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Effect of Water Stress on Morphology, Anatomy, and

Molecular Characteristic of Maize (Zea mays L.)

PLANT ECOPHYSIOLOGY
Lecturer : Dr.agr. Nunun Barunawati, SP. MP.

Fathiyyah 176040200011003
Bagus Sulistyono Putra 176040200011004
Dellia Rezha Bayu R. 176040200011005
Muhammad Rif’ad Najib 176040200011006
Zaenul Mustaqim 176040200011007
Ani Nurin Ni’mah 176040200011009
Bona Asa Sharon S. 176040200011012

AGRONOMY PROGRAM
FACULTY OF AGRICULTURE
BRAWIJAYA UNIVERSITY
MALANG

2018
PREFACE
Praise be to Allah the Almighty God, for the abundance of His mercy and
grace so that the author can complete this practice report about “Effect of Water
Stress on Morphology, Anatomy, and Molecular Characteristic of Maize
Plant”, which intended to fulfill the task of Ecophysiology Course of Plant.
Thank you to :
1. Dr.agr. Nunun Barunawati, SP.,MP. As a lecturer of Ecophysiology of
Plants who have taken the time to provide guidance and direction during
the preparation of this report and on the knowledge given during the
teaching and learning process in Agronomy Masters Program,
Postgraduate Faculty of Agriculture Brawijaya University.
2. Ardian Candra S., A.Md as a laboratory assistant who has given guidance
and direction during the laboratory process.
3. Mr.Rifad as leader of the group who have given up his time to plant a
sample plant and take care of it.
4. All member who assist in the process of preparing this practice report.
Author realizes that in the writing of his practice report still need input and
suggestion so that what is written in this report will be better.

Malang, May 31, 2018

Author,
DAFTAR ISI
1. INTRODUCTION

Plants are very influenced by environment both in growth and development.


Each plant has a growing requirement on the environment, if the growth
requirements are unappropriate, then the plant will show the physiological
response. Mismatch is a condition of stress on the plant. Crop responses show that
the plant is self-defeating with unsuitable environmental conditions, some stress
conditions in plants include soil nutrient stress, light stress, water stress, and CO2
stress. In this laboratory pratice will be observe plant physiological response to
water stress either in drought or water logging, which is compared with osmotic
stress and control in vitro.
Drought stress one of the factors that affect the plant physology that can also
affect the production of plants. This adverse condition induces osmotic pressure in
plant cells by reducing the availability of water, there by causing the weakening of
cell turgor and the accumulation of reactive oxygen species (ROS) that adversely
affect the growth and development of the plant. To survive in drought conditions,
plants must bind to the intra cellular and physiological signaling networks to
respond quickly and efficiently neutralize stress. This is done in order to survive
the plant.
Water logging stress also affects the growth and development of plants. In
conditions of waterlogged soil, the roots of plants will be deprived of oxygen, the
condition makes the plants in stresess. The high osmotic pressure in the roots
makes nutrients, water, and O2 unable to enter the roots so that the plants can not
metabolize properly. In the stagnant conditions, the plant emits a signal of
ethylene that modulates ABA (Absicic acid) and GA (Gibberellic acid) hormones
that play a role in cell division and photosynt translocation in plants, so that
growth and development of plants will be hampered by stagnant stress conditions.
In vitro Stresses with sorbitol take effect in plants, this condition uses sugar
in large quantities. The concentration of sugar in large quantities is also called
hypertonic. This condition will decrease the plant cell volume and decrease the
pressure of turgor and other solutions present in the cell, so the plasma membrane
is thicker, and more compressed and then plasmolisis. All activities related to
turgor activity may decrease such as, leaf lengthening, roots, stem lengthening. So
that condition can affect plant physiology. The conditions of some of these
stresses can be demonstrated in maize and long bean plants.
2. MATERIAL AND METHOD
This laboratory experiment has been done on Thursday, 31st May 2018. It
held by some laboratories. Biotechnology laboratory held morphology
experimental, Proline analysis and DNA extraction analysis. Anatomy analysis
has been done in Plant Breeding Laboratory.

