1. Micronutrients: physiological role for the plants, visual
symptoms of deficiency and a possibility to prevent or
correct plant mineral status
Topic 4

2. 2
Addition from the previous lecture: “Evaluation of mineral disorders of
plants by visual observation and measurement of photosynthetic
parameters with portable devices”
Chlorophyll fluorometer Chlorophyll meter Portable photosynthesis system

3. 1. Essential microelements in plants. Optimal leaf concentrations of
micronutrients in important crops
Micronutrients (mg/kg)
Fe 300 20 – 600
Cl 100 10 – 100
B 20 5 – 100
Mn 40 10 – 150
Zn 30 10 – 70
Cu 6 6 – 20
Мо 0.1 0.1 – 1.0
Crop and phase B Mo Cu Mn Zn
Wheat (tillering) 6-12 0.1-0.3 7-15 40-150 25-70
Maize (5-6th leaf) 7-15 0.2-0.5 7-15 40-150 30-70
Beans (flowering) 40-80 0.4-1.0 7-15 40-150 30-70
Sunflower (flowering) 35-100 0.3-1.0 10-20 25-100 30-80
Potato (flowering) 25-70 0.2-0.5 7-15 40-200 20-80
40-80 0.3-1.0 6-12 40-150 30-80
Cucumber (flowering) 25-40 0.5-1.0 6-10 30-50 30-80
Pepper (first fruits) 40-80 0.2-0.6 8-15 30-150 20-60
Table 1. Optimal leaf concentrations of micronutrients
(mg/kg=ppm in different crops according to Bergman (W.
Bergmann (1996)
Concentrations
(average and range)
Tomato (first fruits)

4. New leaves –
low mobile or
immobile МЕ
Old leaves –
mobile elements
1. Mobility, reutilization and physiological role of micronutrients in
the plants
Mobility of mineral nutrients
Mobile Low mobility Immobile
Chlorine (Cl) Iron (Fe) Boron (B)*
Manganese (Mn)
Copper (Cu)
Zinc (Zn)
Molybdenum (Mo)
* B mobility is species-specific! B can transport with
polyols in the phloem of the trees
Microelements (Fe, Mn, Cu, Zn and Mo) have relatively low mobility in the plants, with one
exception - (Cl). Specific case is B, which in most of the plants is almost immobile, but in some
tree species has mobility in complexed form with polyols. As usual, the micronutrients have
affinity to form complexes with organics.

5.  Fe is involved in different biological processes in the form of heme or as a Fe-S clusters.
Fe-S clusters
1.1. Micronutrients: Iron (Fe)
Fe in heme form
 Heme is a cyclic structure acting as co-factor in biological systems. Fe in the heme form is
included in the catalytic centers of redox enzymes due to Fe ability to change the valence
from oxidized (Fe3+) to reduced (Fe2+) states and vice versa.
 Fe in the Fe-S cluster is involved in the protein ferredoxin in chloroplasts as well as in the
catalytic centers of many enzymes, such as SOD, glutamate synthase and others.

6.  Briefly, Fe takes part in many important physiological processes. It is: (1) a part of
antioxidant enzymes in plants (eg peroxidase); participates in (2) the prosthetic groups
of enzymes, involved in electronic transport in chloroplasts and mitochondria,
(3) enzymes, related to nitrogen metabolism (nitrate reductase, nitrogenase) as well as
in (4) the biosynthesis of chlorophyll and others.
Iron
Nitrite reductase
NAD(Р)Н+Н+
NAD(Р)+
Nitrate reductase
FAD
FADH2
 Fe in a heme form participates in
the process of nitrate reduction. The
process is located in both cytoplasm
and chloroplasts. Donor of electrons
is NADPH.
 Fe in a cluster form participates in
the prosthetic group of the enzyme
nitrite reductase, which transforms
nitrites to ammonium ions. Donor of
electrons are reduced ferredoxin.

