Ministry of Education and Science of the Republic of Kazakhstan
A. Baitursynov Kostanay State University
Department of Biology and Chemistry
Sultangazina G.Zh.
BIOLOGY OF PLANTS
(Plant Physiology)
Training manual
Kostanay, 2017
2
UDC 581.1(075.8)
LBC 28.57 73
S 91
Reviewers:
Sitpaeva Gulnara Tokbekenovna doctor of biological sciences, Institute of
Botany and Phytointroduction, Committee of Science, Ministry of Education and
Science of the Republic of Kazakhstan
Aydarhanova Gulnar Sabitovna doctor of biological sciences, associate
professor of the Department of Management and Engineering in the sphere of
Environmental Protection, L.N.Gumilyov Eurasian National University
Ruchkina Galiya Adgamovna candidate of biological sciences, associate
professor of the Department of Biology and Chemistry of the A. Baitursynov
Kostanay State University
Author:
Sultangazina Gulnara Zhalelovna, candidate of biological sciences, associate
professor
S 91 Sultangazina G.Zh.
Biology of Plants (Plant Physiology). The training manual is intended for
students of agricultural, biological, and technical specialties. Kostanay, 2017. 99
p.
ISBN 978-601-7387-71-6
The training manual covers the processes of vital activity and plant functions at
the cellular, molecular levels, and at the level of the whole organism. The main
physiological and biochemical methods of studying physiology and biochemistry of
plant cells, water metabolism, photosynthesis, respiration, mineral nutrition, growth
and development, adaptability and plant resistance to unfavorable environmental
factors are represented.
The training manual is intended for students of agricultural, biological, and
technical specialties.
UDC 581.1(075.8)
LBC 28.57 73
It is recommended for publication by educational and methodological council of the
A. Baitursynov Kostanay State University, 26.04.2017, protocol No 3
ISBN 978-601-7387-71-6
Sultangazina G.,2017
3
Content
Introduction ...... 5
1 Physiology of the plant cell .... 7
1.1 Permeability of live and dead cytoplasm for substances of cell sap . 9
1.2 Effect of cations and anions on the plasmolysis form and time ...... 11
1.3 Observation of cap plasmolysis ... 12
1.4 Determination of the seeds viability by a staining method (according to
D.N. Nelyubov) .................
12
1.5 Determination of the isoelectric point of plant tissue by a colorimetric
method .......
13
2 Water metabolism of plants ... 16
2.1 Determination of water content and dry matter in plant material . 18
2.2 Determination of the stomata condition by an infiltration method
according to Molisch .............................................................................................
19
2.3 Determination of the osmotic pressure of cell sap by a plasmolytic method 20
2.4 Determination of the water potential of plant tissue by a method of strips
(according to Lilienstern) ..
22
2.5 Determination of the water potential of leaves by Shardakovs method ...... 23
2.6 Comparison in transpiration of the upper and lower sides of a leaf by a
chlorocobalt method ..
24
2.7 Determination of the rate of transpiration in cut leaves by torsion scales..... 25
2.8 Determination of the rate of transpiration and relative transpiration by
technical scales ...............
27
2.9 Determination of the water-retaining capacity of plants by a "wilting"
method according to A. Arland .....
29
2.10 Determination of the productivity of transpiration and the transpiration
coefficient .....
30
3 Photosynthesis ... 32
3.1 Preparation of an alcohol solution (extract) of pigments ...... 34
3.2 Separation of pigments by Kraus ...... 34
3.3 Saponification of chlorophyll with alkali ......... 35
3.4 Preparation of pheophytin and the reverse substitution of hydrogen by a
metal atom .
35
3.5 Optical properties of pigments .. 36
3.6 Determination of the leaf area ... 38
3.7 Determination of the rate of photosynthesis by the assimilation flask
method (according to L.A. Ivanov and N.L. Kosovich) ...
40
3.8 Determination of the rate of photosynthesis by the accumulation of
organic carbon (I.V. Turin's method, modified by F.Z. Borodulina) ...................
42
3.9 Determination of the net productivity of photosynthesis ............................. 44
4 Respiration of plants ...... 47
4.1 Loss of dry matter during germination of seeds ..... 50
4.2 Determination of the rate of respiration in a closed vessel .. 52
4
4.3 Determination of the respiration rate of germinating seeds in the air
current by an infrared gas analyzer ......
53
4.4 Determination of the respiratory quotient of germinating seeds .. 54
5 Mineral nutrition .... 56
5.1 Study of the effect of nutrients on plant growth . 59
5.2 Growth of wheat roots in a solution of pure salt and a mixture of salts 63
5.3 Microchemical analysis of ash ..... 64
5.4 Determination of the total and working adsorbing surface of the root
system by Sabinin and Kolosov method...........
65
6 Growth and development of plants 67
6.1 Observation of the periodicity of shoot growth . 69
6.2 Determination of the growth force of seeds by morphophysiological
evaluation of sprouts .
70
6.3 Determination of the physiological activity of gibberellins in a biotest with
the extension of sprouts hypocotyls of dicotyledonous plants .....
72
6.4 Establishment of photoperiodic reaction of sarept mustard . 73
7 Adaptation and resistance of plants ... 75
7.1 Identification of the protective effect of sugars on protoplasm 77
7.2 Early diagnostics of plant resistance to wetting ... 78
7.3 Protective effect of sucrose on proteins at negative temperatures .... 79
7.4 Effect of sucrose on the frost resistance of plant cells ... 80
7.5 Method of hardening and determination of frost resistance of winter crops
with the use of exogenous sugars .
81
7.6 Determination of heat resistance of plants (according to F.F. Matskov) . 82
7.7 Effect of high temperature on the permeability of cytoplasm .... 82
7.8 Determination of drought resistance of plants by a starch test method 83
7.9 Determination of the temperature threshold of cytoplasm coagulation
(according to P.A. Genkel) .
84
7.10 Determination of salt resistance by growth processes 85
8 Transformation of organic substances in plants .... 87
8.1 Detection of spare sugars in plant material .. 89
8.2 Detection of spare proteins in plants 90
8.3 Acid hydrolysis of starch .. 92
8.4 Determination of total protein content . 92
8.5 Determination of lipase activity during germination of seeds .. 96
References............................. 99
5
Introduction
Plant physiology is one of the fundamental basis in general biological
education for students of agricultural, biological and technical specialties.
The study of the physiological processes occurring in plants is possible only
with a deep knowledge of the connections between plant physiology and inorganic,
organic, biological and physcolloid chemistry, plant anatomy and morphology, soil
science, agrochemistry, selection, genetics, agriculture, vegetable growing, fruit
growing, plant growing, as well as mathematics, physics, and cybernetics.
Plant physiology, relying on the laws and regularities, improves the theoretical
basis for the growth and development of the plant organism as a whole and its
individual organs, taking into account the soil and climatic features. Life, as a special
form of matter motion, is basically one for both plants and animals.
The ability to reveal the contradictions inherent in physiological processes, to
concretize the physiological phenomena in various plant species and varieties
expands and deepens the possibilities of active human intervention in the
physiological processes of plants, allows them to master these processes, to direct
them according to the goals set. The ability to navigate in the processes occurring in
plants is a prerequisite for every specialist of the agro-industrial complex.
Plant physiology is a dynamically developing science, methods of
physiological and biochemical research are constantly being improved, high-
precision instruments are created, which allows to obtain new information on the
structure and properties of biogenic compounds ensuring participation and
synchronism of the course of metabolic processes in living organisms.
The aim of plant physiology to a greater extent is a logical and complete
explanation of the processes in the plant organism in accordance with known physical
and chemical laws. It requires the use of physical, chemical methods, and actively
developing recent methods of biological statistics.
"Plant biology" is a discipline of the obligatory component established by the
Model curriculum of the specialty 5B080100-Agronomy, and consists of two
sections: botany and plant physiology. The proposed training manual is written on the
second section and allows to form knowledge, creative skills for independent
experimental activity of students in the field of plant physiology.
For students of specialties 5060700-Biology, 5060600-Ecology, 5072700-
Technology of Food Production, 5072800-Technology of Processing Production
"Plant biology (Plant physiology)" can be recommended as an elective basic
discipline.
The training manual on the discipline "Plant biology (Plant physiology)" is
intended for students of agricultural, biological, and technical specialties and in its
content corresponds to the course program used for training of the relevant
specialists.
