3. PHYTOHORMONES
AND
THEIR
PHYSIOLOGICAL
EFFECTS
Three terms used routinely to describe various aspects of the changes that a plant
undergoes during its life cycle are growth, differentiation, and development.
3.1.
Development, growth and differentiation
3.1.1. Development
Development
is an umbrella term, referring to the sum of all of the changes that an
organism goes through in its life cycle – from germination of the seed through growth,
maturation, flowering, and senescence. Development is most readily manifest in changes in
from of the organism or organ, such as the transition from the vegetative to flowering
condition or from leaf primordium to fully expanded leaf. Development may also be manifest
at the subcellular and biochemical levels, such as when chloroplasts appear in leaf cell
brought into to the light and the enzymes of photosynthesis become active.
Development is the sum of growth and differentiation.
3.1.2. Growth
Growth is the quantitative term, related to changes in size and mass. It can be assessed
by a variety of quantitative measures. Growth of cells in culture is sometimes measured as in
increase in cell number or the fresh weight of packed cells. For higher plants, however fresh
weight is not always a reliable measure. Although most plant tissues are approximately 80%
water, water content is highly variable and fresh weight will fluctuate widely with changes in
the water status of the plant. Dry weight, a measure of the amount of protoplasm or dry
matter, is used more often than fresh weight, but even dry weight can be misleading as a
measure of growth.
3.1.3. Differentiation
Differentiation is a qualitative term, referring to the differences other than size that arise
among cells, tissues and organs. Differentiation occurs when a dividing cell gives rise to two
daughter cells destined to assume different anatomical characteristics and functions. In the
earliest stage of development, for example, division of the zygote gives rise to cells that will
become the root or shoot of the plant. Unspecialized parenchyma cells differentiate into
xylem vessels or phloem sieve tubes, each with a distinct of morphology and specialized
function. Differentiation does not lend itself easily to quantitative interpretation but must
normally be described as a series of qualitative changes. Finally, although growth and
differentiation are normally concurrent events, examples abound of growth without
differentiation and differentiation without growth.
Differentiation is a two-way street. Even though plant cells may appear to be highly
differentiated or specialized, they may often be stimulated to revert to a more embryonic
form; that is cells dedifferentiate. It is a though the cells have been genetically
reprogrammed, allowing them to reverse the process and them to differentiate along new and
different paths.
The ability of differentiated cells to regenerate new plants demonstrates that all living
plants cells retain complete a genetic program, even though not all of the information is
actively used by the cell at any given time. This concept is known as totipotency. All that is
required to change the pattern of differentiation is the right input to select the appropriate
genetic information at the right time.
3.1.4. Control of growth and development
The orderly growth and development of complex multicellular organisms require
coordination and are subject to controls at three distinct levels. Intrinsic controls operate at
both the intracellular level and the intercellular level. Typically, intracellular controls
involve changes in gene expression that influence cellular activities through altering the kinds
of proteins in the cell. Intercellular controls focus on hormones and their roles in coordinating
the activities of group of cells. Extracellular controls are extrinsic; that is, they originate
outside the organism and convey information about environment.
3.1.4.1. Genetic control of development
Totipotency of plant cells indicates that all of the information required for the
development of a complete plant is contained within the genetic complement of each cell,
even highly differentiated cells. In other words, cells do not lose genes although many genes
are not expressed or may be turned off as differentiation and development progress. The
orderly development of a plant requires a programmed sequence of gene activations in order
to produce the required gene products, that is, proteins, at the appropriate time. The cells must
also have to capacity to respond to those products.
Genes consist of specific sequences of nucleotides in the deoxyribonucleic acid (DNA)
molecule. A sequence of three nucleotides (a codon) codes for each amino acid. The
nucleotide sequences in the gene thus determine the primary structure or sequence of amino
acids of proteins, principally enzymes that determine the course of cellular metabolism. Gene
expression refers to the synthesis of specific proteins encoded by specific genes. Not all
genes are active all the time, but may be turned on or off depending on the requirements of the
developmental program or in response to changing environmental conditions. Differential
gene expression is thus the principal means for altering the complement of enzymes in the cell
and, consequently, the course of metabolism and differentiation of the cell.