7. Protons diffuse to the site of ATP synthase
Dashed lines represent electron transfer Solid lines represent proton movement
About 60% of leaf Fe is located in the chloroplasts. Each photosystem (PSII and PSI) contains
minimum 20 Fe atoms. Here, Fe is included in the protein ferredoxin, which participates in the
photosynthetic electron transport in the chloroplasts.
Iron

8. Iron
 Summing up, the Fe deficiency may disturb photosynthesis, nitrogen assimilation,
redox homeostasis and many other processes.
 Point out that the biggest reason for plant Fe deficiency is the low content of mobile
Fe form in many soils due to mainly high pH, high carbonate content, etc.
 Plants have 2 possibilities to absorb Fe from the soil: strategy I for all plants without
cereals, which represents an acidification of the root environment and reduction of ferri
(Fe3+) to ferro (Fe2+) in the roots and strategy II - absorption through release of natural
chelates (phytosiderophores).
Strategy I (Fe-reduction in the roots) Strategy II (Fe-chelation)

9. Iron
 Fe deficiency strongly reduces chlorophyll biosynthesis.
 There are about 20 reactions involved in chlorophyll
biosynthesis. One of the first is the synthesis of the co-called
aminolevulinic acid. Fe deficiency retards this reaction as well
as formation of protoporphyrins .
 Fe deficiency induces a drastic reduction of thylakoid number
in the chloroplasts – instead of 10-15 they can be 2 or 3.
 It is important to mention, that in some cases Fe content in the
leaves can be is sufficient, but the bigger part of it to be located
in the appoplast, where it is inactive. In this case, both normal
and Fe-deficient plants may have similar Fe content, but will
have different plant performance.

10. Fe deficiency manifests itself in the form of interveinal chlorosis in the young leaves,
because it is not reutilized. Fe deficiency inhibits chlorophyll synthesis, formation of the
main components of phosynthetic apparatus and many other reactions in plants. The final
result is poor photosynthetic activity, limited growth of plants, fall of flowers, low yields.
- Fe
Iron deficiency

11.  The roots absorb Cu as cation - Cu2+. Usually, it is transported by xylem and phloem
systems in complexed form with different organic compounds.
 More than 50% of leaf Cu is located in the chloroplasts; in the electron carrier
plastocyanin in ETC). The main function of Cu in the plants is to participates in different
redox reactions, which is due to very high ability of Cu to change its valence
(Cu2+ Cu+).
 There are several important proteins, the
activity of which is due to presence of Cu:
(1) Proteins, participating in electron transfer
reactions (plastocyanyn).
(2) Proteins with peroxidase activity, participating
in monophenol oxidation to diphenols as well as
proteins with oxidase activity (ascorbate oxidase).
1.2. Micronutrients: Copper (Cu)
 Having in mind the physiological role of Cu, it is reasonable to expect that its
deficiency will produce disorders in photosynthesis, redox balance of the cells as well
as in the secondary metabolism.

12. Cu deficiency induces leaf tips dying, which can be due to oxidative stress (“burst”). In
cereals, the most often symptoms are retarded plant growth, together with twisting and drying
of the leaf tips. The symptoms usually are shown on acidic and sandy soils. Sensitive to Cu
deficiency crops are wheat, barley, sunflower.
Copper deficiency

13. Another typical symptom of Cu deficiency is lodging of cereal crops.
Copper deficiency
It is due to insufficient lignification of cell walls. It is known that polyphenol oxidase is a
Cu-containing enzyme, involved in lignification of cell walls. Lignification hardens cell walls
and makes them stronger. This process is extremely important for proper xylem formation.
Copper deficiency, even in small degree, reduces polyphenol oxidase activity and reduces
lignification of the xylem, which often results in lodging or breaking of cereal stems.