The paper describes modern theoretical basis of plant physiology. The paper
considers the methods of studying the physiology of plant cells; water exchange;
photosynthesis; respiration; mineral nutrition; metabolism; growth and development
6
of plants, plant resistance to unfavorable external factors. Along with the classical
works, this manual includes the new ones, tested at the Department of Biology and
Chemistry of A. Baitursynov Kostanay State University within a number of years. It
is impossible to perform all the works within the time allocated by the curriculum, so
teacher can choose those works, the fulfillment of which is possible in available
conditions.
Mastering practical knowledge and skills in this discipline allows students to
comprehend physiological processes in a plant organism, methods of their study -
which constitutes the basic knowledge necessary for understanding other disciplines,
like agrochemistry, plant growing, biotechnology, bioengineering, etc.
The skills of experimental work acquired on the lessons can be used in writing
a course or graduate work, in organization of the research work of a student.
The procedure of each laboratory work is preceded by a brief theoretical course
on the topic under study. Each work gives a description of the research principle and
detailed laboratory methods. Students are invited to formulate main conclusions and
answer control questions on the results of the work.
The principal textbooks used in the work:
1 Zitte P., Weiler E.V., Kaderayt J.W., Brezinski A., Kerner K. Botanika. V.2
Fiziologija rastenij. - M.: Publishing Center " Akademija", 2008. - 496 p.
2 Tretyakov N.N., Panichkin L.A., Kondratiev M.N. et al. Praktikum po
fiziologii rastenij. M.: KolosS, 2003. - 288 p.
3 Rogozhin V.V. Praktikum po fiziologii i biohimii rastenij: training manual. -
SPb.: GIORD, 2013. 352 p.
7
1 PHYSIOLOGY OF THE PLANT CELL
Information material. A plant cell is an ordered structure, which is an
elementary functional unit of living organisms. The cell has a certain size and shape,
due to the ordered arrangement of proteins that carry information both about the
structure of the cell and the plant in general. The functioning of the cell is regulated
by the information embedded in the DNA structure, which is localized in the nucleus
of the cell. According to this information, an ordered structure of the plant is formed,
the functioning of the plant is determined, the behavior of the living organism and its
life time are given.
The basis of the cell structure is membranes. Thus, endoplasmic reticulum
(ER) permeates the entire cytoplasm of the cell, expanding into small vacuoles. There
are rough and smooth ER. Ribosomes are located on the outer side of the rough ER
membrane, which are the elements of the protein-synthesizing system. Smooth ER
does not have ribosomes, but contains enzymes that catalyze the synthesis of lipids.
In general, ER performs the role of a cytoskeleton of the cell and a transport system,
connecting different parts of the cell. In addition, ER regulates transport flows of
various substances, and synthesized proteins can move in a certain direction through
the reticulum channels.
The constituent parts of the plant cell are specialized organelles: nucleus,
nucleolus, mitochondria, chloroplasts, Golgi apparatus, lysosomes, peroxisomes,
ribosomes, vacuoles, etc. Various biogenic molecules are involved in the formation
and functioning of the organelles and membranes of the cell: amino acids,
nitrogenous bases, nucleic acids, proteins, lipids, carbohydrates, secondary
metabolites, etc.
A cell nucleus has a double membrane, in the structure of which there are pores
of 10...20 nm in size. Active transportation of biogenic molecules from the nucleus to
the cytoplasm and back is carried out through the pores. Nuclear DNA of plants is
fragmented and is part of chromosomes.
Mitochondria belong to specialized organelles of ATP synthesis. They have a
double membrane. The number of mitochondria in a plant cell can be from 300 to
1000. Mitochondria have their own DNA, as well as RNA and ribosomes. The latter
participate in the synthesis of proteins. Enzymatic complexes are localized in the
inner membrane of mitochondria, which catalyze the reactions of oxidative
phosphorylation. The enzymes of tricarboxylic acid cycle are contained within
mitochondria, in the matrix.
Chloroplasts are specialized structures of plant cells, in which photosynthetic
reactions occur. Chloroplasts have their own DNA, RNA and ribosomes.
Peroxisomes are small organelles, containing oxidoreductases (catalase,
peroxidase, oxidases, dehydrogenases, etc.) that participate in the respiration process.
These enzymes catalyze the oxidation reactions of various substances.
Spherosomes are formed from the membranes of zondoplasmic reticulum; they
mainly contain lipids and enzymes, participating in the processes of synthesis and
decomposition. A high content of spheroids is found in oilseeds. With the
8
germination of seeds, the splitting reactions of redundant lipids are activated in these
organelles.
The Golgi apparatus is made up of a series of compartments consisting of
cisternae and vesicles, surrounded by membranes. The cisternae of the Golgi
apparatus have two ends.
The process of new cisternae formation takes place at one end (the forming end), and
the formation of vesicles happens on the other end (the secreting end). These
processes proceed continuously and consistently. Meanwhile, the formation of
vesicles occurs simultaneously with the formation of new cisternae. The functioning
of the Golgi apparatus provides the plasmolemma and the cell membrane with plastic
material.
Lysosomes are the organelles, formed from the membranes of endoplasmic
reticulum or the Golgi apparatus, where the hydrolytic enzymes are mainly contained.
The latter catalyze the splitting reactions of proteins, nucleic acids, carbohydrates and
lipids.
Vacuole is a cavity that fills 90% of the space of an adult plant cell. Vacuole
contains amino acids, proteins, carbohydrates, pigments, ions of various salts and
acids. Vacuole is involved in maintaining of the osmotic pressure of the cell,
regulating the flow of incoming water and water circulation inside a plant. The
products of metabolic processes and secondary metabolites are accumulated in a
vacuole, which may later participate in the metabolism of the cell again.
Non-membrane structures of the plant cell are ribosomes and microtubules.
The latter have the form of a tube with a channel inside. The walls of the tubules
consist of globular proteins - tubulins. Microtubules can degrade and re-emerge in
plant cells.
The main function of microtubules is their participation in the formation of a
cell cytoskeleton and a structure of the cell wall. Ribosomes are involved in the
synthesis of a polypeptide chain. They are complex nucleoprotein complexes,
consisting of two parts (small and large).
The basis of a ribosome is ribosomal RNA. Most of ribosomes attach to the
membranes' surface of the endoplasmic reticulum and participate in the process of
protein biosynthesis in the structure.
Methodological recommendations on the studying the topic. While working
out this program section, it is necessary to pay attention, firstly, to the structural and
functional organization of the cell, as well as the physical and chemical basis of its
energy, since the cell carries life. Consider the structure of the cell membrane and
membranes, their role in metabolism. Analyze the functions of nucleus and
cytoplasm, structural basis of cytoplasmic permeability and dependence of
permeability on internal and external factors. It is necessary to find out what the
heterogeneity of cytoplasm is.
Particular attention should be paid to the study of the chemical composition of
the plant cell cytoplasm and the functional role of its main components.
9
The enzyme system is derivative to the basic protein structure of the cellular
organelles. Nucleotides take an active participation in the implementation of
enzymatic transformations in the cell.
It is necessary to pay attention to the structure of RNA and DNA, their
physiological role in the biosynthesis of proteins, to the localization of protein-lipoid
compounds in peripheral cytoplasmic layers, and to the physiological role of
plasmatic membranes.
References: 2, pp 7-81; 3, pp 11-85; 4, pp 39-53; 5, pp 3-19.
Questions for self-examination:
1. What disciplines is the plants physiology connected with?
2. The objectives of plant physiology at the present stage.
3. Physical and chemical basis of the plant cell energy.
4. Structural organization of a plant cell.
5. Functional organization of a plant cell.
6. Composition, structure and physiological role of membranes in the vital activity of
the cell.
WORK IN A LABORATORY
The laboratory work performance for students consists of three main stages:
Preparatory stage: Students independently study methodological
recommendations on the laboratory work performance. Each laboratory work has a
list of materials and equipment, a brief theoretical explanation, a description of the
procedure, instructions on the work performance.
Main stage: With the guidance of a teacher and a laboratory assistant student
perform Tasks assigned to them (work out the educational questions). In this case, the
laboratory work is considered performed only after the recording of its results, which
should contain a description of the observed changes during an experiment and a
brief analysis of the data obtained.
Final stage: After completing the laboratory work, students prepare the results,
formulate conclusions and report to the teacher. There is no description of the
expected results or ready conclusions in the manual, for this technique develops the
independence of students and promotes a better lasting learning of the studied
material.
Summing up the lessons. When the training questions are done, teacher
summarizes the results of the laboratory study, evaluates the work and assesses each
student.
1.1 Permeability of live and dead cytoplasm for substances of cell sap
External cytoplasmic cell membrane (plasmalemma) separates the cell from the
environment, controls transportation of substances into and out of the cell.
