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Cytokinins induce hyperhydricity in leaves of in vitro grown Arabidopsis thaliana
I
Cytokinins induce hyperhydricity in leaves of in vitro
grown Arabidopsis thaliana
Ziqi Zeng
Reg. Num: 911208986090
Msc thesis Plant Breeding
Department of Plant Breeding, Wageningen UR
PBR-80424
Supervisors:
Dr. Frans Krens
Nurashikin binti Kemat
Examiner:
Dr. Frans Krens
Nurashikin binti Kemat
Wageningen UR Plant Breeding,
Droevendaalsesteeg 1, 6700 AA Wageningen,
The Netherlands
August 2016
II
Abstract Tissue culture is a useful and powerful tool for plant breeding and vegetative
propagation. However, physiological disorder hyperhydricity (HH) that leads to
morphological abnormalities such as translucent, thick and brittle leaves in tissue
culture seriously affect the quality and multiplication rate of micropropagation. It is
known that hyperhydricity is resulted from flooding of apoplast and might be due to
deficiency in lignin synthesis. Cytokinins as a growth regular are vital hormone for
multiplication rates in tissue culture that we cannot do without. In order to study th
effect of different types of cytokinins on HH development of Arabidopsis, we
examined different types of cytokinins. We have found that all cytokinins induced HH.
The highest amount of apoplastic water in the apoplast was found on TDZ at
concentration of 0.5µM on Arabidopsis thaliana (col-0). Besides, there were
significantly different between the cytokinins types and their concentration on
stomata aperture, but there were no difference in stomatal density. Using Zeatin or
BAP at low concentration of 0.1µM resulted less HH symptoms in all treatments.
These results indicate that adenine type of cytokinin produce less HH than phenylurea
type.
Keywords: Apoplast, Arabidopsis thaliana, hyperhydricity, cytokinins, lignin,
stomatal, water accumulation.
III
TABLE OF CONTENT
INTRODUCTION ................................................................................................................... 1
MATERIALS AND METHODS ............................................................................................ 3
PLANT GROWTH AND TREATMENTS ...................................................................................... 3
ESTIMATION OF APOPLASTIC WATER AND APOPLASTIC AIR VOLUMES IN LEAVES ............... 3
MEASUREMENT OF STOMATAL APERTURE ............................................................................ 4
RESULTS ................................................................................................................................. 5
CULTURING ON GELRITE AND/OR CYTOKININS INDUCED HH IN ARABIDOPSIS THALIANA
(COL-0) SEEDLINGS ................................................................................................................ 5
APOPLASTIC WATER AND APOPLASTIC AIR VOLUMES IN ARABIDOPSIS THALIANA (COL-0)
SEEDLINGS ............................................................................................................................. 6
STOMATAL APERTURE AND DENSITY IN ARABIDOPSIS THALIANA (COL-0) SEEDLINGS ........... 7
APOPLASTIC WATER VOLUMES IN ARABIDOPSIS THALIANA MUTANTS (REF1-4, REF3-3 AND
FLP) SEEDLINGS ..................................................................................................................... 8
DISCUSSION ........................................................................................................................... 9
CAUSES OF HH IN ARABIDOPSIS THALIANA SEEDLINGS .......................................................... 9
CONSEQUENCE OF HH IN ARABIDOPSIS THALIANA SEEDLINGS ............................................ 12
CONCLUSION ...................................................................................................................... 14
REFERENCE ......................................................................................................................... 15
APPENDIX ............................................................................................................................. 20
1
Introduction Tissue culture is a useful and powerful tool for plant breeding and vegetative
propagation both in horticulture and agriculture (Van Den Dries et al. 2013). However,
during tissue culture, plants are undergoing unnatural and extreme conditions, which
may result in physiological disorder, for example hyperhydricity (HH) (Van Den
Dries et al. 2013; Debergh et al. 1981; Rojas-Martinez et al. 2010). HH causes
translucent, thick and brittle leaves of the explants that are visible to naked eyes,
seriously reducing the quality and multiplication rate of commercially vegetative
propagation in tissue culture (Van Den Dries et al. 2013; Gaspar et al. 1995; Sadhy
Saher et al. 2005; Kevers et al. 1987). It was reported that HH occurs in many plant
species (Van Den Dries et al. 2013; Wu et al. 2009; Chakrabarty et al. 2005). In
Arabidopsis, Delarue et al. (1997) found that a mutant (cril) severely formed HH in
vitro cultured with 0.7% (w/v) agar.