14. With severe Cu deficiency, the generative processes in the plants are also disrupted. The
most sensitive is the process of microsporogenesis - the formation of pollen. In this case,
incomplete grain spikes are formed, often completely “white” ears with a small number of
grains.
Copper deficiency
Cu deficiency

15.  The plants absorb manganese (Mn) as Mn2+ cation. Manganese has similar to iron (Fe)
and copper (Cu) physical-chemical characteristics. The most important among them is its
variable valence, allowing it to participate in redox reactions. The dominant form in the
plants is Mn2+, but it can be in the forms of Mn4+ and Mn3+.
1.3. Micronutrients: Manganese (Mn)
 Mn2+ is cofactor of more than 30 enzymes, including several enzymes of the Krebs cycle,
therefore Mn-induced deficiency decreases of cell respiration, which diminishes nitrate
reduction and many other reactions.
 The average content of Mn in plants is about 40 ppm.
Usually, its content in the roots is higher than in the
shoots. It was established that up to 40% of Mn is located
in the cell walls. The highest Mn content is in the
chloroplasts. The most important Mn functions are the
following:

16. НАДФ+
АДФ
АТФ
НАДФН
Цикъл
на
Калвин
 Manganese participates in the photolysis of water, a process involved in
photosynthesis. Mn deficiency can disturb photosynthesis.
Мn
The donor of electrons for the
photosynthetic electron trans-
port is water, which is split
during photolysis in OEC
(oxygen evolving complex) in
PS2.
OEC contains 4 Mn, 1 Ca and
1 Cl as cofactors. In fact, this
is light-driven process including
four steps and leading to
production of protons, electrons
and molecular oxygen.

17. 4e–
4 Photons
2 H+
2 NADP+
2 NADPH
Lower
Higher
Photosystem I
Ferredoxin
+
4e–
4 Photons
4e–
Photosystem II
4 H+
PQ
PC
P700
ATP
produced via
proton-motive force
Cytochrome
complex
Pheophytin
P680
+ O2
2 H2O
 Mn is involved in the catalytic center of one of the isoforms of superoxide
dismutase (Mn-SOD).
When electron transport within ETC is disturbed by any kind of stress factors, the levels of
active oxygen forms increase leading to oxidation of important molecules – proteins,
nucleic acids, etc. One of the enzymes involved in oxidative radicals quenching is the
enzyme Mn-SOD. Mn2+ + O2
.- + 2H+ Mn3+ + H2O2
O2 •O2
–
H2O2
Mn-SOD (superoxide dismutase

18. Manganese deficiency
Mn deficiency leads to chlorosis, which is similar to iron chlorosis as well as magnesium
deficiency, because the veins remain green. In cereals, curled leaves are observed.

19.  Zn plays an important role in nucleic acid metabolism. It
participates in the regulation of gene expression. It forms
the so-called “Zn fingers“ - small protein structural
motifs containing 10-15 amino acids. These motivs
interact with DNA, allowing it to activate or inhibit
transcription of specific genes.
 Zn stabilizes and activates over 100 enzymes. It does
not participate directly in redox processes, but forms
complexes with O, S and N, through which it performs a
catalytic and structural role.
The concentrations of Zn in the plants are in the range (10-80 ppm) and are usually 10 times
more than Cu and 10 times less than Fe. Zinc is not a transition metal. It plays an important
role in the metabolism of nucleic acids, the synthesis of amino acids and proteins, as well as
cell division.
1.4. Micronutrients: Zinc (Zn)

20. Maize plants often suffer from Zn deficiency. Visually,
Zn-deficient C-4 plants, in addition to weaker growth,
show specific chlorosis. It begins at the base of the leaves
around the vascular tissues (veins). This is due to the
destruction of chlorophyll in the bundle sheath cells.
Later, whole spots of dying leaf tissues appear.
In the maize (C-4 type of plant), photosynthesis absorbs
not CO2 but HCO3
- (bicarbonate anion), which is
obtained by hydration of CO2. This reaction is catalyzed
by carbonic anhydrase (CA), which is a Zn-containing
enzyme (Zn concentration in the enzyme 0.3%). Under Zn
deficiency, the activity of CA and photosynthesis
decreases.
Role of Zn and Zn deficiency in maize