10
The main property of membranes is their selective permeability. Selective
permeability of a membrane is maintained as long as the cell remains alive. After its
death, the membranes become completely permeable. The presence of membranes is
bound to a number of intracellular processes and cell functions:
- membranes divide cells and organelles into compartments, in which
oppositely directed processes are carried out;
- localization of the enzymes or the intermediates of metabolism on a
membrane determines the sequence of reactions;
- selective permeability of the outer membrane of the cell forms a certain
composition of its internal contents;
- low permeability of membranes for protons underlies the processes,
associated with the synthesis of macroergic compounds and with the use of ATP for
the transportation of substances.
Selective permeability is a property of a living cytoplasm to maintain the
constancy of the intracellular environment (homeostasis). If the cell is damaged, the
cytoplasm loses this property, and the substances in the cell exit freely. The degree of
damage correlates with the amount of the anthocyanin pigment, released into the
aqueous medium. The intensity of the cell substances' lost serves as a criterion for its
damage.
Task: Identify the differences in membranes' permeability of the living and
dead cells and make a conclusion on the causes of these differences.
Procedure. From the peeled root of red beet cut four even-sized slices about 2
cm in length and 0.5 cm in width (the core must be fresh, i.e. it must have a good
turgor, since the experiment with the faded material can't give clear results). Put the
slices into a porcelain cup and rinse them repeatedly with tap water until the leak of
pigmented juice from the cut cells stops. Put the slices into 4 test tubes. Fill two test
tubes with water (up to 1/2 volume) and boil one of them for 1-2 minutes. Fill the
third test tube with water and add 5 drops of chloroform, fill the fourth test tube with
30% solution of acetic acid. Observe the changing of color of the liquid in the test
tubes within 1-2 hours, shaking the contents from time to time.
Record the results into Table 1 by the form given.
Table 1 - Permeability of live and dead cytoplasm for substances of cell sap
Variant of experience Speed of coloring of the liquid
Water at room temperature
Boiling
Water + chloroform
30% acetic acid solution
Materials and equipment: stove, test tubes, test tube rack, test tube holder, porcelain
cup, beaker, measuring cylinder, root of table beet, filter paper.
Reagents: chloroform, 30% acetic acid solution.
11
1.2 Effect of cations and anions on the plasmolysis form and time
Plasmolysis a cytoplasm's peelling away from the cell wall, placed in a solution
with a higher concentration than the concentration of cell sap (hypertonic solution).
During plasmolysis, the shape of a cytoplasm changes: firstly, it shrinks, and after a
complete loss of turgor, protoplast peels away from the cell wall at the corners
(angular plasmolysis), then in many places (concave plasmolysis) and, finally,
protoplast is rounded (convex plasmolysis).
Plasmolysis time is the period from the moment of immersing a plant tissue
into the plasmolithic solution until the convex plasmolysis. This index can
characterize the viscosity of a cytoplasm: the longer the plasmolysis time, the higher
the viscosity of the cytoplasm.
The following solutions can be chosen for each cell:
1) hypotonic, an osmotic pressure of which is less than the osmotic pressure of
the cell sap,
2) isotonic, an osmotic pressure of which is equal to the osmotic pressure of the
cell sap,
3) hypertonic, an osmotic pressure of which is greater than the pressure of the
cell sap.
Selective permeability of membranes ensures the passage of water molecules
through them, prevents the penetration of substances, dissolved in water, and causes
the phenomenon of plasmolysis when a hypertonic solution affects on the cell.
Deplasmolysis occurs in a result of a gradual penetration of the dissolved
substance into the cell, changing of the water potential from the outside and inside,
and also the flow of water into the cell from the outer solution along the gradient of
the water potential.
Cations and anions of salts have a specific and diverse effect on cytoplasm.
One of the notable external manifestations of this effect is the changes in the degree
of swelling and viscosity of the cytoplasm, observed by the plasmolysis time. When
comparing the viscosity of cytoplasm in the solutions of potassium and calcium salts,
it can be noted that potassium ions, penetrating the cytoplasm, increase its
hydrophilicity, reduce viscosity and promote its rapid separation from the cell wall.
Task: Draw the forms of plasmolysis. On the basis of the obtained results,
make a conclusion on the cations and anions' affect on the cytoplasm viscosity.
Procedure. Put an epidermis segment from the convex surface of red onion's
peel into a drop of the test salt's solution, cover with a cover slip and examine under
microscope immediately. Observe the change of plasmolysis forms. Determine the
time of plasmolysis in each salt.
Record the results of the experiment into Table 2 by the form given.
Table 2 - The effect of cations and anions on the plasmolysis form and time
Variant Salt Concentration
of a solution,
Time of the
immersion
Time of
occurrence
Plasmolysis
time, min
12
mol/L of tissue in
solution
of the
convex
plasmolysis
1 Ca(NO3)2 0.7
2 KNO3 1.0
3 KCNS 1.0
Materials and equipment: microscopes, slides and cover slips, safety razor blade,
scalpel, needle, bulb of red onion, filter paper.
Reagents: 0.7 Ca(NO3)2 , 1 KCNS, 1 KNO3.
1.3 Observation of cap plasmolysis
Cap-plasmolysis occurs with the hypertonic solutions of salts, penetrating
through plasmalemma, and not passing or very weakly passing through the tonoplast.
Such salts cause the swelling of mesoplasma, the decrease of its dispersion degree,
the change of the structure. Cap-plasmolysis manifests itself in the formation of caps
from the swollen cytoplasm on the narrow sides of a vacuole.
Task: Draw one cell with a cap plasmolysis in the full view. On the basis of
the observations, make a conclusion on the properties of cytoplasmic membranes.
Procedure. Put an epidermis segment from the convex surface of pigmented
onion's peel on the microscope slide containing a drop of 1 M of KCNS solution and
cover with a cover slip. Observe the formation of capsular plasmolysis immediately,
firstly, with low and then medium magnification.
Materials and equipment: microscopes, slides and cover slips, safety razor blade,
scalpel, needle, bulb of red onion, filter paper.
Reagents: 1 KCNS.
1.4 Determination of the seeds viability by a staining method (according to
D.N. Nelyubov)
The method of seeds coloring to determine their germination is based on the
impermeability of the living cytoplasm for certain colorants (indigocarmine, acid
fuchsin), whereas a dead cytoplasm is colored easily. There are the cases, when an
embryo is dead and yet, when the seed is immersed into the colorant solution, it is not
colored, due to the fact that the surrounding parts of the seed do not permeat the
colorant. In this regard, it is necessary disclose the embryo firstly: for the seeds with
endosperm, extract the embryo or cut the seed along, and for the seeds without
endosperm, remove the seed cover.
Keep the seeds, prepared in the way described above, in the colorant solution
for 1 to 3 hours (depending on the species of a plant) and evaluate the viability of the
seeds: the seeds with fully colored embryos or with colored roots are considered non-
germinant; the seeds with uncolored or partially colored cotyledons are considered
13
viable.
This method is used for a quick germination assessment of the seeds of peas,
beans, lupine, flax, hemp, pumpkin.
Task: Evaluate the viability of the seeds of peas, beans, lupine.
Procedure. Count, without choosing, two portions of 10 swollen pea seeds.
Put one portion into a glass of water and boil for 5 minutes (with control). Carefully,
without damaging the cotyledons, peel the seeds of both portions with the aid of a
needle, place them into porcelain beakers, pour indigocarmine solution and leave for
1 hour, then drain the paint back into the bottle and rinse the seeds from the excess
dye.
Mark the coloring of seeds killed by boiling. In the experimental portion count
the number of colored, partially colored and uncolored seeds. To check the
germination, put all 10 seeds into a glass with wet sawdust (squeeze excess water out
the sawdust before filling the glass), place it into a dark cupboard and water daily. A
few days later, count the amount of germinated seeds.
Record the results of the experiment into Table 3 by the form given.
Table 3 - Determination of the seeds viability
Object Number
of seeds
taken,
pcs
Number of seeds, pcs
colored
completely
partially
colored
uncolored germinated ungerminated
Materials and equipment: seeds of peas, beans, lupine, soaked in water for 10-15
hours before classes, beaker, stove, porcelain beakers, razor blade, needle, sawdust,
filter paper.
Reagents: 0.1% indigocarmine solution (1 g per litre of distilled water), acid fuchsin.
1.5 Determination of the isoelectric point of plant tissue by a colorimetric
method
Amino acids and proteins of the cytoplasm are amphoteric substances. In
solution they dissociate both as acids and as bases. The higher the concentration of
hydrogen ions in the medium, the more acidic dissociation is suppressed, and the
protein acquires a greater positive charge. The higher the concentration of hydroxyl
ions in the medium, the stronger the suppression of the basic dissociation, and the
protein acquires a greater negative charge.