2.1. Instrument and Material


The instrument and material for this practice are:
a. Morphology
 Plant
 Ruler
 Cloth
 Cutter
 Scissor
 Camera
b. Anatomy
 Slice of maize leaf 2 x 2 cm
 Nail Polish
 Cover slide
 Cover Glass
 Preparat glass
 Microscope Olympus BX-51
 Selotipe
 Scocth tape
 Label
 Tape
c. Proline Analysis
 Material Preparation
 Ninhidrin Acid
 30 mL Glacial Acetate Acid
 20 mL Phosphate Acid.
 0.5 g Fresh leaf
 liquid nitrogen
 3% sulfosalisilat Acid
 whatman paper no.1
 2 mL ninhidrin acid
 2 ml Glacial Acetate Acid
 reaction tube
 gelaspiala
 ice water
 4 mL toluene than
 Pipet
 spectrophotometer
d. DNA Extraction and PCR
 Thermofisher DNA isolation Kit
 Electrophoresis Chamber
 Gel doc By BIO-RAD
 Liquid nitrogen
 PCR kit (primer SSR, Taq polymerase, dH2O,
2.2. Laboratory Practice Protocol
a. Morphology
1. Prepare tool and materials
2. Put the maize plant on top the paper or cloth
3. Measure the Leaf length and root length by ruler
4. Count the leave and root number
5. Take their picture using a mobile phone camera
b. Anatomy
1. Prepare tools and materials.
2. Cut leaves of maize on three stresess treatment.
3. Smear the nail polish on the top and bottom of the leaves.
4. Wait until it dries from nail polish.
5. Put the tape on the nail polish has dried on the leaves.
6. Pull the tape and dry nails from the leaves, and put on preparat glass
7. Give labels on preparat glass according to treatment and type.
8. Than observed under microscope number of stomata, stomata area,
and density stomata
c. Proline Analysis
1. Material Preparation
2. Make Ninhidrin Acid solution by warm 1.25 g of ninhidrin in 6 M of
30 mL Glacial Acetate Acid and 20 mL Phosphate Acid.
3. Pour 0.5 g Fresh leaf by liquid nitrogen in mortar than grinding it to
be a pounding.
4. Make 10 ml solution of the fresh leaf pounding with 3% sulfosalisilat
Acid.
5. Filter the fresh leaf pounding and 3% sulfosalisilat Acid solution with
whatman paper no.1
6. Make a reaction of 2 ml filtrate, 2 mL ninhidrin acid and 2 ml Glacial
Acetate Acid in reaction tube about 1 hour in 100° C.
7. End the reaction by inserting reaction tube into gelaspiala which filled
by ice water.
8. Extract the cold solution with 4 mL toluene than centrifuge with
vortex around 15-30 seconds until two layer of solution was separated
9. The top of red toluene layer contains proline. Suck the up layer using
pipet. Count the absorbance degree byspectrophotometer with 520 nm
wave.
10. Proline contains count base pure proline solution curva standard
formula.
d. DNA Extraction

e. PCR Analysis
 Prepare the eight tube of PCR @25 µl
 Put into tube Enzyme Taq polymerase 12, 5 µl
 Primer SSR Forward (5’-TCA TAC GGA ACG GAT GCT GG-
3’)
 Primer SSR Reverse (5’-CAC CTC GAT ATC CCC TCC CA-3’)
 dNtps
 Water deionized dH2O 7,5 µl
The run the thermal cycler of PCR 35 cycle With 3 step per cycle
Denaturation 94 oC 30 s, Annealing 52 oC 30 s, Elongation 72 oC
3. RESULT AND DISCUSSION

3.1 Water Stress Impact on Non Green House Maize

This observation was on Non Green House maize. The maize was cultivated
in field. The observation was 3 weeks after planting. There were three treatments
of water stress. The first was control (C) by daily irrigation. The second was water
logging (WL) treatment by flooding. The last was Drought (D) treatment by
without irrigation supply for a week. Table 1. showed morphological impacted by
water stress.