The water content in hyperhydric plant is usually high. This is because plants that are
affected by HH are not capable of keeping water balance correctly (Rojas-Martinez et
al. 2010). It was observed that hyperhydric plants have excess water in apoplast
(Gribble et al. 1996; Gribble et al. 1998), which is the intercellular space. Water
accumulation in apoplast may result in physiological disorder by reducing gas
exchange (Gribble et al. 2003), because diffusion rate of gases is much more lower in
water than in air in plant tissues as it would be expected (Jackson 1985).
There are several factors that will result in HH. First of all, as plants are cultured in a
sealed vessel, the gas exchange is affected and the humidity is relative high, which are
the major contribution to the formation of HH (Ivanova & van Staden 2010; Debergh
et al. 1992). Furthermore, other factors such as the type and concentration of gelling
agent (Debergh et al. 1992; Ivanova & Van Staden 2011), cytokinins (Ivanova & Van
Staden 2011; Kadota & Niimi 2003) may also play a significant role in the
development of HH. Besides, it was reported that deficiency of cellulose and lignin
may also result in HH since symplast may take up more water by reducing cell wall
pressure in hyperhydric plants (Kevers et al. 1987; Van Den Dries et al. 2013). In
addition, malformed stomata was also found in leaves of hyperhydric plants
(Apóstolo & Llorente 2000), and stomatal closure in hyperhydric leaves may impair
transpiration and thereby aggregate HH (Van Den Dries et al. 2013).
2
Many researches have been done on HH, however, the phenomenon and underlying
mechanism of HH remain poorly understood (Van Den Dries et al. 2013; Ziv 1991;
Gribble et al. 2003). The present research examined on how the apoplast is flooded.
One such condition may due to plant hormone effect which are necessary for
multiplication, cytokinins. The objectives of this study were: (1) To evaluate the
effect of different cytokinins on morphological and physiological characteristics in
leaves of in vitro grown Arabidopsis plants. (2) To study on the effect of lignin and
stomata on development of HH.
3
Materials and methods Plant growth and treatments
Arabidopsis thaliana (col-0) seeds were sterilized with 70% (v/v) ethanol for 1 min
and 2% (w/v) sodium hypochlorite for 15 min. After then, the seeds were rinsed three
times with sterilized distilled water for 10 min. Subsequently, sterile seeds were
transferred to a Petri dish with half-strength Murashige and Skoog (MS) basal salt
mixture including vitamins (Murashige & Skoog 1962) with a supplement of 1.5%
(w/v) sucrose and solidified with 0.7% (w/v) Micro-agar (all of them are from
Duchefa Biochemie, Haarlem, The Netherlands). Seeds were first stratified in dark for
3 d at 4 0C and subsequently germinated in a growth chamber with 16h light/8h dark
period (30µmol m-2 s-1, Philips TL33) at 21 0C. 7-days-old wild type Arabidopsis
thaliana (col-0) seedlings were transferred and cultured in high-sided Petri dish with
ten seedlings per dish solidified with 0.2% (w/v) Gelrite and/or 0.7% (w/v)
Micro-agar. Four types of cytokinins were supplied (zeatine, 6-Benzylaminopurine
(BAP), 6-(3-hydroxybenzylamino) purine (meta-topolin) and Thidiazuron (TDZ))
with three levels of concentration (0µM, 0.1µM and 0.5µM respectively). Each
treatment replicated three times.
7-days-old lignin mutant (ref1-4 and ref3-3) and stomata mutant (flp) Arabidopsis
thaliana seedlings were transferred and cultured in high-sided Petri dish with five
seedlings per dish solidified with 0.2% (w/v) Gelrite and/or 0.7% (w/v) Micro-agar.
Each treatment replicated three times.