At a certain pH value of the medium, the number of positive and negative
charges balances. Ampholyte (a molecule of an amphoteric substance in the state of
dissociation) becomes electrically neutral. This pH value of the medium is called the
isoelectric point (IET).
Each ampholyte has its own IET value. In proteins and amino acids, it depends
14
on the amount of free acidic (carboxyl) and basic (amine) groups. Knowing the IET
of proteins, one can judge on the ratio of acidic and basic amino acids in their
composition. If the dissociation in the solution of amphoteric compound follows the
basic type (in the media with a pH below the IET), the positive charge binds the
anions, but if it follows the acidic type (in the media with a pH above the IET), the
negative charge connects the cations.
To determine the IET of plant tissue, use an acidic colorant - eosin, where the
anion has a bright pink color, and the basic colorant - methylene blue (MB), the color
of which is determined by the cation. In the process of coloring an amphoteric
compound with these colorants and plunging into the media, which pH is below the
IET, mainly the anions of eosin are bound. Therefore, the plant tissue acquires the
pink color. In the medium, where the pH is above the IET, the plant tissue mainly
retains methylene blue cations and turns blue. In the medium with the pH equal to the
IET, the color of the plant tissue ampholytes is intermediate between pink and blue -
violet, since in this case the number of positive and negative charges is the same.
The cytoplasm contains a mixture of amphoteric substances, so the transition
zone from pink to blue is gradual.
Task: Study the colorimetric method of determining the isoelectric point;
determine the IET of a plant tissue.
Procedure. Prepare weighing bottles with 10 ml of buffer solutions at the pH
values in accordance with the table below. At a distance of 0.5 cm from the tip of a
pea sprout make strictly transversal cuts (not less than 24) with the aid of a razor and
place them into a porcelain cup with 70% solution of ethyl alcohol for 5 minutes for
fixation.
Pour 2-3 ml of 0.1% eosin into one porcelain cup and in the same amount of
0.02% methylene blue into another. Using a brush replace the cuts from the alcohol
into eosin solution for 10 minutes. All the cuts will be pink. Then, without washing
replace the cuts from eosin into methylene blue solution for 8-10 minutes and they
will become blue.
Transfer colored cuts with the aid of a brush into the buffer solutions with
different concentrations of hydrogen ions, three cuts per each solution and leave there
for 1-1.5 hours. Then take out the cuts, place them on the microscope slide in the
prescribed order and examine under microscope at low magnification. In the
solutions with a pH below IEP the color of the tissue will be pink, in a pH above IEP
- blue. For bark and central cylinder the color gradation from pink to blue will take
place at different pH values (violet color). Consequently, IEP of these tissues is not
equal, which indicates different cytoplasm composition in the cells of these tissues.
Record the results of the experiment into Table 4 by the form given. Depending
on the amount of proteins, containing acidic amino acids, the pH of IEP varies from 3
to 6 in different tissues of the root.
Table 4- Preparation of buffer solutions with different pH and the color of the plant
tissues at the specific pH value
15
0.2
NaHPO4
solution, ml
0.1 citric acid
solution, ml
Color of the tissue IEP
Bark Central
cylinder
Bark Central
cylinder
2.2
3.0
3.6
5.0
5.4
6.0
7.0
8.0
0.20
2.05
3.22
5.15
5.57
6.31
8.23
9.72
9.80
7.95
6.78
4.85
4.43
3.69
1.77
0.28
Materials and equipment: germinated seeds of peas and beans, safety razor blade,
brushes, needles, porcelain beakers, weighing bottles, microscopes, slides and cover
slips.
Reagents: 0.1 citric acid solution, 0.2 NaHPO4 solution, 0.1% eosin, 0.02%
methylene blue, 70% solution of alcohol.
16
2 WATER METABOLISM OF PLANTS
Information material. In biological systems water has a wide variety of
functions. Thus, in a liquid state, water is capable of ensuring the maximum solubility
of the biogenic molecules of polar nature. It can serve as a medium, in which optimal
conditions for the formation of individual structures of biogenic molecules (proteins,
lipids, enzymes, nucleic acids, etc.) are created. Water participates in the formation of
ordered structures of protein-lipid complexes, the membranes of organelles and
plasmalemma.
Water takes part in enzymatic reactions, catalyzed by hydrolases (lipases,
peptidases, nuclease, etc.). It is able to provide transportation of ions, biogenic
molecules and gases (O2 and CO2). Having a high thermal capacity, water serves as a
temperature regulator, ensuring the maintenance of a stable temperature in plants.
Due to thermal effects, water provides the energy needs of a plant organism, and the
presence of the transpiration mechanism ensures the directed movement of water
within a plant. A limited dissolution of gases (oxygen, nitrogen, CO2, etc.) occurs in
water, which transfers the gases to various organs and tissues of a plant.
Water comes into plants from soil and spreads along the ascending and
descending paths in the plant organism. Directed movement of water in various parts
of plants is provided due to the active work of stomatal apparatus of leaves, which
determine the functioning of transpiration mechanisms.
The water content in plants depends on the species, age and functional state of
the plant organism. Plants can contain 60-90% of water. Seeds and spores contain the
least amount of water. For example, the grains of cereals contain from 8-10% of
water in the period of forced rest. The high activity of oxidases, including peroxidase,
in the wheat grains may indicate the participation of these enzymes in maintaining
their viability in the period of forced rest. Thus, for example, peroxidase is able to
catalyze the reactions of oxidase and peroxidase oxidation of the organic compounds
in plant tissues. In these reactions oxygen is consecutively reduced to water and, due
to this, during the seeds rest period, the need in water for an embryo is satisfied.
Generation of water in the resting grains is a consuming process, since it is
associated with the oxidation of biogenic compounds. However, due to the fact that
peroxidase substrates can be a variety of biogenic molecules (carbohydrates, amino
acids, phenols, etc.), the oxidation of these compounds does not cause significant
damage to the cells of the embryo. The involvement of biogenic molecules in oxidase
and peroxidase reactions is determined by their affinity to the active center of
enzyme. In peroxidase reactions there can be observed a substrate-substrate
activation, which promotes the acceleration of oxidation of some biogenic molecules
in the presence of other molecules, causing an acceleration of water generation.
It is possible to distinguish two forms of water in biological systems: free (with
initial physical and chemical properties) and bound (with altered physical and
chemical properties, due to the interaction with various biogenic molecules). Water is
a good solvent for polar organic compounds, containing amino, carboxy, sulfhydryl
and hydroxyl groups. Therefore, carbohydrates (mono- and oligosaccharides),
17
alcohols, aldehydes, ketones, amino acids and volatile carboxylic acids are well
soluble in water. The solubility of these compounds is explained by the fact that their
polar groups are able to form hydrogen bonds with water molecules. Most salts are
soluble in water, the ions of which are present in the hydrated form in a solution.
Polarity of water molecules causes the solubility of polar and charged
molecules in water. Hydrophobic compounds, containing ultimate hydrocarbon
radicals, are insoluble in water and, therefore, escape from the contact with its polar
molecules, locating mainly on the water surface. Some compounds, containing both
hydrophobic and hydrophilic groups, are capable of forming aggregates in water,
forming micellar structures. In this case, the hydrophilic groups of these compounds
contact with the water molecules, while the hydrophobic radicals are exposed
inwardly to the micelles.
Thus, the stability of micelles, formed in a polar medium, is maintained mainly
due to the weak hydrophobic interactions. In this case, water-soluble compounds are
able to change the physical properties of water. The osmosis energy ensures a water
supply to the seeds during the swelling period, as well as an active water movement
into the tissues and organs of plants during their growth period.
Methodological recommendations on the studying the topic. Water plays a
crucial role in the life of plants. Therefore, it is necessary to know how water is
absorbed and released by the cell, what the water exchange of plants is, what the
water content and water distribution in the cell is, what the thermodynamic
parameters of the water regime of plants are - water activity, chemical potential,
methods for their determination; as well as the consistent parts of the water potential -
osmotic potential, matrix potential, pressure potential, gravitational potential.
While studying the root system as an organ of water absorption, it is necessary
to pay attention to what forms of water are present in the soil and are absorbed by the
roots of plants, what the constants of soil moisture are. Difine, what the ascending
flow in plants means, its path, speed, driving forces, and what the engines of the
water flow are. Find out the role of the intermediate engines in water raising, the
physiological significance of the water movement in plants and the renewal of its
stock.