Tabel 1 Effect Water Stress On Maize Morphology. PH: plant height, ƩL: Leaf
Number, SL: segment length, LA: Leaf Area, ƩR: Root Number, RL:
root length, SPAD: Soil Plant Analysis Development, C: Control, WL:
Lodging water, D: Drought stress.
Treatment PH ƩL SL LA ƩR RL(cm) SPAD
(cm) (cm)
C 14 4.5 5.5 45 8 8 26.6
WL 11.75 3.5 7.25 20 5.5 9.55 23.05
D 10.5 4.5 6 26 4.5 7 20.08
Corn samples show different plant height, in drought stress treatment (D)
plants 11,9% shorter than plants in water logging treatment (LW) and 33,3% than
control (C). This value is related to the result of segment length of maize
(poybag), segment length of maize with the water logging treatment (LW) has a
31,8% and 20,8% longer length segment compared with other treatment,
respectively.

Plant Hight of NGH (cm) Internode Lenght of NGH


15 (cm)
10
10

5 5

0 0
C LW D C LW D
Leaf Number of NGH Leaf Area of NGH (cm2)
6 60.0

4 40.0

2 20.0

0 0.0
C LW D C LW D

Fig 1. Effect of water stress on plant PH, SL, ƩL and LA of maize, : NGH:
Non-Green House maize,C: control, LW: lodge water, D: drought
stress.

Segment length value of the plant also affected plant height, the longer
segment plant result in higher maize plant. Shoot apex serves as the primary
source of auxin for the entire plant, polar transport has long been believed to be
the principal cause ofn an auxin gradient extending from the shoot tip to the root
tip. The longitudinal gradient of auxin from the shoot to the root affect various
developmental process, including stem elongation (Taiz and Zeiger, 2000+).

Fig 2. Morphology of Maize (Non-Green House) on water stress treatment.C:


control, LW: logging water, D: drought.
b
a

Fig 3. Morphology of Maize (Non-Green House) (a) leaf, (b) stem and (c) root on
water stress treatment. C: control, LW: logging water, D: drought.

Number of leaves value in logging water treatment (LW) is 28,6% lower than
in control treatment (C). While in drought stres treatment (D) number of leaves is
28,6% higher than in logging water treatment (LW). This data is supported by the
result of plant leaf area where in drought stress treatment (D) has a 42% smaller
leaf area compared to control treatment (C). Decreased leaf area is an early
adaptive response to water deficit/ drought stress, as the water content of the plant
decrease, its cells shrink and the cell walls relax. This decrease in cell volume
result in lower turgor pressure and the subsequent concentration of solutes in the
cells. The plasma membrane becomes thicker and more compressed because it
covers a smaller area than before. Because turgor reduction is the earliest
significant biophysical effect of water stress, turgor-dependent activities such as
leaf expansion are the most sensitive to water deficits (Taiz and Zeiger, 2000).
Root Lenght of NGH (cm) Ʃ. Root of NGH
15.0 10.0

10.0
5.0
5.0

0.0 0.0
C WL D C WL D

Fig 4. Effect of water stress on plant RL and ƩR, : NGH: Non-Green House
maize, C: control, LW: lodge water, D: drought stress.

Actually, Taiz (2000+) assume that Water uptake decreases when roots are
subjected to low temperature or anaerobic conditions, or treated with
respiratoryinhibitors (such as cyanide). These treatments inhibit root respiration,
and the roots transport less water. The exactexplanation for this effect is not yet
clear. On the other hand, the decrease in water transport in the roots provides an
explanation for the wilting of plants in waterlogged soils: Submerged roots soon
run out of oxygen, which is normally provided by diffusion through the air spaces
in the soil (diffusion through gas is 104 times faster than diffusion through water).
The anaerobic roots transport less water to the shoots, which consequently suffer
net water loss and begin to wilt.

Fig 5. Effect of water stress on plant Root, C: control, LW: lodge water, D:
drought stress.

On this result, root length (RL) and root number (ƩR) of water logging
treatment was higher than root length (RL) and root number (ƩR) of drought
treatment even the root of water logging treatment was in anerob condition. We
assume that, the both of stress treatment condition had not been maximum. So,
the root regulation of water logging treatment had not decreased as an anaerobic
root treatment regulation. Also, the root of drought treatment had not been in
stress maximum condition which did not allow the root to do elongation to water
supply.