Estimation of apoplastic water and apoplastic air volumes in leaves
The apoplastic water was extracted from leaves tissues with mild centrifugation
(Terry & Bonner 1980; Van Den Dries et al. 2013). Leaves were excised from plants,
weighed and subsequently put into a spin mini filter microcentrifuge tube (Starlab,
Ahrensburg, Germany), centrifuging at 3000g for 20 min at 4 0C. After centrifugation,
leaves were weighted again. The formula: Vwater = [(FW - Wac) x ρH2O] / FW was
used to calculate the apoplastic water volume (Vwater) in µl g-1 fresh weight (FW). FW
represented the fresh weight of leaves in mg, while Wac represented the weight of
leaves after centrifugation and ρH2O represented water density that was taken as
equal to 1 g ml-1.
The apoplastic air volumes in leaves was assessed with the help of a pycnometer with
4
a stopper (Raskin 1983; Van Den Dries et al. 2013). Leaves were excised, weight and
subsequently put into the pycnometer, which was then filled with distilled water and
stoppered. Filter paper was used to remove any excess water on the exterior of the
pycnometer. The combined weight of the full pycnometer and leaves was measured.
The pycnometer, with both water and leaves, underwent a vacuum (about 500 mm Hg)
for 5 min in order to remove air from the apoplast and replace it by water. If needed,
this vacuum treatment was re-did till all air was supposed to be removed out of the
apoplast and the leaves had sunk to the bottom of the pycnometer. After vacuum
infiltration, the pycnometer was refilled, dried and then weighted again. The formular:
Vair = [(Wbv – Wav) x ρH2O] / FW was used to calculated rge apoplastic air volume
(Vair) in µl g-1 FW. Wbv represented the weight in mg of the pycnometer including
both leaves and water before vacuum infiltration, while Wav represented the weight of
the pycnometer including both leaves and water after vacuum infiltration. FW
represented the fresh weight of the leaves, and ρH2O represented the water density
that was taken as equal to 1 g ml-1.
Measurement of stomatal aperture
The stomatal aperture was explored by making epidermal impressions of adaxial leaf.
Leaves were excised from plants, and then adaxial surface were placed onto
impression material and then solidified. The impression material was
polyvinylsiloxane-based high precision President Light Body impression material
(Coletene/Whaledent AG, Altstatten, Switzerland) (Geisler et al. 2000). Subsequently,
leaves were removed. Transparent nail polish was applied to the epidermal imprints
leaving over. After drying, the nail polish were gently and carefully peeled and placed
on a microscope slide. The leaf stomata impressions were then shown under an
Axiophot light microscope (Zeiss, Oberkochen, Germany). Images were subsequently
captured using AxioCam ERc5S digital camera (Zeiss), and AxioVision software
4.8.2 (Zeiss) was used to measure the stomatal aperture. Guard cell lengths (µm) were
measured to the nearest micrometer viewed at 40x magnification. In addition,
stomatal density was measure by counting the number stomata per field (200x200µm)
of view at 40x magnification. Sample size was three leaves per plant and three fields
per leaf, replicated twice.
5
Results Culturing on Gelrite and/or cytokinins induced HH in Arabidopsis thaliana (col-0) seedlings
It is shown in figure 1 that on days 14 after transfers, the control seedlings
(non-hyperhydric) has no symptoms of HH (fig.1A). The seedlings grown on
Gelrite, gelrithe plus cytokinins, and agar plus cytokinins showed HH symptoms with
thick, wrinkle and translucent leaves, as well as elongated petioles (fig.1). Overall,
Gelrite-grown (Gelrite and Gelrite plus cytokinins) seedlings apparently developed
severer HH than agar-grown (agar and agar plus cytokinins) seedlings, comparing
figure 1B to figure 1A. Besides, either grown on Gelrite or agar, it is shown that TDZ
induced more HH symptoms while meta-topolin and BAP were moderate, and zeatine
induced the least severe HH (fig.1). Increasing cytokinins concentration induced more
HH, as it can be seen that seedlings from 0.5µM cytokinins had thicker, curler leaves
and anthocyanin production, compared to those from 0.1µM for all four types of
cytokinins (fig.1).