3.1.4.2. Hormonal regulation of development
Interaction between cells is an exceedingly complex affair. Much of this kind of
behavior can be interpreted in terms of hormones, chemical messengers that carry information
between cells. Several classes of hormones are known, which promote or inhibit various
developmental responses, either singly or in combination. The details of hormones and their
actions in regulating plant development will be discussed in the following chapters.
3.1.4.3. Environmental regulation of development
A variety of external or environmental stimuli can at various times be involved in
regulating plant development. Most environmental stimuli are physical parameters. Light,
temperature, and gravity have the most obvious and dramatic impact. Other parameters such
is magnetic field, sound, and wind (a mechanical stimulus) may have more subtle effects, but
these have been difficult to establish experimentally. Other environmental factors such as soil
moisture, humidity, and nutrition may also influence development in some cases. More
recently it has become evident that a variety of air and water pollutants represents a
significant environmental challenge to plants and may significantly modify developmental
patterns.
Because environmental signals originate outside the plant, plants must have some means
of perceiving the signal and converting, or transducing, the information into some permanent
metabolic or biochemical change. It is becoming increasingly evident that most, if not all,
environmental stimuli act at least in part through modifying gene expression or hormonal
activities.
3.2. The role of hormones in plant development
Hormones are naturally occurring, organic substances that, at low concentration, exert a
profound influence on physiological processes. In addition, hormones, at least in animals, are
1. synthesized in a discrete organ or tissue, and
2. transported in the bloodstream to a specific target tissue where they
3.control a physiological response in a concentration- dependent manner.
While there are many parallels between animal and plant hormones, there are also some
significant differences. Like animal hormones, plant hormones are naturally occurring organic
substances that profoundly influence physiological processes at low concentration. The site of
synthesis of plant hormones, however, is not so clearly localized. Although some tissues or
parts of tissues may be characterized by higher hormone levels than others, synthesis of plant
hormones appears to be much more diffuse and cannot always be localized to discrete organs.
For example, there is good evidence that auxin, the prototypic plant hormone, is synthesized
in the tip of a grass coleoptile but influences the elongation of cells lower down in the same
organ. Another plant hormone, cytokinin, is synthesized in the root and transported to the
leaves where it influences metabolic activity and delays senescence. Yet there are many other
examples where plant hormones appear to act within the same tissue or even the same cell in
which they are synthesized.
Table 3.1. The influence of plant hormone groups on different categories of development. An x
indicates a demonstrated effect of that hormone group on one or more aspects of that developmental
category. The absence of an x does not mean that the hormone is ineffective, only that an effect has not
been reported in literature. (Reprinted from G. Hopkins: Introduction to Plant Physiology. John
Wiley et Sons, New York, 1995.)
There are currently five recognized groups of plant hormones: auxins, gibberellins,
cytokinins, abscisic acid, and ethylene. In addition to the five principal hormones, two other
groups sometimes appear to be active in regulating plant growth, the brassinosteroids and
polyamines. Each of these groups will be briefly introduced at this time, followed by a
general discussion of its physiological role. The influence of plant hormone groups on
different categories of development we can see in Table 3.1.
3.2.1. Physiological action of auxins
Auxins were the first plant hormones to be discovered. The principal auxin in plants is
indole-3-acetic acid (IAA). IAA is produced mainly in the shoot apex bud and young leaves
of plants. Others meristematic tissue, flowers, fruits and young seeds have also been shown to
be sites of this hormone production. The amino acid tryptophan is an intermediate in the
synthesis of IAA. The IAA movement is strictly polar from the apex to the organ base, i.e.
basipetal. This polarity in transport is a manifestation of cell polarity. The rate of transport in
the coleoptile is 10 – 20 mm h-1, which cannot be explained by simple diffusion. This process
must be an active process (two partial processes at the plasma membrane of neighbouring
cells: (1) IAA uptake, driven by an electrochemical gradient, and (2) secretion by an IAA
efflux carrier localized only at the base of the cell.
Fig. 3.1. The effects of auxin
concentration on the growth of
different organs.
Other
natural
occurring
auxins are also based on the indole
ring (e.g. indole acetonitrile acid
and indole pyruvic acid). However
the indole group is not essential
for auxin activity as is shown by
the auxin activity of certain
synthetic compounds, e.g. naphthalenacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid
(2,4-D). The concentration of auxin is very important in determining the nature of the growth
response and the optimum auxin concentration differs for different organs (see Figure 3.1.).