3.2 Water Stress Impact on Green House Maize

3.2.1 Character of Morphology


This observation was on Green House maize. The maize was grown in Green
House. The observation was 6 weeks after planting. There were three treatments
of water stress. The first was control (C) by daily irrigation. The second was water
logging (WL) treatment by flooding on 5 weeks after planting. The last was
Drought (D) treatment by irrigated once a week. Table 2. Showed morphological
impacted by water stress on green house maize.

Tabel 2. Summary of Maize Morphological Character in Green House


SL
Treatments PH (cm) ƩLN LA ƩRN RL (cm) SPAD LFW
(cm)
C 32,0 7,0 1,5 286,5 18,0 71,0 21,5 3,2
WL 56,5 7,5 3,0 265,6 27,5 61,8 28,3 2,3
D 27,8 4,5 2,3 147,4 14,5 27,3 26,9 1,3
IS - 5,0 5,0
Note: Effect water stress on maize morphology. PH: plant height, ƩL: Leaf
number, SL: segment length, LA: leaf area, ƩR: root number, RL: root
length, SPAD: Soil Plant Analysis Development, LFW: Leaf Fresh Weight,
C: control, WL: lodging water, D: drought stress, IS: Invitro Sorbitol

Maize plant samples on Green House show different plant height, in drought
stress treatment (D) plants 13,1% shorter than plants in control treatment (C) and
56,7 % than water logging (WL).The value is related to leaves number
observation parameters, where the number of plant leaves in the treatment is less
than normal plants caused by the higher the plant, the more number of stem
segments will be followed by the increasing number of leaves on the plant. Under
severe water deficiency, cell elongation of higher plants can be inhibited by
interruption of water flow from the xylem to the surrounding elongating cells.
Plant High (cm) Leaf Number
60 8
6
40
4
20
2
0 0
C LW D C LW D

Segment Length (cm) Leaf Area


4 400.0

3 300.0

2 200.0
1 100.0
0 0.0
C LW D C LW D

Fig 6. Effect of water stress on plant height of maize,. C: control, LW: lodge
water, D: drought stress.

Fig 7. Morphology of Maize height stem (green house) on water stress


treatment C: control, LW: logging water, D: drought.

Drought caused impaired mitosis; cell elongation and expansion resulted in


reduced growth of plant height (Hussain et al., 2008). Leaf area on water stress
also decreased compared with the normal leaf area of maize plant, water stress
caused a reduction in the whole plant leaf area by decreasing the initiation of new
leaves,withno significant changes in leaf size of leaf abscission.
Fig 8. Morphology of Maize leaves (green house) on water stress treatment. C:
control, LW: loggingwater, D: drought.

Fig 9. Morphology of Maize segment length (green house) on water stress


treatment. C: control, LW: loggingwater, D: drought.

In water logging conditions, plant height, leaves number and length of


segment higher than other treatments. This is caused by the water logging
treatment in this experiment has not entered the tense phase so that there has been
no decrease in morphology of maize, whereas in the water logging condition there
is a decrease in plant growth due to lack of oxygen so the plants show wilting
even when enclosed by excess of water, which affect nutrient and water uptake.

Fig 10. Morphology of Maize roots (green house) on water stress


treatment. C: control, LW: logging, water, D: drought.

Fig 10. Effect of Water Stress on Morphology of Maize roots. C: control,


LW: logging, water, D: drought.

The result of root in maize which was placed in Green House has similarity
with root in maize which was placed in Non-Green House. The root length (RL)
and root number (ƩR) of water logging treatment was higher than root length (RL)
and root number (ƩR) of drought treatment. Even thought, the root of water
logging treatment was in anerob condition.
The fact is several genes encoding glutathione-S-transferases (GSTs), a class
of proteins stimulated by various stress conditions, are induced by elevated auxin
concentrations.Likewise, ACC synthase, which is also induced by stress and is the
rate-limiting step in ethylene biosynthesis, is induced by high levels of auxin
(Taiz, 2000).
Initiation of lateral (branch) roots and adventitious roots is stimulated by high
auxin levels. Lateral roots arecommonly found above the elongation and root hair
zone and originate from small groups of cells in thepericycle. Auxin stimulates
these pericycle cells to divide. The dividing cells graduallyform into a root apex,
and the lateral root grows through the root cortex and epidermisauxin is involved
in stress responses, such as wounding by auxin (Taiz, 2000).