It is noticed in figure 2A that on days 7 after transfer, the control seedlings were
healthy, whereas those on agar plus cytokinins (0.5µM BAP, 0.5µM TDZ) started to
show HH symptoms with anthocyanin production (fig 2A). The seedlings grown on
Gelrite, and Gelrite plus cytokinins (0.5µM BAP, 0.5µM TDZ) developed symptoms
of HH with curled leaves, anthocyanin production and elongated petioles, and it was
apparently severer on Gelrite (without cytokinins) (fig. 2A). After 12 days, it is shown
that Gelrite-grown seedlings grew faster than agar-grown seedlings (fig.2B). The
control seedlings were healthy, whereas seedlings grown on agar plus cytokinins
started to show HH symptoms (fig. 2B). The seedlings grown on Gelrite and Gelrite
plus cytokinins apparently developed severer HH than those on days 7 (fig. 2A). After
19 days, the control seedlings were healthy (fig. 2D), whereas the HH symptoms of
seedlings grown on agar plus cytokinins were severer than those on days 15 (fig. 2C).
The HH symptoms was even more severer in seedlings grown on Gelrite and Gelrite
plus cytokinins, and the seedlings already gave signs of highly stress with
anthocyanin production and leaves necrosis (fig. 2D). After 25 days, the control
seedlings were still healthy (fig. 2E). The seedlings grown on agar plus cytokinins
developed severer HH than those on days 19 (fig.2D) but they were still alive (fig.
2E). The seedlings grown on Gelrite and Gelrite plus cytokinins had mostly necrosis,
6
which indicated the later stage of HH (fig. 2E).
Apoplastic water and Apoplastic air volumes in Arabidopsis thaliana (col-0) seedlings
After 14 days, it is shown in figure 3A that overall Gelrite-grown (Gelrite and Gelrite
plus cytokinins) seedlings accumulated remarkably more water in apoplast than
agar-grown (agar and agar plus cytokinins) seedlings. Either grown on agar or Gelrite,
the apoplastic water was significantly different (p-value <0.05) for both cytokinins
types (zeatine, meta-topolin, BAP and TDZ) and cytokinins concentration (0µM,
0.1µM and 0.5µM) (fig. 3A). When grown on agar, seedlings from agar (the control,
without cytokinins) accumulated the lowest amount of apoplastic water, comparing to
agar plus cytokinins. Regarding the cytokinins types added, seedlings from agar plus
TDZ had the largest amount of apoplatic water, whereas seedling from agar plus
zeatine had the less effect. Regarding concentration, seedlings from 0.5µM cytokinins
accumulated significant larger apoplastic water than those from 0.1µM for all four
types of cytokinins. When grown on Gelrite, seedlings from Gelrite (without
cytokinins) accumulated the significantly largest amount of apoplastic water,
comparing to those from Gelrite plus cytokinins. Regarding cytokinins types, seedling
from Gelrite plus TDZ showed the significantly largest amount of apoplastic water,
whereas seedlings from Gelrite plus zeatines showed the least. Generally, seedlings
from 0.5µM cytokinins accumulated significant larger apoplastic water than those
from 0.1µM for all four types of cytokinins.
On the apoplastic air, it is shown in figure 3B that overall agar-grown seedlings had
substantially more volume of apoplastic air in apoplast than Gelrite-grown seedlings.
Either grown on agar or Gelrite, the apoplastic air was significantly different (p-value
<0.05) for both cytokinins types and cytokinins concentration (fig. 3B). When grown
on agar, the control seedlings had significantly larger volume of apoplastic air
compared to seedlings from agar plus cytokinins. As for cytokinins types added,
seedlings from agar plus zeatine had the significantly largest volume apoplastic air,
whereas seedlings from agar plus TDZ had the lowest. The seedlings from 0.5µM
cytokinins showed significantly larger volume of apoplastic air than those from
0.1µM for all four types of cytokinins. A similar pattern of apoplastic air was also
observed for seedlings grown on Gelrite and Gelrite plus cytokinins (fig. 3B).
