762.
Li, L., Santerre-Ayotte, S., Boivin, E.B., Jean, M. and Belzile, F. (2004) A novel
reporter for intrachromosomal homoeologous recombination in Arabidopsis
thaliana. Plant J. 40: 1007–1015.
Puchta, H., Swoboda, P. and Hohn, B. (1995) Induction of homologous DNA
recombination in whole plants. Plant J. 7: 203–210.
Ries, G., Heller, W., Puchta, H., Sandermann, H., Seidlitz, H.K. and Hohn, B.
(2000) Elevated UV-B radiation reduces genome stability in plants. Nature
406: 98–101.
Sancar, A., Lindsey-Boltz, L.A., Unsal-Kacmaz, K. and Linn, S. (2004) Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu. Rev. Biochem. 73: 39–85.
Schuermann, D., Molinier, J., Fritsch, O. and Hohn, B. (2005) The dual nature
of homologous recombination in plants. Trends Genet. 21: 172–181.
Swoboda, P., Gal, S., Hohn, B. and Puchta, H. (1994) Intrachromosomal homologous recombination in whole plants. EMBO J. 13: 484–489.
West, G., Inze, D. and Beemster, G. (2004) Cell cycle modulation in the
response of the primary root of Arabidopsis to salt stress. Plant Physiol. 135:
1050–1058.
(Received January 20, 2006; Accepted March 31, 2006)