The role of transpiration is great in plants' water exchange. With this in mind, it
is necessary to clarify the biological significance of transpiration, its dependence on
external factors and the state of stomata, their number and allocation in leaves.
It should be clarified, how to determine the intensity and productivity of
transpiration, the transpiration coefficient, as well as the phase, biological and
commodity coefficients of water consumption, how they are used to explain the water
balance of plants, the total water consumption by phytocenoses and the irrigation
regime of crops (irrigation norms, watering methods and periods).
References: 1, pp 71-93; 2, pp 83-116; 3, pp 276-304: 4, pp 183-189; 5, pp 38-70.
Questions for self-examination:
1. How does the plant absorb and release water?
2. What is a chemical potential of water and a water potential of the cell?
18
3. What biological significance does the transpiration have?
4. What physiological indicators can be used to optimize the water regime of
agrophytocenoses?
5. What are the main functions of water in the regulation of plant growth and
development?
WORK IN A LABORATORY
2.1 Determination of water content and dry matter in plant material
The degree of water content is an important indicator of water availability in
plants. Concentration of the cell sap, water potential of the individual plant organs, its
relation to soil and atmospheric drought are associated with water content.
Determination of water content in leaves makes it possible to clarify ecological and
physiological characteristics of plants, to reveal the mechanisms of their adaptation to
the environmental conditions.
Water content in plant tissues is usually calculated as a percentage of dry or
raw mass. In the leaves of the majority of temperate zone plants, depending on
weather conditions and stages of the ontogenesis, water contents is 65-82% of the
raw mass. The plants with unequal drought resistance differ in the nature of water
exchange. The plants of hygrophilous species and varieties contain a lot of water with
a sufficient amount of it in the soil. However, when the water content of the soil
decreases, they lose water quickly. In more drought-resistant forms, the water content
of plants is generally lower, but its amount is more stable.
Task: Calculate the water content as a percentage of raw and dry mass of the
material; make a conclusion on dependence of the water content in leaves on their
location on a plant.
Procedure. The amount of water and dry material in the leaves is measured by
the weight method. Experiment is done in two versions with the leaves of the upper
and lower tiers. Choose normally developed, green leaves without obvious traces of
damage and drying. Each measure of raw leaves of at least 5 grams is done three
times. Mark exactly, which leaves are considered to the lower tier and which to the
upper, follow the established order for all experimental plants.
Firstly, measure the weight of absolutely dry weighing bottle. For this put a
clean bottle with a cap vertically on the shelf of drying cabinet at a temperature of
100-150C. After 1 hour, take the bottle with crucible forceps and put in an opened
state into desiccator for 30 minutes for cooling, then close the cap and weigh on
analytical scales. Place the bottle again into drying cabinet for 20-30 minutes, cool in
desiccator and weigh repeatedly. If the weight of the bottle does not change, you can
put a sample in it.
Weigh the bottle with a plant sample on analytical scales, place it for 5 hours
into the cabinet heated until 105C, then cool in desiccator (the bottle must be
opened) and weigh again. However, to remove all the moisture from the plant within
5 hours can be not enough, therefore after weighing, open the bottle and place into
19
drying cabinet at the same temperature. Then weigh the bottle cooled in desiccator
again. Repeat the procedure until the mass of the bottle with the sample becomes
constant or a subsequent mass becomes slightly bigger than the previous one.
When working follow the rules. The raw material must lie in the bottle loosely.
Do not keep it in the cabinet longer than 5 hours. Put the bottle into cabinet at 105C.
The temperature in different parts of cabinet is unstable; therefore it is desirable to
place the bottles at the same level with thermometer. Do not place the bottle close to
the cabinet walls, since the temperature there can be higher than thermometer shows.
Take the bottle with forceps with rubber bands at the ends, because the weight can
change, if touched.
Subtract the mass of dried material from the mass of the initial plant material to
get the mass of water in the taken sample. Calculate the percentage of water content
in raw and dried materials; make an inference about the water content in leaves
depending on their location on the plant.
Record the results of the experiment into Table 5 by the form given.
Table 5 - Determination of water content in the leaves
Crop
Leaf tiers
Repeatability
Number of weighing bottle
Weight of weighing bottle, g
empty
with a raw material
with a dry material
Raw mass, g
Dry mass, g
Water content
in grams
% of the raw mass
% % of the dry mass
Materials and equipment: fifteen-days old sunflower or corn plants, analytical
balance, forceps, drying cabinet, weighing bottles, desiccator.
2.2 Determination of the stomata condition by an infiltration method
according to Molisch
The cause of stomatal motions may be the effect of light, changes in tissues,
temperature, and concentration of C2 in intercellular spaces. In conditions of
insufficient water supply, there occurs a hydroactive closure of the stomata.
Therefore, the degree of stomata openness can serve as a physiological indicator for
determining the water supply in plants and establishing the watering periods.
20
Intercellular spaces are usually filled with air, so when looking at a leaf in the light, it
is matt. In case of infiltration, i.e. if intercellular spaces are filled with some liquid,
the corresponding sections of the leaf become transparent.
Determination of the stomata condition by an infiltration method is based on
the ability of liquids, moistening cell walls, to penetrate capillarity through open
stomatal gaps to the nearest intercellular spaces and displacing air from them. It is
can be easily verified by the appearance of transparent spots on the leaf. Liquids
penetrate into stomatal slots depending on their width: petroleum ether - through
weakly open stomata, xylene - through medium open, and ethyl alcohol - only
through widely open ones.
Task: Examine the leaves kept in different conditions (fresh and wilted, lighted
and darkened, etc.). Examine 2-3 leaves per each variant. Write the results down into
the table, marking the penetration of liquid with the "+" sign, and the absence of
penetration with the "-" sign.
Procedure. Drop benzene, xylene and ethanol sequentially on the neighboring
sections of the lower surface of a leaf. Keep the leaf in horizontal position until the
drops disappear completely, which can either evaporate or penetrate inside the leaf.
Examine the leaf in transmitted light. If the liquid penetrated into the intercellular
spaces of the leaf, transparent spots will appear on it.
On the basis of the obtained data, make conclusion on the different degree of
stomata opening, keeping in mind, that they are slightly opened when infiltrated only
by xylene, by xylene and benzene - medium-opened, by xylene, benzene and alcohol
- strongly-opened.
Record the results of the experiment into Table 6 by the form given.
Table 6 - Determination of the degree of stomata opening
Object Terms of
experience
Benzene Xylene Alcohol Stomata
condition
Make conclusions about the influence of external conditions on stomatal movements.
Materials and equipment: droppers, ten-fifteen-days old sunflower plants, geranium.
Reagents: alcohol, benzene, xylene.
2.3 Determination of the osmotic pressure of cell sap by a plasmolytic
method
Osmosis is a diffusion of water or other solvent through a semipermeable
membrane. A plant cell can be considered as an osmotic system, in which the role of
an osmotically active substances solution is played by the cell sap, and the role of a
semipermeable membrane is the cytoplasmic membranes.
21
Cell sap is an aqueous solution of various organic and inorganic substances.
The potential osmotic pressure depends on the number of particles in this solution,
i.e. the concentration and dissociation degree of dissolved molecules. The potential
osmotic pressure expresses the maximum ability to absorb water. The value of this
indicator shows the possibility of a plant growth on the soils of different water-
holding strength. The increase in osmotic pressure of the cell sap in drought is a
criterion for dehydration and necessity to water the plant.
This method is based on the selection of such an external solution
concentration that causes initial (angular) plasmolysis in the cells of the tissue under
examination. In this case, the osmotic pressure of the solution is approximately equal
to the osmotic pressure of the cell sap. Such external solution is called isotonic.
Task: Determine the degree of the cell plasmolysis in each solution and find
the isotonic concentration. Determine the value of the cells' osmotic potential.
Procedure. Prepare 10 ml of solutions in weighing bottles according to the
table. Mix the solutions thoroughly. Close the bottles with caps to prevent
evaporation and put in a decreasing concentration sequence.
With the aid of the safety razor's blade cut thin sections from the convex
surface of the onion peel about 25 mm2 in size from a middle well-colored area.
Put 2-3 sections into each bottle, starting from the one with high concentration,
with an interval of 3 minutes. 30 minutes after immersion into the first bottle,
examine the sections under microscope. Then, after every 3 minutes, examine the
sections from the following bottles. This way is optimal to achieve an equal length of
staying of the sections in plasmonolytic solutions. The sections are examined under
microscope in a drop of solution from the bottle, from which they were taken.