Root elongation is particularly sensitive to auxin. IAA will promote the growth of
excised root sections and intact roots, but only at very low concentrations (10-8 M or less).
Higher concentrations of auxin, in the range that normally stimulates elongation of shoots (105
to 10-6 M), caused a significant inhibition of root growth. The inhibition of growth at higher
auxin concentrations may be due to the auxin-promoted synthesis of ethylene, which inhibits
cell elongation. Such inhibitory effects have been exploited in the production of herbicides
based on 2,4-D or 2,4,5-T.
The effects of IAA in the plant are extremely varied (see Figure 3.2.).
Fig. 3.2. Multiple effect of auxin in
higher plants. The hormone is
formed mainly in the tip of the
shoot and is transported from there
into the different organs. Following
the predetermined competence for
auxin, different site react differently
with specific processes of growth
and differentiation (from Hopkins
1995, after Steward 1964).
Fig. 3.3. Auxin stimulated adventitious root
development on American holly (Ilex opaca)
cuttings. Row A: Treated with a 0.01%
solution of indolebutyric acid solution for 17
hours before being rooted insand. Row B:
Untreated controls. (after T.E.Weier et al.
1982,
reprinted
from
G.
Hopkins:
Introduction to Plant Physiology. John
Wiley et Sons, New York, 1995.)
Although auxin commonly inhibits root elongation, high auxin concentrations will
promote initiation of secondary, or branch, roots. Conversely, removal of young leaves or
buds, both sources of auxin, will often reduce the number of secondary roots formed. These
results indicate that secondary root initiation is normally controlled by auxin supplied by the
shoot. Auxin also promotes adventious root formation on stems (Figure 3.3.). Certain
synthetic auxins (e.g. NAA and indole butyric acid or IBA) are widely used as rooting
compounds.
Abscission has been correlated with low levels of auxin in the organ concerned and
auxins have thus been used to prevent premature fruit drop. Other phenomena in which auxins
have been implicated include apical dominance, phototropism, and epinasty.
Fig. 3.4. (1)The control of apical dominance by auxin. Experimental material: Field bean
(Vicia faba). After removal of the apical terminal bud (a) side buds grow in the lower leaf
axils (b). If the cut apical surflace is covered by an agar block containing IAA, lateral buds
remain inhibited (c). A control block (without IAA) has no effect (d). IAA is therefore able to
replace the end bud with respect to apical dominance. (from Mohr et Schopfer 1995, after
Bonner et Galston 1952)
Fig. 3.4. (2) Apical dominance in broadbean
(Vicia faba) (A) Control plants. (B) Removal
of the stem apex, a source of auxin, promotes
axillary bud growth. (C) Dominance can be
restored by applying auxin (in lanolin paste)
to the cut stem surface. (Reprinted from G.
Hopkins: Introduction to Plant Physiology.
John Wiley et Sons, New York, 1995.)
Fig. 3.5. Auxin (IAA) and cytokinin (kinetin) as factors limiting mitotic activity and formation
of organs in a tissue culture. Experimental material: Explant from stem pith of a tobacco
plant (Nicotiana tabacum). Relatively high concentrations of IAA and kinetin lead to the
formation of a callus after a few weeks. Development can be redirected to root or shoot
formation by either increasing or decreasing the ratio of IAA/kinetin. (from Mohr et Schopfer
1995, after Ray 1963)
Apical dominance is the inhibition of the development of some or all of the lateral buds
by the terminal (apical) bud of shoot. Removal of the terminal bud releases some of the lateral
buds from inhibition. This implies that a substance produced at the apex, most probably auxin,
is responsible for the inhibition (see Figure 3.4.). Cytokinins have been shown to promote the
growth of lateral buds.
Many effects of auxins are brought about by the combined action of auxin with other
growth substances. For example, the stimulation of cambial activity, the induction of
parthenocarpy, and the enhancement of internode elongation are all more effectively
promoted by a combination of auxin and gibberellin than by either substance alone. Similarly
appropriate concentrations of auxin and cytokinin are needed in culture media to promote cell
division in tissue explants. Depending on the relative concentration of each, root meristems
(high auxin: low cytokinin) or shoot meristems (low auxin: high cytokinin) may be initiated
(see Figure 3.5.).