3.2.2 Character of Anatomy


Tabel 3 : Effect water stress on maize stomata
Density Density
Stomata
Adaxial Abaxial ∑ Adax ∑ Abax
Treat ment Area
Stomata Stomata Stomata Stomata
(µm)
(mm) (mm)
Control 37.4 46.8 51.5 64.5 10335.3
Drought 38.1 70.0 52.5 96.5 12170.2
Water Logging 50.4 77.6 69.5 107 14642.5

110.0
100.0
90.0
80.0
70.0
60.0
50.0
40.0
30.0
Density Adax Density Abax ∑ Adax Stomata ∑ Abax Stomata
Stomata (mm) Stomata (mm)

Control (C ) Drought (D) Water Logging (WL)

Fig 11. Effect of Water Stress on Maize Stomata. C: control, LW: logging,
water, D: drought.
a b c

Fig.12 Micrograph photos of abaxial stomata in maize in a:Normal, b: Water


Lodge, C: drought

Anatomical observations of stomata maize stomata in treated stress showed


that the density of stomata adaxial and abaxial of maize plants in water logging
(WL) stack increased by 34.76% and 65.81% compared to control (C),
respectively. This in line with the statement of Lestari (2006), that in the condition
of excess water the plant will have stomata with a higher density compared to
water deficit plants. At drought stress (D) the density of abaxial and adaxial
stomata increased by 1.87% and 49.57% compared to control (C), respectively.
Plants experiencing drought stress and water logging have entered the generative
phase so it is assumed that stomatal density is higher than control plants that are
still in the vegetative phase.
Stomatal density was positively correlated with stomata. It was observed that
the number of stomata adaxial and abaxial of maize plants in the waterlogging
stress condition increased by 34.95% and 65.89% compared to control,
respectively. Whereas in drought stress the number of stomata adaxial and abaxial
of maize increased 1.94% and 49.61% compared to control. The number of
stomata and stomatal density is higher in maize plants in stagnant stagnation, it is
assumed that the large amount of stomata in the leaves can help increase the
transpiration of the plants, so the plant will release a lot of water to reduce water
saturation in the body, thereby increasing plant tolerance to stress.
Maize stomata extent indicated that in the condition of stagnant water
logging, the area of plant stomata increased 41.67% higher than control. While the
dread stress condition of stomata area increased 17.75% higher than control. The
leaf area is also obtained in the treatment of stagnant stagnation, it is assumed also
that larger and more open stomata can reduce the saturation of water in the crops
through the transpiration process, whereas in stomatal plants with less drought
stress and more closed it is assumed that the crops reduce excessive evaporation
resulting lack of water in the plant body, so the plants defend themselves by
reducing the number, size, and density of stomata so as not to lose water in large
quantities, because the plants in a state of water deficit.

3.2.3 Proline Contain

Tabel 4. Proline Contain of maize water stress.


Proline Content
Treatment Fresh Leaf Weight (g)
(µmol g-1 FW)
Control 0,197 3,2
Water Logging 0,238 2,2
Drought 0,867 1,3