Define the degree of cells plasmolysis in each solution and find isotonic
concentration as an arithmetic average between the concentration, at which
plasmolysis begins and the concentration that no longer causes plasmolysis.
Record the results of the experiment into Table 7 by the form given.
Table 7 - Determination of the potential osmotic pressure
Concentration
of a sucrose
solution,
mol/L
Length of staying of the
sections in solution
Degree of
plasmolysis
Isotonic
concentratio
n, mol/L
Potential
osmotic
pressure, kPa immersion
time
observatio
n time
0,7
0,6
0,5
0,4
0,3
0,2
The value of the potential osmotic pressure (in kPa) is calculated by the
formula 1:
22
P = R T c i 101.3 (1)
where R is the gas constant, 0.0821 l atm/deg mole;
T is the absolute temperature (273 + room temperature);
c is the isotonic concentration in moles;
i is the isotonic coefficient of Vant Hoff;
101.3 is a multiplier to transfer atmospheres into kilopascals.
The Van't Hoff coefficient characterizes the ionization of solutions and for
nonelectrolytes (sucrose) it equals to 1.
Materials and equipment: scalpel, razor blade, needle, microscope, slides and cover
slips; pencil on glass; filter paper, test tubes (weighing bottles).
Reagents: 1 sucrose solution.
2.4 Determination of the water potential of plant tissue by a method of
strips (according to Lilienstern)
The water potential () characterizes the absorbing power of the plant tissue.
The value of the water potential depends on the difference in the chemical potentials
of the water in the cell and in pure water. The water potential always has a negative
sign. The lower the water potential, the more dehydrated the plant cell is, so this
indicator is determined in order to catch the signs of plant dehydration in time and to
choose the right time for irrigation. Optimal values of the water potential are
established for specific cultures of different soils and climatic zones. This enables to
irrigate plants in optimal time, according to reference data.
This method is based on the selection of an external solution of such
concentration, that a strip of plant tissue does not change its length when immersed
into it.
If the osmotic potential of the external solution exceeds the water potential of
the tissue, the solution takes water from the cells, and, as a result, their volume and
the length of the strips decrease.
If the osmotic potential of the solution is less than the water potential of the
tissue, the cells, taking the water from the solution, increase in volume and the length
of the strip becomes larger.
In the solution, where the osmotic potential is equal to the water potential of
the tissue, the length of the strip does not change.
Task: Determine the value of the water potential in a potato tubers tissue.
Procedure. Prepare 0.6 M; 0.5 M; 0.4 M; 0.3 M; 0.2 M; 0.1 M of sucrose
solutions in test tubes of 10 ml. Cut a potato tuber into ten strips of 4-6 cm long and
of about 4 mm2 in cross section. Cut the ends of the strips obliquely. Perform quickly
to avoid the drying of the strips. Measure their length accurately with a millimeter
ruler and place two pieces into each tube. After 20 minutes, remove the strips, dry
them with filter paper and measure the length again. To calculate the quantity of the
23
water potential, take the concentration at which the length of the strips did not
change.
The quantity of the water potential () is calculated by the formula 2:
= - Psolution = - R T C i 101.3 (2)
Record the results of the experiment into Table 8 by the form given.
Table 8 - Determination of water potential
Materials and equipment: test tubes, pipettes, glass stick, potato, millimeter ruler,
filter paper, distilled water.
Reagents: 1 sucrose solution.
2.5 Determination of the water potential of leaves by Shardakovs method
The method is based on the determination of change in the concentration of the
solution after keeping the plant tissues in it. Shardakov's method is based on the
comparison of densities of the initial (control) solution with the same solution after
keeping the tissue in it. The solution's s, which has not changed its density, is q.
Task: Determine and calculate the water potential of tissues. Explain, in what
cases a drop of a colored solution will float, drown, or stay in the same place.
Procedure. Place the tubes into a desk set in two rows: five at the top and five
at the bottom. Prepare 10 ml of sucrose solutions of 0.5M; 0.4 M; 0.3 M; 0.2 M; 0.1
M in the upper row by diluting 1M of sucrose solution with distilled water.
Transfer 0.5 ml of the solution from the upper test tubes into the test tubes of
the lower row and close them with caps. Drill out 10 discs from a leaf. To do this,
turn the leaf with its underside upwards, put a rubber plate under it and drill discs
between the large veins. Plunge two discs into each tube of the lower row for 40
minutes. Shake the test tubes with the discs every 10 minutes. Then remove the discs
with the aid of a glass stick and color test solutions in the test tubes of the lower row
with a small amount of methylene blue (at the tip of a wire). Shake the contents to
Concentration
of sucrose,
length of the tissue strips, mm Concentration at
which the length
of the strips did
not change,
Water
potential, kPa before the
immersion
into a
solution
after the
immersion into a
solution
0.6
0.5
0.4
0.3
0.2
0.1
24
color the solution evenly. Take the colored test solution with the aid of a 0.5 ml
pipette. Plunge the end of the pipette into the corresponding initial solution into the
tubes of the upper row, the liquid level in the pipette should exceed the level of the
solution in the tube. Eject the liquid from the pipette slowly into the initial solution,
marking the movement direction of the squirt. If the concentration and, consequently,
the density of the colored solution increases in comparison with the initial one, the
squirt will go down, if the concentration decreases, the squirt will go up. In case of
equal concentrations, the squirt distributes evenly inside the tube with the initial
solution.
The quantity of the water potential is calculated by the formula 3:
= - Psolution = - R T i 101.3 (3)
Record the results of the experiment into Table 9 by the form given.
Table 9 - Determination of water potential
Concentration
of sucrose,
Movement
direction of the
squirt
Concentration of an
external solution remained
unchanged
Water potential,
kPa
0.5
0.4
0.3
0.2
0.1
Materials and equipment: test tubes, distilled water, pipettes, glass stick, plant
leaves, millimeter ruler, filter paper, distilled water, crystalline methylene blue.
Reagents: 1 sucrose solution.
2.6 Comparison in transpiration of the upper and lower sides of a leaf by a
chlorocobalt method
Stahl's cobalt chloride sample method is based on a filter paper's change of
color, moistened with cobalt chloride, when it absorbs water vapor, evaporated by the
surface of a leaf. The time needed for changing blue color (the color of a dry
chlorokobalt paper - Co12) to pink (the color - CCl26H2O) is a criterion for a plant
transpiration.
The chlorokobalt method of determining the transpiration of leaves, not
separated from the plant, is very simple and accessible. However, its use is limited
only by comparative experiments, since it does not allow determining the absolute
values of transpiration intensity. There are quantitative modifications of this method,
based on chlorokobalt paper weighing before and after a certain exposure of it on the
leaf, but they are inaccurate.
25
Task: Compare the stomatal and cuticular transpiration.
Procedure. Put discs from chlorokobalt paper on a celluloid substrate on the
top and bottom sides of a leaf and strengthen the substrate with the aid of a paper
clip. Observe after how many minutes the paper on the top and bottom sides of the
leaf will turn pink. According to the coloring speed, define from which side of the
leaf evaporation goes faster.
At the end of the experiment, examine the epidermis of the top and bottom
sides of the leaf under microscope and count the number of visible stomata. For this,
look through three-five magnifications on three materials of each variant and
calculate an average arithmetic value.
Sketch the epidermis of the top and bottom sides of the leaf. Make conclusions
on the causes of different intensity rate of evaporation from the sides of the leaf of the
plant and on the correlation between stomatal and cuticular transpiration.
Record the results of the experiment into Table 10 by the form given.
Table 10 - Comparison in transpiration of the upper and lower sides of a leaf
Side of
the leaf
Observation period The time
during which
the paper will
turn pink,
min
Number of stomata in the
microscope field of view
Beginning of
the
experiment
End of the
experiment
Individual
calculations
Average
arithmetic value
Materials and equipment: three-weeks old bean plants. Discs from chlorokobalt
paper on a celluloid substrate, paper clips, watch, microscopes, slides and cover slips,
tweezers, droppers with water, safety razor blade, needles.
Preparation of chlorokobalt paper. Take a uniformly thick filter paper or thin
filter strips and soak it (them) in a cuvette, filled with a solution of cobalt chloride
prepared according to Kamerlingh's method (dissolve 6.7 g of Co(NO3)2 and 2.64 g of
NaCl in 100 ml of water) for 1 minute, then dry in a suspended state on glass sticks
until a blue color appears. From the paper cut out the circles with 1 cm in diameter
and with the aid of a polyethylene tape with a sticky layer glue two circles per a
celluloid substrate.
Celluloid chambers with chlorokobalt paper are stored in the desiccator above
calcium chloride.