21. Zinc deficiency
Zn deficiency is characteristic mainly on carbonate-rich soils. It manifests itself in the form of
various chloroses, light brown or whitening necrotic spots, etc. on the leaves of the plants.
The plants, produced from Zn-deficient seeds, are with slow growth. The leaf of Zn-deficient
trees are smaller as compared to normal ones.
Severe Zn deficiency
in barley
Severe Zn deficiency
in plums

22. Boron is a microelement with different functions in plants. It is taken from plants as boric
acid B(OH)3. B in dicotyledonous plants is in higher concentrations than other
microelements. Much of B content in cells is localized in the cell walls. In healthy plants, the
B content is highest at the growing points.
 One of the functions of B is to
increase the cell wall elasticity. This is
due to its ability to form complexes
with hydrocarbons, which control the
direction of the cellulose fibrils
located in the walls. If the plants are
deficient in B, the walls become less
elastic, cells stretch slightly, and stems
often break (example sunflower).
Sucrose is moving faster
in a complex with B.
1.5. Micronutrients: Boron (B)
Another function of B is to accelerate the flowering of plants due to a
better supply of flowers with sucrose. Sucrose in a complex form with
B moves faster along the phloem.

23. Boron (B)  The lack of B delays the germination of pollen grains and
causes a lack of pollination and fertilization. For example,
B-deficient sunflower head contains empty of seeds areas.
 B deficiency disturbs the phloem transport due to
coagulation of the cytoplasm in the sieve tubes as well as
deformation of xylem vessels. All this leads to the death of
growth points, chlorotic spots and necrotic points on the
leaves, "hollow stem" symptom in cabbage and others.

24. NAD(P)Н+Н+
NAD(Р)+
Nitrate reductase
FAD
FADH2
 Molybdenum is the element with the lowest concentration in plants from the group of
microelements. Its average content in plants is less than 1 ppm, but may vary
depending on the crop, phase, organ and soil. In some legumes crops, the Mo content
can reach 5 and even more ppm.
 Molybdenum has a regulatory role in plants due to its participation in enzymes
associated with nitrogen metabolism - nitrate reductase and nitrogenase. The Mo itself
has no catalytic function, but in biological systems it forms molybdo-cofactors. In
these cofactors, it is in bound form with pterin.
1.6. Micronutrients: Molybdenum (Mo)

25. Nitrogenase is an enzyme, through which legumes do symbiotic nitrogen fixation with bacteria
from the genus Rhizobium. The process occurs primarily in root nodules.
Molybdenum (Mo)
This enzyme consists of 2 components, Mo participates as a cofactor in one of them. The process
requires a significant amount of energy (30-40 molecules of ATP per 1 molecule of fixed N2).
Symbiotic nitrogen fixation uses solar energy to reduce the inert N2 gas to ammonia at normal
temperature and pressure, while industrial production needs both high temperature and pressure.

26. Molybdenum deficiency
Cauliflower
Mo deficiency occurs on acidic soils, which is a specific feature, different from the other
elements, which are more mobile in acidic than alkaline environments. Mo deficiency is more
pronounced on old leaves in the form of chlorosis, some of which subsequently turns into
necrosis. Legumes and vegetables are the most sensitive to Mo deficiency. In cauliflower, the
leaf surface is greatly reduced under Mo deficiency.