3.2.2. Physiological action of gibberellins
Gibberellins are produced by the both fungi and higher plants. There are produced in the
root, mainly in the root apex, and youngest leaves of plants. Seeds have also been shown to be
sites of this hormone production.
Gibberellins (GAs) were originally discovered as phytotoxins in 1926 by Japanese
phytopathologist. The pathogenic fungus Gibberella fujikuroi (= Fusarium moniliforme)
attack rise plants and secretes an agent which causes pathological longitudinal growth
(Bakanae, “mad seedling disease”). Between 1935 and 1938 Japanese scientists isolated and
crystallised the active substance, which was called gibberellin, chemically gibberellic acid.
The first was gibberellin GA3. Intensive research in the second half of twenty century showed
clearly that gibberellins are also formed by higher plants and are very important in the
regulation of growth and in differentiation processes.
The exogenous application of gibberellins causes hyperelongation of intact stems.
Gibberellins are also prominently involved in seed germination and mobilization of
endosperm reserves during early embryo growth, as well as flower and fruit development.
More than 100 gibberellins are now known and additional members are added almost
every year. All gibberellins are demonstrated to be naturally occurring.
The basic structure of gibberellic acid and most other compounds with gibberellin-like
activity is the gibbane carbon carbon skeleton. Gibberellin acid is a terpenoid and is
synthesized from mevalonic acid. Other gibberellins are synthesized by the same pathway and
there is probably much interconversion in the plant between the different gibberellins.
Gibberellins can overcome certain forms of genetic dwarfism (see Figure 3.6.) and
dwarf varieties are often used in bioassays for gibberellin.
Fig. 3.6. (A) Dwarfism in a GA deficiency mutant and its reversal by GA application.
Experimental material: Dwarf-5 mutant (D-5/d-5) and wild type of maize (Zea mays). The
mutant received in total 250 μg GA3 applied to the apex from the seedling stage at intervals
of 2 to 5d. From left to right: Wild type, untreated; wild type, treated; mutant, untreated;
mutant, treated. Analysis shows that stunted growthis based on the failure of one gene, which
controls the cyclisation of copalylpyrophosphate to ent-kaurene. The phenotypic defect can
therefore be reversed by application of ent-kaurene. (after Phinney and West 1960, from
Mohr et Schopfer 1995)
Fig. 3.6. (B) The effect of gibberellic acid on dwarf
pea. Left: Control, showing reduced internode
elongation. Right: Gibberellin treated. Enhanced
internode elongation following a foliar-drench
with 5 x 10-4 M gibberellin. (after Hopkins 1995)
Environmentally limited rosette plants such
as spinach (Spinacea oleracea) and cabbage
(Brassica sp.) generally do not flower in the rosette
form. Just before flowering, these plants will
undergo
extensive
phenomenon
known
internode
as
elongation,
a
Bolting
is
bolting.
normally triggered by an environmental signal,
either photoperiod (as in spinach) or a combination of low temperature and photoperiod (as in
cabbage). It is sufficient to note here that, under conditions normally conducive to the rosette
habit, spinach, cabbage, and many other rosette plants can be induced to bolt by an exogenous
application of gibberellic acid (see Figure 3.7.). They can partly or completely replace the
photoperiod or cold requirement necessary to some species for flowering.
Fig.
3.7.
(A)
Gibberellin-
stimulated stem growth in a
rosette genotype of Brassica
napus. Treatments were (from
left): 0, 0.5, 1.0, 10.0 ng GA3
per
plant,
applied
to
the
meristem. (from Hopkins 1995)
Fig. 3.7. (B) Induction of internodal growth
in
rosette
plants
by
application
of
gibberellin. Experimental material: White
cabbage (Brassica oleracea, var. capitata).
The apex of the right-hand plant was
treated, from the seedling stage, at regular
intervals with GA3 solution; left untreated
control. In this case GA induces not only
internodal
growth
formation,
i.e.
but
also
physiological
flower
reactions
which also occur during natural bolting of
the plants. However, it cannot be deduced
automatically that these processes are
caused by increased endogenous GA. (after
Galston 1961, from Mohr et Schopfer 1995)
Gibberelins have also been found to
break dormancy in buds and seeds that
normally have a light or chilling requirement. Gibberellin levels are high in young leaves, and
if applied to ageing leaves, can delay senescence. Levels are also high in developing seeds
and fruits. Gibberellins application can induce parthenocarpy and this has been put to
commercial use in the production of seedless varieties of fruit.