7
Adding the apoplastic water and apoplastic air volumes, the total apoplast was
calculated. According to figure 4, it is shown that when seedlings were grown on
Gelrite and Gelrite plus cytokinins, the apoplastic water accounted for more than 90%
(fig.4A) whereas apoplastic air account for less than 10% (fig.4B), indicating that the
apoplast was saturated by water. However, when seedlings were grown on agar and
agar plus cytokinins, it is shown that the control seedlings had the lowest percentage
of apoplastic water (fig.4A) and highest percentage of apoplastic air (fig.4B) (11.6%
and 88.4 %, respectively), followed by agar plus zeatine. On the other hand, agar plus
TDZ showed the highest percentage of apoplastic water and lowest percentage of
apoplastic air (fig. 4). Overall, seedlings from 0.5µM cytokinins accounted for larger
percentage of apoplastic water and smaller percentage of apoplastic air than those
from 0.1µM for all four types of cytokinins (fig.4). These results indicate that the
increase of apoplastic water in hyperhydric seedlings were at the expense of
decreased apoplastic air volume.
Figure 5 shows that from days 7 to days 15 the apoplastic water in hyperhydric
seedlings grown from Gelrite, Gelrite plus cytokinins (0.5 BAPµM and 0.5µM TDZ)
and agar plus cytikinins (0.5µM BAP and 0.5µM TDZ) increased along with the
development of HH, and Gelrite-grown seedlings accumulated remarkably more
water in apoplast than agar-grown seedlings. From days 15 to days 19, the increasing
of apoplastic water in hyperhydric seedlings from Gelrite and Gelrite plus cytokinins
slowed down, indicating that the apoplast was close to saturated. After days 19, the
apoplastic water in hyperhydric seedlings from agar plus cytokinins continually and
slowly increased, whereas those from Gelrite and Gelrite plus cytokinins started to
decline because of necrosis of leave tissues. On the other hand, the apolastic water in
the control seedlings remained more or less constant during the whole period, and it
was at least three times less than that of hyperhydric seedlings on days 25.
Stomatal aperture and density in Arabidopsis thaliana (col-0) seedlings
It is shown in figure 6 that after 14 days of culture the stomata in the leaves were
partially or fully closed in hyperhydric seedlings compared to the control seedlings,
and the opening of stomata decreased along with the development of HH. It is shown
in figure 7 that either grown on agar or Gelrite, the stomatal aperture in seedlings was
8
significantly different (p-value <0.05) for cytokinins types (BAP and TDZ) and
cytokinins concentration (0µM, 0.1µM and 0.5µM). Overall, the control seedlings had
the significantly largest value of stomatal aperture than that of hyperhydric seedlings.
Hyperhydric seedlings grown on Gelrite (without cytokinins) had the significantly
lowest value of stomatal aperture than those grown on Gelrite plus cytokinins and
agar plus cytokinins. In addition, hyperhydric seedlings from 0.5µM cytokinins had
significantly smaller stomatal aperture than those from 0.1µM for both BAP and TDZ.
However, no significant difference (p-value >0.05) in stomatal density between
hyperhydric and non-hyperhydric (the control) seedlings were observed (fig. 7).
Apoplastic water volumes in Arabidopsis thaliana mutants (ref1-4, ref3-3 and flp) seedlings
It is shown in figure 8 that on days 14 after transfers, wild type seedlings grown on
agar were healthy, whereas those on Gelrite developed HH. The mutants (ref1-4,
ref3-3 and flp) seedlings grown on agar and Gelrite showed HH symptoms with thick,
wrinkle and translucent leaves, as well as elongated petioles (fig.8). Overall,
Gelrite-grown seedlings apparently developed severer HH than agar-grown seedlings
(fig.8).
After 14 days, it is shown in figure 9 that overall Gelrite-grown seedlings
accumulated remarkably more water in apoplast than agar-grown seedlings.
Apoplastic water was significantly different (p-value <0.05) for wild type and mutants.
Either grown on agar or Gelrite, wild type (col-0) seedlings accumulated significantly
less apoplastic water than mutants (ref1-4, ref3-3 and flp), but the apoplastic volumes
within mutants were not significantly different (fig.9).