28. Foliar fertilization is now widely accepted as a supplement to soil
fertilization. It is an important tool for the sustainable management of
crops, and is of significant commercial importance worldwide. The
rationale for the use of foliar fertilizers is well motivated:
 when soil conditions limit availability of soil applied nutrients;
 in conditions when high loss rates of soil applied nutrients may
occur;
 when the internal plant demand and the environmental conditions
may limit delivery of nutrients to critical plant organs.
Foliar
fertilizer
The foliar fertilization can be applied before appearance of visual mineral deficiencies as
well as after that. In the first case, it may prevent physiological disorders and in the second
case, it may decrease the plant disorders.
Foliar fertilization as a means to prevent or to correct deficiency of
micronutrients in the plants

29. Now, modern plant nutrition technologies use not only
"pure" foliar fertilizers, but also a number of fertilizing
substances which are able to expand their physiological
action. They are part of a new group of products called
plant biostimulants.
Plant biostimulants are fertilizing products that, when
applied to seeds, rhizosphere or vegetative plant part, can
increase: (1) both nutrient availability and efficiency of
mineral nutrition;(2) plant tolerance to abiotic stress and
(3) quality of plant production.
.
1. Foliar products, supplying mineral nutrients to plants
Plant biostimulants can have different origin – they may be protein hydrolysates, humic and
fulvic acids, seaweed extracts, microbial and combined products, etc. They can be applied in
different phases of plant development.
Foliar fertilizers and plant biostimulants

30. 2. Biological aspects of foliar feeding of plants by nutrients
There are many biological an technological questions, which have to considered when
someone would like to apply foliar fertilization. Point out to several of the them.
How exactly are minerals and organic matter (amino acids, carbohydrates, etc.)
absorbed - through the stomata, cuticle or both?
So, only stomata entrance could not be sufficient for fast uptake of ions, but uptake through
cuticle depends on its state – swollen or compressed. When it is swollen, the water pore size
in the cuticle is about 0.40-0.50 nm, which is close to the size of large hydrated ions - K+ and
PO4
3-.
The stomata cells are about 10 µm
wide. They are a big entrance for
nutrients, but stomatal area is just
1-2% of leaf lamina.
The pores in the swollen cuticle are
about 1 nm or less wide, which is
about 1000 times less than the
stomata pores, but at the same time
they are million times more in
number.

31. Mineral ions in hydrated form have size ranging
from 0.5 to 1 nm, which is comparable with
several organics, which easily are taken by
plants. For example:
Sucrose has 1 nm size and molecular weight =
342, Glucose has 0.6 nm size and usually pass
easily trough the leaves.
Free amino acids have similar or even less
molecular weight (75 to 204). They can pass
through the water pores in the cuticle.
Small peptide
К+
Free amino acid
Hydrated potassium ion
The question is what is the
case with small peptides. How
they pass? – use of pinocytosis
as well as endophytic
microorganisms.
2. Biological aspects of foliar feeding of plants by nutrients

32.  It has been proven that the mineral elements
applied on the leaves can be transported along
the phloem to flowers, grains, fruits and other
organs and parts, as well as to the roots.
 Imported nutrients and organic substances
activate the photosynthetic activity of the
leaves.
 Foliar fertilizers increases the outflow of
photoassimilates to the roots. The roots begin to
excrete more organic substances that activate
microbial activity and as a result the mobile
forms of the mineral nutrients in the soil
increases.
 Summing up, the foliar nutrition may correct
plant mineral deficiencies, but may also helps
the root absorption of mineral elements.
Foliar
fertilizer
2. Biological aspects of foliar feeding of plants by nutrients

33. Many factors can influence the performance of foliar nutrient sprays, but for simplicity they
may be grouped in 3 categories:
 physico-chemical properties of the formulation;
 the environment under which sprays are applied;
 the characteristics of the plant to which the spray is applied.
3. Technological aspects of foliar fertilization
The main physico-chemical properties, playing major role for
the efficacy of nutrient uptake by the foliage are the following:
 molecular size of the active substance;
 solubility;
 electric charge and pH;
 surface tension;
 retention and spreading, etc.