2.7 Determination of the rate of transpiration in cut leaves by torsion
scales
The work of the upper-end engine is connected with water evaporation from
the leaves surface - transpiration. An absorbing effect of transpiration is given to the
roots in the form of hydrodynamic tension, which connects the work of both engines.
26
The work of the upper-end engine, based on the use of solar radiation as an energy
source, is automatically regulated (the loss of water reduces the water potential of the
evaporating cells, which leads to increased water intake). The plants with many
leaves have a greater absorbing force of transpiration than the root pressure force.
The amount of water evaporated by a plant from the leaf surface per a unit of
time is called transpiration intensity.
In plants the main role in regulating water evaporation is played by the
stomata. Therefore, the intensity of transpiration largely depends on the degree of
their openness. In addition, a plant can reduce transpiration, reducing the evaporation
of water from the cell surface to the intercellular spaces by increasing the water-
holding capacity of protoplasm and cell walls.
The method is based on taking into account the changes in the weight of the cut
transpiring leaf in short time intervals, which makes it possible to observe
transpiration in the water saturation state of the leaf growing on the plant.
An interval between the weighings should not exceed 5 minutes, because with a
longer exposure the water content in leaves decreases and the transpiration rate
decreases too. For quick weighing it is convenient to use torsion scales.
Task: Determine the intensity of transpiration by the weight method.
Procedure. Install the torsion balance strictly horizontally with the aid of two
screws on the balance stand. Check the zero point, set the mass indicator with the
tension lever in position 0, empty the beam of the balance by moving the fastening
lever to the left.
Then proceed weighing. Hang another hook on the hook of the beam, which is
located on the side of the balance in a closed chamber and weigh its mass. For this,
empty the beam of the balance by moving the fastening lever to the right. Turn the
mass indicator with the tension lever to the left until the balance pointer coincides
with the balance line. In this position, the mass indicator shows the weight of the load
on the scale. Turn the fastening lever to the left, the arrow shows "closed" and return
the mass indicator to zero on the scale.
Then calculate the intensity rate of transpiration. Cut a leaf, put it on the hook
and hang on the balance beam. Weigh quickly and place the leaf on the needle. In the
same way weigh the leaves of the same tier from ten plants. 5 minutes after weighing
the first leaf, reweigh all the leaves in the initial order.
Calculate the weight of the leaves by subtracting the hook mass from the
indicators of the scale. The loss in weight of the leaves during the time between the
first and second weighings shows how much water evaporated during the period.
Provide all calculations on the total weight of ten leaves of each variant.
Calculate the amount of water, evaporated from 1g of raw leaves per 1 hour.
Determine the intensity rate of transpiration in the room conditions (under control)
and in dry warm wind (use a hair dryer).
Record the results of the experiment into Table 11 by the form given.
27
Table 11 - Determination of the rate of transpiration in cut leaves
Variant Weight of the
leaves, mg
Total weight of
10 leaves, mg
Loss in water
of 10 leaves,
mg
Rate of
transpiration,
g/(2h)
Control
Dry warm
wind
Materials and equipment: ten-days old oats or wheat sprouts, torsion balance, hair
dryer, scissors, hanging leaves stands.
2.8 Determination of the rate of transpiration and relative transpiration
by technical scales
Intensity of transpiration is the amount of water, evaporated from the leaf
surface per a unit of time. The value depends on the intensity of external factors, the
time of day and ranges from 15-250 g/m2 h.
The main method for determining the intensity of transpiration is a weighting
method, based on taking into account the loss of water during evaporation. This
method can reveal the transpiration of an entire plant or its individual parts. The work
with whole rooted plants has considerable difficulties, therefore cut shoots or leaves
are often used. To ensure that during the experiment, the water content of the tissues
does not decrease, the samples are placed into the Veska device filled with water.
Relative transpiration is the ratio of the transpiration intensity to the
evaporation rate from a free water surface under the same conditions. This indicator
characterizes the ability of plants to regulate transpiration and is usually expressed in
the figures 0.1-0.5 rising sometimes to 1 and dropping in some well-protected from
the water loss leaves to 0.01 and lower.
Task: Determine the intensity of transpiration and relative transpiration by the
weight method.
Procedure. Cut a leaf together with a petiole from a sunflower plant. Fasten
the petiole is tightly with fleece in the hole of rubber stopper. Cut the lower end of
the petiole diagonally underwater to about 1 cm to restore the water strands in the
conducting vessels. Put the stopper with the leaf into the Veska device filled with
room temperature water, so that the leaf petiole is immersed in water. The Veska
device must be completely dry, tightly closed: the stopper must touch water, and the
leaf petiole must be immersed in water.
In this way, prepare two Veska devices, weigh them on technical scale and,
labeled, place one in a dark chamber, the other one in direct light. After an hour,
weigh again. By the difference with the initial mass, determine the amount of water
that the leaf evaporated during the experiment.
28
According to on the results, calculate the intensity rate of transpiration, i.e. the
amount of water in grams, which a unit of leaf surface evaporates (1 m2/) per time (1
hour).
To make such calculation, you need to know the area of the leaf taken for the
experiment. You can use the weight method. Cut a area of 100 cm2 (10x10 cm) from
paper and weigh. On another sheet of the same paper put the examined leaf, outline
its contour carefully with a sharply sharpened pencil, cut it and weigh also. From the
data obtained make up the proportion and find the area of the leaf. If the paper area of
100 cm2 has a mass g, and the contour of the leaf of unknown area - g, then the
required area of the leaf is found in the following way:
S =
*100 (4)
The intensity rate of transpiration (g/m2 h) is calculated according to formula 5:
I = tS
*
*10000 (5)
where C is the loss in mass during the experiment, g,
S is the area of the leaf, cm2,
t is the duration of the experiment, h.
Simultaneously, in the same conditions calculate the evaporation from free
water surface. For this, take into account the amount of water evaporated within 1h
from the surface of the Petri dish. With the inner diameter, calculate its area by the
formula:
S = r2
(6)
Calculate the intensity rate of evaporation from free water surface, using
formula 5, and calculate the relative transpiration :
rel = E
I m (7)
Compare the obtained data and make conclusions about the dependence of the
intensity rate of transpiration and relative transpiration on lighting conditions and on
the plants' ability to regulate transpiration.
Record the results of the experiment into Table 12 by the form given.
Table 12 - Determination of the rate of transpiration and relative transpiration
Transpiration
Mass of the device with the leaf, g
at the beginning of the experiment
at the end of the experiment
29
Loss in mass, g
Area of the leaf, sm2
Duration of the experiment, h
Rate of transpiration (g/m2 h)
Continuation of table 12
Evaporation
Mass of the Petri dish with water, g
at the beginning of the experiment
at the end of the experiment
Loss in mass, g
Area of the evaporating surface, sm2
Duration of the experiment, h
Rate of evaporation (g/m2 h)
Relative transpiration
Materials and equipment: technical scales, three-weeks old sunflower plants,
geranium, scissors, scalpel, bottom half of a Petri dish, millimeter paper.
2.9 Determination of the water-retaining capacity of plants by a "wilting"
method according to A. Arland
In plants regulation, a significant role belongs to their water-holding forces,
connected mainly to the content of osmotically active substances in the cells and the
ability of colloids to swell.
The water-holding capacity of cells depends on the conditions of growing
plants. In particular, the nutrition conditions are of great importance. Under optimum
conditions, the water-holding capacity increases. The determination of water-holding
capacity according to Arland is based on taking into account the loss of water by the
fading plants.
Task: Determine the water-holding ability of tradescantia and geranium, make
a conclusion on the water-holding capacity of these plants.
Procedure. Take fifteen-days old oat plants grown in sand with fertilization
(experiment) and without fertilizers (control). Take out carefully from the sand 20
plants of each variant and separate their upper part from the roots. Then cover a part
of the stem, which was in the sand, covered with paraffin to exclude its participation
in water evaporation. For this, plunge the lower etiolated parts of the stem into the
expanded paraffin, colored with Sudan III, with a temperature not higher than 50C.
Weigh all the plants together on technical scale. Repeat the weighing after 30
minutes, 1h, 1h 30 minutes, 2 hours. The loss in mass shows the absolute amount of
water, that the test plants lose in 30 minutes. Using the obtained data, calculate the
amount of evaporated water and the percentage to the initial weight of the evaporated
mass within 30 minutes; 1; 1,5; 2 hours. Represent graphically the dynamics of water
30
loss, make a conclusion about the water-holding capacity of plants of different
species.
Record the results of the experiment into Table 13 by the form given.