In barley seeds gibberellin has been found to stimulate the synthesis of the enzyme αamylase. It does this by making available the messenger RNA responsible for α-amylase
synthesis. This effect has proved of use in the brewing industry where α-amylase activity is
essential for the production of malt. Gibberellin can even promote the enzyme's activity in
inviable seed.
Gibberellins interact with other hormones in various ways. There is evidence that
abscisic acid reduces gibberellin levels, hence the antagonistic effects of these hormones.
Gibberellin is believed to interact with auxin in the control of sex expression in dioecious
plants. Gynoecious plants usually have low levels of gibberellin and high levels of auxin.
Application of gibberellin can induce the formation of male flowers. This effect has been put
to use in the cucumber industry where certain hybrid varieties are naturally gynoecious. Since
pollen production is necessary for fruit set some of the plants are sprayed with gibberellin to
produce the necessary male flowers.
3.2.3. The physiological roles of cytokinins
Cytokinins are produced mainly in the root, and in the buds of plants. They were
discovered during work on tissue culture media when it was found that cells of tobacco pith
explants could be stimulated to divide by adding the purine adenine to the medium.
Subsequently various adenine derivatives, e.g. kinetin (discovered 1955 by American
researches), were found to have even greater effect on cell division. However these effects
are not seen in the absence of auxin. Moreover, by changing the proportion of cytokinin to
auxin, different types of meristematic activity may be induced. The first evidence of a native
cytokinin was found in 1963 with the isolation of zeatin from maize caryopses (Miller et
Letham 1964). Later, it was shown that zeatin is also present in many other plants, either in its
free form or bound as riboside (or ribonucleotide). About 50 cytokinins are now known.
Cytokinins are probably active in most aspects of plant growth and development.
Example of this is Figure 3.8. There are shown the effect of cytokinins on growth and
development of cereal plants.
Fig. 3.8. The effects of cytokinins on growth and development of cereal plants. (After Kamínek
et al. 2003, reprinted from Macháčková et Romanov (eds.): Phytohormones in Plant
Biotechnology and Agriculture. Kluwer Academic Publishers, Dordrecht, 2003).
Generally the most obvious effects of cytokinins include delay of senescence mature
leaves. Senescence is characterized by the breakdown of protein, nucleic acids and other
macromolecules, a loss of chlorophyll, and the accumulation of soluble nitrogen products
such as amino acids. Cytokinins also induce of flowering in certain species, and cut across of
dormancy in axillary buds and some seeds. Endogenous cytokinins are found in the greatest
concentrations in embryos and developing fruits, e.g. in the 'milk' of the coconut. The
application of cytokinins stimulates also release of axillary buds from apical dominance,
thus antagonizing the effect of auxins.
3.2.4. The physiological roles of abscisic acid
Abscisic acid (ABA) is a sesquiterpene compound; like gibberellin, its synthesis
therefore starts from mevalonic acid (mevalonate). ABA shares early biosynthetic step with
carotenoids. It can be formed in many parts of the plant, mostly in the roots, mainly in the root
apex, in mature leaves, and ripening fruits. Small amount of this hormone is produced by
many others plant organs, also. The hormone is secreted by cells into the apoplast and can
thus be relatively easily transported. Evidence of the hormone has been found in the xylem
sap and phloem sap.
This hormone was discovered in about 1960 by two groups whose research was based
on two different physiological functions: (1) “abscisin” causing the abscission of fruits in
cotton, and (2) “dormin” inducing the dormancy of buds in birch trees. It was found in 1965
that “abscisin” and “dormin” were the same substances, these terms were abandoned and
replaced by the new term abscisic acid.
ABA is active, possibly in association with gibberelic acid, in the promotion of leaf and
fruit abscission and the control of dormancy; there is a positive correlation between ABA
concentration and the intensity of dormancy. It prevents cell elongation and shoot growth and
also inhibits seed germination. At physiological concentrations, ABA is not toxic to plants.