9
Discussion In many previous researches on HH (Debergh et al. 1981; Rojas-Martinez et al. 2010;
Gaspar et al. 1995; Sadhy Saher et al. 2005; Gribble et al. 2003; Ziv 1991), the extent
of HH was visually accessed and evaluated based on the malformation and
morphological abnormality of hyperhydric plants. However, seldom of them gave
research on the underlying mechanism of HH. Gribble et al. (1996; 1998; 2003)
assumed that HH resulted from accumulation of apoplastic water that interrupt and
reduce gas exchange in apoplast of plant tissues, but the volume of apoplastic water
and air were not adequately quantified. The studies of Van den Dries et al. (2013), for
the first time, assessed the severity of HH by measurement of apoplastic water and
apoplastic air volume in hyperhydric plant leaves tissues. In the present study, we
followed the methods of Van den Dries et al. (2013) and quantified the apopalstic
water and air volume in both hyperhydric and non-hyperhydric Arabidopsis thaliana
seedlings. We found that in hyperhydric seeldings, the volume of apoplastic water
were significantly larger than that of non-hyperhydric ones (fig.3), which
accumulated over time (fig.5) and was at the cost of the volume of apoplastic air
(fig.4). It was calculated that in hyperhydirc Arabidopsis thaliana seedlings, the
percentage of apoplastic water accounted for more than 90% in total apoplast (fig.4).
These findings are in lines with the study of Van den dries et al. (2013).
Causes of HH in Arabidopsis thaliana seedlings
Obviously, the main cause of HH is the flooding of apoplast that affects the water
balance, reduces gas exchange in plant tissues and eventually leads to physiological
disorders (Rojas-Martinez et al. 2010; Gribble et al. 1996; Gribble et al. 1998; Gribble
et al. 2003; Van Den Dries et al. 2013). According to figure 3 and figure 4, it is shown
that generally the seedlings grown on (0.2%) Gelrite accumulated significantly more
apopalstic water than those grown on (0.7%) agar. This can be expected because
Gelrite are supposed to release much more water than agar over time, increase the
water uptake of plants and generate HH in many plant species such as Calophyllum
inophyllum,Prunus avium and Aloe polyphylla other than Arabidopsis thaliana
(Turner & Singha 1990; Franck et al. 2004; Ivanova & Van Staden 2011; Van Den
Dries et al. 2013). On the other hand, the less severe of HH on medium with agar
compared with Gelrite may be due to the sulphated galactan in agar that has the
ability to control HH, according to Nairn et al. (1995).
10
It was reported that cytokinins could induce HH in vitro propagation (Kadota & Niimi
2003; Bairu et al. 2007; Debnath 2009). However, cytokinins-induced HH may be a
difficult problem to overcome because cytokinins as a growth regular are vital
hormone for multiplication rates in tissue culture that we cannot do without (Huang et
al. 1998). Therefore, we need to establish how exactly cytokinins induce HH and to
maintain the multiplication factor. In the present study, four types of cytokinins were
used to induce HH, which are zeatine, meta-topolin as well as BAP from adenine
derivatives, and TDZ from phenylurea derivatives. Three level of concentration were
applied, which were 0µM, 0.1µM and 0.5µM. It is found that TDZ generated the
severest HH among others, whereas zeatine has the least effect (Fig. 1 and 3).
Generally, phenylurea derivative has higher activities in terms of growth promotion,
shoot proliferation and regeneration than adenine derivative (Kadota & Niimi 2003).
Although TDZ has powerful cytokinins activities regarding yield and multiplication
(Ružić & Vujović 2008; Pawlicki & Welander 1994; Briggs et al. 1988), its shoots
quality is usually lesser because of compact hyperhydric growth (Briggs et al. 1988).
It is reported that TDZ has a stronger effect on inducing HH compared with zeatine,
meta-topolin and BAP. This is exemplified in the work undertaken by Kadota &
Niimi (2003) in which HH in explants of Pyrus pyrifolia was found to be more
affected by TDZ than by BAP. Another example of this is the study carried out by
Debnath (2009) in which Rhodiola rosea L showed rapid proliferation with HH
symptoms at more than 0.5µM TDZ but produced normal shoots within four weeks of
culture after transfer to 1-2µM zeatine. Furthermore, it is observed in figure 1 and
figure 3 that zeatine generate less HH than meta-toploin and BAP. The study of Bairu
et al. (2007) found that when culturing Aloe polyphylla in vitro, there were more
hyperhydric shoots generated on medium with BAP than with meta-topolin or zeatine.