Table 13 - Determination of the water-retaining capacity of plants
Object Weight of
plants, g
Amount of evaporated
water, g
Loss of water to the initial
weight, %
initial
after 30 min
after 1 hour
after 1 hour 30
min
after 2 hour
Materials and equipment: fifteen-days old oats or wheat plants, paraffin colored
with Sudan III, a test tube racks, technical scales, scissors, and water bath.
2.10 Determination of the productivity of transpiration and the
transpiration coefficient
In the cultivation of crops, the efficiency of water use by plants is of great
importance, and the index of it is a transpiration coefficient. The value of the
transpiration coefficient is influenced by the conditions of mineral nutrition, water
availability, light intensity and other factors. The degree of water use by a plant can
be increased, creating optimal conditions of water supply and nutrition for it.
Regularities of water exchange of plants are important to consider when developing
agrotechnical techniques, aimed at obtaining high yields.
To characterize the water exchange of plants in a full state, it is necessary to
know the indicators of the effectiveness of water consumption: the productivity of
transpiration, i.e., the amount of dry matter (g) formed when 1 l of water evaporates,
and the reciprocal value - the transpiration coefficient.
When 1 g of dry matter is formed, in average, 300-500 g of water is consumed.
Millet cereals have lower values of the transpiration coefficient, while flax and
perennial grasses have higher values. The conditions for growing plants have a
significant effect on the efficiency of the water use: the better the conditions for
mineral nutrition and the water supply of plants, the higher the yield and the less
water consumption is done for a unit of mass creation.
Water productivity indicators are usually determined during the vegetation
period. However, one should remember, that during ontogeny they change. Thus,
spring wheat during shooting has a greater transpiration coefficient, then it decreases
and reaches a minimum in the end of tillering, increases again during forming a tube,
reaches a maximum in the phases of earing and flowering, and then decreases.
Task: Determine the indicators of the effectiveness of water consumption by
31
wheat in the phases of tillering and earing.
Procedure. For work in a sand culture at 0.5 norm of the Hoagland-Snyders
nutrient mixture grow three and five-weeks old spring wheat plants. Pick six vessels
with equalized plants of each planting period.
From the three vessels of each variant, carefully remove the plants, wash the
roots from the sand, dry with filter paper and weigh the initial mass of the air-dry
material in each vessel in separately. For this, ground the plants and place them in
open boxes from tracing paper into a drying cabinet preheated to 105C; at this
temperature there occurs a complete inactivation of all enzymes, which prevents
subsequent changes in the dry mass. Then dry the material in air or in drying cabinet
at 60C and weigh on technical scales with precision to the second sign.
Number the remaining three vessels of each option, water through a drain pipe
until the constant mass (60% MC of humidity) and within a week mark the amount of
water consumed by the plants.
Correct account is possible only if the evaporation of moisture by the root-
inhabited part is excluded. For this, pour molten (not hot!) paraffin the sand surface,
which after hardening makes a waterproof layer. You can replace paraffin with a
layer of non-absorbent cotton wool or mulch the surface with the aid of a non-
hygroscopic granular foam. After 1 week, weigh the air-dry mass of the plants in
each vessel. Carry out the work in the same sequence as in the first observation.
Based on the data on the water amount consumed by the plants in each vessel
and the accumulated dry matter in this period, calculate the productivity of
transpiration and the transpiration coeffect. Compare the results of calculations
according to the variants.
Record the results of the experiment into Table 14 by the form given.
Table 14 - Determination of the indicators of the effectiveness of water consumption
Plant
age,
week.
Initial
air-dry
mass, g
Water amount
consumed in
1 week, g
Accumula
ted air-dry
mass, g
Transpiration
coefficient
Productivity
of
transpiration
Materials and equipment: three- and five-weeks old wheat plants are grown in liter
culture vessels in sand culture. Technical scales, drying cabinet, crystallizers, tracing
paper, filter paper, beakers.
32
3 PHOTOSYNTHESIS
Information material. Photosynthesis is a process, in the result of which the
light energy absorbed by an autotrophic organism is transformed into the chemical
energy of biogenic plant compounds. In this case, the light energy is mainly used to
initiate the 2 reduction reactions before the formation of various monosaccharides.
In the process of photosynthesis, other compounds can be reduced to form sulfates,
nitrates, hydrogen. In addition, the energy of light is expended on the transportation
of substances through membranes and on the processes of biosynthesis.
In general, photosynthesis is an oxidation-reduction process of 2 and 2O
interaction, which occurs with the participation of chlorophyll, carrying the light
energy.
Photosynthetic reactions occur in chloroplasts. Chloroplasts of higher plants
usually have a spherical or a disk-shaped form (1-10 microns). In the cells of higher
plants, there are several dozens of chloroplasts, the total total surface of which can
exceed the area of leaves in dozens or hundreds of times. Outside, chloroplasts are
surrounded by a continuous protein-lipid membrane, consisting of a two-layer
membrane (inner and outer membranes). The inner part of chloroplast is represented
by lamellae, which are immersed in a stroma. Most of the lamellae are densely
packed into individual granules, laid across chloroplast. Each grainule is a pile of
discs, which are called thylakoids in granules. Thylakoids have a membrane
structure.
The granules contains a bulk of chlorophylls, all pigments of photosynthesis, as
well as functional proteins, lipids and enzymes. The coupled membranes of thylakoid
granules serve to trap the light quanta.
Plant chloroplasts have their own DNA (chlDNA), which is a closed circular
double-stranded molecule, formed from sequentially bound nucleotides. The structure
of the primary DNA structure can have 1.3 105 up to 1.6 105 pairs of nitrogenous
bases. At present time, the primary structure of the tobacco and rice DNAs has been
studied. 130 genes display their selectivity in the structure of chlDNA.
The DNA of chloroplasts contains the information about approximately 40
proteins of the thylakoid membrane, involved into photosynthetic activity of
chloroplasts, which is about a half of the total protein composition of thylakoids. It
should be noted that there is a gene in chlDNA, containing the information on a large
subunit of ribulose-1.5-diphosphate carboxylase. The enzyme catalyzes the limiting
step in the Calvin cycle.
The study of photosynthesis made it possible to identify the presence of two
consecutive stages that occur in the presence and the absence of light. Under the
influence of the light, photolysis reactions of water take place, which occur with the
participation of pigments and catalytic proteins of photosynthetic systems. As a result
of the light stage of photosynthesis, NADPH is synthesized, the reaction is catalyzed
by the ferredoxin-NADP+-reductase enzyme.
Thus, photosynthesis is a collection of physico-chemical processes initiated by
the light, which results in water splitting process, ensuring the reactions of high-
33
energy molecules synthesis (ATP, GTP, CTP, etc.) and the reduced form of NADP+,
used in the further dark reactions of photosynthesis. At the same time, adenosine
triphosphoric acid is consumed in biological reactions, determining the direction of
the biological processes course, participating in the synthesis of various biological
molecules, while the reduced nicotinamide adenine dinucleotide phosphate is used in
the reactions of the Calvin cycle.
The light reactions of photosynthesis occur in thylakoids, in the membranes of
which the components of photosynthetic systems are located. These are, first of all,
light-gathering pigments and functional proteins, as well as electron-transport
complexes, a NADPH-synthesizing enzyme and an ATP-synthesizing complex. The
latter carries out the phosphorylation of ADP with the formation of ATP.
The dark stages of photosynthesis take place in the stroma of chloroplasts and
represent a set of biochemical processes that result in the reduction of 2 to
carbohydrates.
Methodological recommendations on the studying the topic. While
studying this topic, it is necessary to understand, that photosynthesis is the basis of
bioenergy on the globe, to determine the nature of the main photosynthetic reactions,
its physico-chemical essence, the energy balance components, the distribution of
absorbed light to the pigments of green cell, the stages of ideas development about
the process of photosynthesis.
It is also necessary to study the primary processes of photosynthesis in 3 -, 4-
and CAM-plants, the ways of energy migration, the structure and the functions of the
electron-transport chain of photosynthesis, quantum flow and quantum yield, cyclic
and noncyclic photophosphorylation, the main regularities of the electron-transport
chain functioning in connection with the energy exchange reactions.
It is necessary to pay attention to the systems of photosynthetic regulation. It is
necessary to study the intensity of photosynthesis, the methods of its determination,
the dependence of this indicator on illumination and spectral composition of the light,
the influence of external and internal factors on the intensity of photosynthesis, the
compensation points, the interaction of factors and the regulatory role of
photosynthesis. To note possible ways of increasing the photosynthetic activity in
crops.
It is necessary to know about the international biological program
"Photosynth