34.  The main environmental factors, which affect the uptake and translocation of foliar
nutrient sprays are relative air humidity, temperature and light.
 In addition, fertilization and biostimulation efficacy is influenced by several plant and
species characteristics, such as leaf shape, cuticle composition, surface wax
architecture, the presence of leaf hairs, plant phenological stage, the mobility of the
nutrient within the plant, etc.
3. Technological aspects of foliar fertilization

35. Low humidity (< 60%) and high T (> 28 ºC) concentrate the droplets and reduce the
the fertilizer hydration and its absorption. The high concentration makes diffusion
difficult and causes burns on the leaves!
Foliar fertilization should be applied at higher humidity and lower T - early in
the morning or late in the evening!
Relative air humidity Evaporation
High concentration
of nutrients
Low concentration
of nutrients
Low to high concentration of nutrients
3. Technological aspects of foliar fertilization

36. Foliar fertilizers contain not only mineral elements but also additional substances.
Some of them buffer the solutions (adjust the pH), improve wetting, expand the
physiological action. Adhesives, for example, reduce the surface tension, which
increases the contact of the droplet with the leaves, force disintegration of droplets
to suitable sizes and better cover the leaf surface.
The effectiveness of foliar feeding depends on the retention of drops on the leaves.
This is determined by their size, as well as the presence of adhesives in the working
solution. It is recommended that the droplet size be about 200-250 microns.
3. Technological aspects of foliar fertilization

37. In most cases, leaf products contain a complex of mineral elements, not just one.
Different types of interactions can occur between these elements - competitive,
chemical reactions, etc., due to which they are complexed or chelated with sinthetic
(EDTA) or organic substances (eg, milk waste products).
Synthetic chelate
Milk acid, amino acids, etc.
(organic chelates)
Synthetic or organic
chelate?
3. Technological aspects of foliar fertilization

38. Influence of foliar fertilizers on maize
Experimental design:
1. Control (plants grown on Hoagland
solution)
2. Induced Zn deficiency (plants grown on
Hoagland solution without Zn)
3. Induced Zn deficiency, followed by
foliar application by ZnO
4. Induced Zn deficiency, followed by
foliar application by ZnHN
4. Results from experiments with foliar products
Krasimir Ivanov, Andon Vassilev, Anyo Mitkov, Nguyen Nguyen, Tonyo Tonev, 2021.
Application of Zn-containing Foliar Fertilizers for Recovery the Yielding Potential of Zn-
deficient Young Maize Plants (in print)
(Lab and field experiment)

39. Results
Variants
Parameters
A E gs
(1) Control (all nutrients) 12.21 ± 1.00 0.76 ± 0.05 0.05 ± 0.00
(2) Zn deficiency (- Zn) 6.26 ± 0.88 0.49 ± 0.06 0.03 ± 0.01
(3) - Zn + foliar ZnO 9.30 ± 0.79 0.56 ± 0.02 0.04 ± 0.01
(4) - Zn + foliar ZnHN 11.14 ± 0.20 0.76 ± 0.01 0.05 ± 0.00
Table 1. Influence of Zn deficiency and foliar application by Zn-containing fertilizers on
leaf gas exchange parameters of young maize plants. A – net photosynthetic rate (µmol CO2
m-2 s-1),E – transpiration rate (mmol H2O m-2 s-1), gs – stomatal conductance (mol m-2 s-1)

40. Results
Experimental design:
1. Control (plants grown on
Hoagland solution)
2. Induced Zn deficiency
(plants grown on Hoagland
solution without Zn)
3. Induced Zn deficiency,
followed by foliar application
by ZnO
4. Induced Zn deficiency,
followed by foliar application
by ZnHN
1 2
3 4

41. Visual Zn deficiency of maize due to low
temperature in the early growth.
-Zn
Results from the practice
Problem
Solution
After foliar
application of Zn-
containing
fertilizer the
problem is solved.
Example 1. Correction of Zn deficiency in maize

42. Well expressed Mo deficiency in sunflower due
to low pH (acid reaction) of the soil.
06.06.2013 г.
-Mo
Problem
Solution
Ten days the foliar application of
Mo-containing fertilizers the deficiency
symptoms are missing and the growth is
recovered.
Example 2. Correction of Mo deficiency in sunflower
Results from the practice

43. Thanks for the attention !
43