Besides, Ivanova & Van Staden (2011) also discovered in their studies on Aloe
polyphylla that zeatine induced less hyperhydric shoots than BAP. These findings are
in consistent with our results.
It is shown in our results that overall increasing the concentration of cytokinins
aggravated the severity of HH. This is supported by the study of Bairu et al. (2007),
which found that the occurrence of HH increased with an increasing concentration
(0.5µM - 15µM) of zeatine, meta-topolin and BAP in Aloe polyphylla’s tissue culture.
Debnath (2009) found that in roseroot (Rhodiola rosea L) tissue culture HH was
induced in high concentration (4µM) of TDZ, but the hyperhydric symptom was
11
almost absent in lower concentration (1µM) of TDZ. In addition, it was also reported
that the higher concentration of cytokinins, the easier to induce HH in many plant
species such as Olearia microdisca (Williams & Taji 1991), Lavandura vera
(Andrade et al. 1999), Malus sylvestris (Phan 1991) etc. All these findings support our
results.
It was suggested that due to its physical structure, Gelrite enhance the absorption of
cytokinins (Williams & Taji 1991; Franck et al. 2004; Ivanova & Van Staden 2011),
which is expected to generate sever HH than Gelrite only. According to our result
(figure 1 and 3), it shows that Arabidopsis thaliana seedlings grown on Gelrite
develop severer HH and accumulated significantly more apoplastic water than
seedlings grown on Gelrite plus cytokinins, which seems to be contradictive with
what we expect. However, it is shown in figure 4A that the apoplast water (%) of all
the seedlings grown on either Gelrite and Gelrite plus cytokinins account for more
than 90% out of total apoplast. This implies that the fact seedlings on Gelrite appeared
to develop severer HH than those on Gelrite plus cytokinins does not indicate Gelrite
was a stronger competitor. As a matter of fact, the apoplast of the seedlings grown on
Gelrite plus cytokinins were also saturated by water.
Less lignin and reduced lignification were constantly considered to be one of the
causes of HH (Piqueras et al. 2002; Shady Saher et al. 2005). Our results show that
both lignin mutants (ref1-4 and ref3-3) developed severer HH than wild type (fig. 8
and 9). Lignin quality and quantity is influenced by certain ref Arabidopsis mutants
(Ruegger & Chapple 2001). It was reported that ref1-4 and ref3-3 Arabidopsis
mutants accumulated less sinapate esters than wild type Arabidopsis (Ruegger &
Chapple 2001; Nair et al. 2004). Mutation that affected sinapate ester biosynthesis
may negatively impact lignin biosynthesis and thereby reduce lignification of plants
(Ruegger & Chapple 2001). This was exemplified in the work undertaken by
Schimiller et al. (2009) and Ruegger & Chapple (2001) who found that there was less
lignin content in ref1 and ref3 Arabidopsis mutants compared to wild type. Kevers &
Gaspar (1986) and Kevers et al. (1987) suggested that due to the deficiency in lignin,
hyperhydric plants may have reduced cell wall pressure, which consequently make
the symplast uptake more water and aggravate HH. Furthermore, Kevers et al. (1987)
confirmed that lignin accumulated over time in normal tissues in carnation, but there
was a deficiency in lignin content in hyperhydric tissues. Therefore in our study, the
development of HH in ref1-4 and ref3-3 Arabidopsis mutant may be resulted from
12
deficiency in lignin synthesis.
Figure 8 and 9 showed that the stomata mutant developed HH in both agar and gelrite.
This result complies with Yang & Sackr (1995) which found that flp Arabidopsis
mutant displays abnormal stomata including paired stomata and a small amount of
unpaired of guard cells in cotyledons and the stomata were partially close compared
to wild type. The closure of the stomata evidently reduces transpiration and thereby
contributes to the flooding of the apoplast which hyperhydricity (Yang & Sackr
1995).