The results showed that there was an increase in proline content along with
the flooding treatment of 0,238 μmol g-1 FW and dryness of 0,867 μmol g-1 FW.
Whereas the control treatment produces a low of proline content of 0,197 mol g-1
FW. At the time the plants are treated for stress, both inundation and dryness have
increased the proline content by 4,80% and 29,40%, respectively. Similarly, in the
fresh leaf weight parameters, the control treatment contained the highest leaf fresh
weight of 3,2 g, while the decrease of wet leaf weight in the treatment of flooding
and drought, respectively 2,2 g and 1,3 g. In Fig 1 the plants treated in drought
show physiologic response with decreasing fresh leaf weight and increasing leaf
proline content.
The mechanism of drought resistance one of which is the ability to produce
compounds such as prolineosmotikum function in osmotic adjustment process.
The results of the analysis leaf proline content levels increased in the treatment of
flooding and drought. This is because the proline serves to regulate osmotic
pressure and accumulation in cells that play a role in maintaining water balance.
The accumulation of proline under stress conditions lowers the water pressure of
the cells so that it will help the plant remain able to absorb more water and
nutrients from the soil. In addition to functioning as a pressure within the cell,
proline also plays a role in reducing free radicals in the cells so that damage
caused oxidative stress can be prevented.
3.5

Proline Content (µmol/g FW)


3

2.5

1.5

0.5

0
Control Water Lodging Drought

Proline Fresh Leaf Weight

Fig.13 : Effect of water stress on Proline Contain of maize

Fig.14. Sample of Maize Leaf Proline extraction.


In Figure 14, the proline content of the proline seen in the treatment of
drought stress, the more reddish the proline content the higher. Plants that in the
stress will do the protection by forming enzyme antioxidant compounds and
reduce the level of detoxification Reactive Oxygen Species (ROS) directly or
indirectly in plant cells. These formed enzymes will be protected by the proline of
denaturation, so that the prevention of damage can take place optimally.
3.2.4 Molecular Analyzing

Fig.15 Electrophoresis of Total DNA (1). Maize control, (2). Maize Water
Logging, (3). Maize Drought, (4). In Vitro maize control, (5) In vitro
maize sorbitol stress

From this picture, extraction dna with Termofisher Genom DNA Purification
Kit produce the high quality genom DNA. The electrophoresis of total DNA every
samples, showed that high DNA concentration about 100 ng. If compared with
CTAB method the thermofisher genejet extraction kit more powerful to produce
DNA with high concentration, because the kit have a proportional buffer and
spesific tube extraction with filter which is can separate the pellet and supernatan
in centrifuge step more quickly and clean. So, from this result we can run to the
next step, it can called PCR analysis. In PCR analysis, DNA genom from the
extraction step, will be reacted with specific SSR primer tagged site like glyphos
detection, Taq Polymerase and dntps in the thermal cycler with 35 cycle. Then
after the PCR, product was separated with agarose gel electrophoresis until 20
minutes and load into EtBr solution then documentation with Gel doc by BIO-
RAD.
Fig.16 The electrophoresis of PCR product with spesific SSR primer tagged
Glyphos (1). Maize control, (2). Maize Water logging, (3). Maize Drought,
(4). Maize control in vitro, (5). Maize Sorbitol Stress in vitro, M: Dna
Ladder 1000 bp

From this picture above, all the maize sample succesfully amplified with this
spesific SSR primer. It was signed about the all sample assosiate with glyphos
character site. Between the three sample of field stress and in vitro stress looks
similar but intensity of fluorescence little bit different. It was indicate the level of
stress given impact the quality of DNA. The size band of amplifying product of
PCR with this primer above the 1000 bp. So we can conclude, the sequence of
nucleotide with coding the stress resistance properties like epsps gene appear in
each sample but with different thicknesss. The fluorescence in in vitro sample
(4,5) more strong among the other but the sample 5 with sorbitol stress most
strong. That was definitly when the plant under pure stress controlled in
laboratory can produce the quantity of specific site with primer complement more
higher.
4. CONCLUSION
1. Proline concentration in plant with abiotic stress more higher than normal
plant control
2. The water lodge stress affect the maize root more thicker
3. The water drought stress affect the maize root more longer
4. The stomata in maize leaf with drought stress more narrow and small than
the other
5. DNA sample succesfully amplifying with SSR primer glyphos site
6. The fluorescence band of DNA sample on plant stress more strong
REFERENCE
Lestari, E.G. 2006. Hubungan antara Kerapatan Stomata dengan Ketahanan
kekeringan pada Somaklon Padi Gajahmungkur, Towuti dan IR 64.
Biodiversitas.7 (1): 44-48