Generally, ABA plays the role of stress hormone in the plant. A large proportion of
ABA is synthesized in the cytoplasm of leaf mesophyll cells and accumulated in chloroplasts.
Its synthesis can be triggered by various stress-inducing environmental factors, especially
water shortage. It is best known effect of ABA on the inhibition of stomatal opening in many
species. In leaves of plants that have been grown to ensure minimum endogenous level of
ABA, exogenous ABA at concentrations of 10-3 to 10-4 M will induce complete stomatal
closure. This appear to be a means for regulation water balance in the plant since the
endogenous level of ABA in leaves is generally very low if the plants are well watered.
Subjecting leaves (e.g. wheat, barley) to a water deficit, however, will induce as much as a
fortyfold increase in the ABA level within as little as 30 minutes.
Stomatal closure does not always rely on the perception if water deficits and signals
arising within the leaves. In some case it appears that the stomata close in response to soil
desiccation before there is any measurable reduction of turgor in the leaf mesophyll cells.
Several studies have indicated a feed-forward control system that originates in the roots and
transmits information to the stomata. There is a significant increasing in ABA content of the
roots in the dry soil, and subsequently in the leaf epidermis. These results provide reasonably
good evidence that ABA is involved in a kind of early warning system that communicates
information about soil water potential to the leaves. ABA overrides the normal diurnal pattern
of stomatal opening and closure and causes the stomata to close during the day. This response
decreases water loss by transpiration in times of drought.
3.2.5. The physiological roles of ethylene
Ethylene is a gaseous hydrocarbon (ethane, C2H4). It is produced in small quantities in
various parts of many plants and has a special role as a phytohormone. The biosynthesis of
ethylene in higher plants is from amino acid methionine (precursor). The concentrations
effective in triggering physiological effects are usually very low, mostly in the range 0.01 –
10 μl l-1. The normally ethylene is hardly exchanged between plant organs.
Though it was known for many years that, for example, coal gas containing ethylene has
drastic effects on various developmental processes in plants, only in 1935 was it demonstrated
that ethylene is a natural product of plant metabolism, occurring in physiologically effective
concentrations and secreted by plants.
The physiological functions of this hormone are apparent in at least two areas.
1. Acceleration of fruit ripening and other senescence processes. In many fleshy fruits,
e.g. apples and tomatoes, the level of ethylene increases sharply after growth stops. This
induces specific ripening processes, e.g. breakdown of chlorophyll, increase of respiration,
enzymatic dissolution of cell walls and formation of sugars, aromatic substances and
pigments. These effects are caused by the induced synthesis of certain “ripening
enzymes”; it is thus possible to block fruit ripening by inhibitors of protein synthesis. The
function of ethylene as a ripening hormone is widely used commercially in the storage of
fruits after harvesting: apples, bananas and other fruits can be kept for a long time in an
unripe condition in an ethylene-depleted CO2-rich atmosphere. Application of ethylene
causes fruit ripening within a few days. Stimulation of abscission of fruits or leaves by
ethylene is also sometimes used in harvesting. Cotton plants, for example, are sprayed
with (2-chlorethyl phosphonic acid (ethephon), a substance which releases ethylene after
it has been taken up by the plant.
2. Triggering of stress reactions. Various stress factors, e.g. flooding (O2 deficiency),
wounding or infection by disease leads to formation of “stress ethylene” in plants.
Other effects mediated by ethylene include: the induction of epinasty; the induction of
root hairs; the stimulation of seed germination in certain species; and the inhibition of auxin
transport.
3.2.6. Hypothetical plant hormones
Studies on flowering clearly indicate the transmission of a diffusible chemical signal
from the leaf to the apex .The existence of a flowering hormone, called florigen, has been
postulated, but it has never been isolated. Similarly, the hormone vernalin has been
postulated to account for the effect of low temperature on the flowering behavior of winter
cereals and biennials. Vernalin remains as elusive as florigen. Phenomena as complex and
precice as flowering no doubt require participation of some regulatory substances. Failure to
isolate the responsible factors could result from several factors. For example, the molecule or
molecules could be extremely labile and readily broken down during extraction. On the other
hand, we may simply lack an appropriate system for testing biological activity. “Florigen”
might also be a complex interaction between several regulatory molecules, rather than a single
hormone.