Consequence of HH in Arabidopsis thaliana seedlings
HH of Arabidopsis thaliana seedlings mainly results from the flooding of the apoplast
within plant tissues, and the consequence is that it impairs gas exchange and leads to
physiological disorders in plants (Van Den Dries et al. 2013). Visually, hyperhydric
plants exhibited morphological abnormalities such as thick, curled and translucent
(watery and glassy appearance) shoots as well as anthocyanin production (fig. 1 and 2)
(Gaspar et al. 1995; Kadota & Niimi 2003; Sadhy Saher et al. 2005; Van Den Dries et
al. 2013). The reduced gas exchange in hyperhydric plant tissues may issue in
hypoxic stress that may consequently lead to oxidative damage (Olmos et al. 1997;
Saher et al. 2004; Chakrabarty et al. 2005; Vergara et al. 2012; Van Den Dries et al.
2013), or issue in accumulation of gaseous compounds like ethylene in plant cells
(Voesenek et al. 1993; Van Den Dries et al. 2013). It is shown in figure 1 and figure 2
that hyperhydic Arabidopsis seedlings also exhibited elongated petioles, which was
likely caused by accumulated ethylene, according to Millenaar et al. (2005).
It was reported that hyperhydric plants also exhibited anatomical defects such as
epidermal discontinuity (Olmos & Hellin 1998; Apóstolo & Llorente 2000),
chlorophyll deficiency (Franck et al. 1998), low lignification (Kevers & Gaspar 1986;
Kevers et al. 1987), malformed non-functional stomata (Werker & Leshem 1987;
Apóstolo & Llorente 2000; Picoli et al. 2001; Olmos & Hellin 1998) etc. In the
present study, it is observed that there was a reduction of stomatal aperture in
hyperhydric Arabidopsis thaliana shoots (fig. 6 and 7A), which is consistent with the
study of Van Den Dries et al. (2013). Besides, Ziv & Ariel (1994) also observed
stomatal closure in his research on HH in carnation. The reasons for stomatal closure
may be due to the increasing stress triggered by, for example flooding of (shoots)
apoplast (Van Den Dries et al. 2013) or flooding of plants roots (Atkinson et al. 2008;
13
Else et al. 2009), in hyperhydric plants. Furthermore, it is shown that the stomatal
densities were not significantly different for agar, agar plus cytokinins, Gelrite and
Gelrite plus cytokinins (fig. 7B). This result is contradictive with the study of
Apóstolo & Llorente (2000) that found a decreasing stomatal density in hyperhydric
Simmondsia chinensis, and the stomatal density is expected to be lower since the size
of epidermal cells is larger in hyperhydric leaves (Olmos & Hellin 1998; Mossad et al.
2013). However, the study of Van Den Dries et al. (2013) also did not find significant
change in stomatal density between hyperhydic and non-hyperhydric Arabidopsis
thaliana seedlings, which is in lines with our results.
14
Conclusion In the present study, the effect of cytokinins types and concentration on HH in wild
type Arabidopsis thaliana (col-0) in vitro was investigated. Applying cytokinins
induced HH. Hyperhydric plants displayed malformation such as thick, brittle, curled,
glassy leaves as well as petioles elongation. In hyperhydric seedlings, apopalstic
water volume was significantly large and accumulated over time, which was at the
expense of apoplastic air volume. Beside, stomatal closure was also observed. In our
research, cytokinins concentration of 0.1µM resulted less HH symptoms than 0.5 µM
in all treatments. TDZ generate significantly severer HH than BAP, meta-topolin and
zeatine, which makes it unsuitable for multiplication of Arabidopsis thaliana.
Although zeatine induces the least sever HH among others, it was rather expensive.
Furthermore, it shows that meta-topolin has a similar effect with BAP on inducing
HH. Therefore giving the fact that BAP is more common in tissue culture, it is likely
that BAP would be the most suitable cytokinins for Arabidopsis thaliana, which
guarantees adequate proliferation of seedlings and keeps the damage of HH under
critical level. In addition, the effect of lignin and stomata on development of HH in
Arabidopsis thaliana mutants was also studied. It was found that both lignin mutants
(ref1-4 and ref3-3) and stomata mutant (flp) developed HH that maybe due to lignin
deficiency and reduced transpiration, and their apoplastic water volume was
significantly large than wild type (col-0). Future research can be carried out to further
investigate the effect of HH on lignification. Also, molecular study on HH can
provide more details into the disorder and help to create new treatment to prevent it.
15
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