Physiological and Biochemical changes during senescence

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UNIVERSITY OF HORTICULTURAL SCIENCES
BAGALKOT COLLEGE OF HORTICULTURE, BAGALKOT
Presentation on Physiological and Biochemical
changes during Senescence
By, Ms. Ayeeshya H. Kolhar
Ph.D. (I yr), Dept. of PHT
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Senescence is a genetically regulated process
that involves decomposition of cellular
structures and distribution of the products of
this degradation to other plant parts.
Three phases may be distinguished in a typical
senescent process
1. Storage mobilization A phase of selective
degradation of certain molecules. This
degradation does not cause a major impairment of
the physiological functions. The mobilized
molecules may be considered as nutrient storage
materials.
2. Generalized breakdown Extension and
generalization of breakdown to components which
are central in maintaining physiological
function. As a result of this breakdown a
physiological function is consequently lost.
During this phase the senescence process becomes
irreversible and death of the cells becomes
inevitable.
3) Abscission The final phase of senescence is
abscission and death.
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Physiological and Biochemical changes
1. Loss of membrane compartmentation
2. Ultra structural changes in chloroplast
3. Chloroplasts are converted into Chromoplasts
4. Loss of Chlorophyll content
5. Reduction of Soluble protein content
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5. RuBisCO activity decreased
6. Reduction of Photosynthetic rate
7. Rate of Respiration generally decreases
8. Nucleic Acid content of leaves declines
9. Biosynthetic Enzymes activity decreases
10. Hydrolytic Enzymes activity increases
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11. Increased content Ethylene and ABA
(Deteriorative Hormones)
12. Decreased content Auxin and Cytokinin
(Growth promoting Hormones)
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Physiological changes..
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Respiration

• Fruit was the first organ where the rate of
  respiration during senescence was described

• Climacteric and nonclimacteric

• Increased rate of respiration in climacteric
  fruits due to their ability to produce and
  response to ethylene. It appears that the rise in
  respiration is a consequence of ethylene action
  and not of senescence as such. The main reason
  for this conclusion is that inhibition of both
  the biosynthesis and action of ethylene
  eliminates the rise in respiration without
  preventing eventual senescence

• Respiration rate decreases as senescence
  pronounced

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Respiratory pathways of senescence plants include
glycolysis, pentose pathway, TCA cycle and the
Electron transport pathway, where some changes
have been described but also an alternative
oxidase pathway which is enhanced during
senescence. It has been suggested that the
alternative pathway is activated when the
cytochrome pathway is saturated or limited,
allowing the TCA cycle to function using up
excess carbohydrates.
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• Mitochondria play crucial roles in programmed
  cell death and aging. Different stimuli activate
  distinct mitochondrion-dependent cell death
  pathways, and aging is associated with a
  progressive increase in mitochondrial damage,
  culminating in oxidative stress and cellular
  disfunction.
• Mitochondria that remain intact until late after
  senescence onset, are in turn degraded when the
  energy demand decreases .

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Photosynthetic changes

• Decrease in the rate of photosynthesis with the
  age of a leaf to decline in dry weight of leaves
  and their eventual death are the symptoms of leaf
  senescence.
• The rate of photosynthesis starts declining as
  soon as the leaf reaches its full size or becomes
  mature.
• There is a gradual loss of chlorophyll and
  carotenoids, which results in yellowing of leaves
  and or cotyledons particularly during the later
  stages of senescence.

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Leaf conductance and CO2 assimilation
Senescence produces closure of stomata leading to
a decline in transpiration. It has been suggested
that stomata aperture may control the rate of
leaf senescence. The main entrance for CO2 are
stomata and insufficient CO2 supply could be the
cause of the decreased photosynthetic
assimilation observed during senescence.
Experimental measurement of CO2 concentration
in the substomatal cavity, suggests that CO2 does
not limit photosynthetic assimilation. Hence
stomatal closure may be more a consequence than a
cause of lowered photosynthetic activity,
according to the optimal variation hypothesis,
which proposes that stomatal conductance adapts
to the photosynthetic capacity of a leaf.
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Hormonal levels
Hormone level changes may be a primary cause or
a secondary effect of senescence in leaves. In
most plants cytokinins are anti senescence
compounds that can postpone plant death,
gibberellins also have similar effect, but in
fewer plant species. Auxins influence
senescence and abscission in very complex
manners, apparently depending on the auxin source
or depending on the age and receptivity of the
tissue. When applied to the leaf auxin often
retards senescence, but paradoxically when auxin
enters the leaf from stem, it sometimes promotes
senescence.
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Ethylene
Little work has been done to examine the
involvement of ethylene in whole plant
senescence perhaps that is because it appears
not to play very important part. The senescence
in flower petals is accompanied by the increase
of production of ethylene. Production of ethylene
in leaves generally rises slowly until the leaves
become senescent and abscide. Flowers also
synthesize ethylene, especially just before they
fade and wither and in most species this gas
causes their senescence and abscission.
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Abscisic acid

• Exogenous application of ABA promotes leaf
  senescence. Genes involved in the key steps of
  ABA biosynthesis and signaling are upregulated
  during leaf senescence in arabidopsis.
• The endogenous concentrations of ABA rises
  during the rapid growth stage of fruit or seed,
  peaks at the point of maximum fresh and dry
  weight of fruit, and then declines rapidly with
  senescence of the plant and with ripening or
  drying of the fruit.

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The function of ABA as a promoter of flower
tissue senescence, including such parameters as
wilting, colour fading of blueing and protein
loss has been established. Here, ABA may act
through hastening of autocatalytic ethylene
production. In some cases, ABA actually exhibits
a senescence retarding effect. ABA may cause
short term inhibition of senescence of cut
flowers by closing the stomata, there by water
loss. Acceleration of RNA and protein loss in
senescing tissues by ABA has been reported in
many cases.
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Mechanistic evidence for a positive regulatory
role of ABA in senescence comes from two
studies. 1. The role of the receptor-like kinase
1 (RPK1) in leaf senescence. RPK1 acts as an
upstream component of ABA signaling, whose
expression was found to increase in an
ABA-dependent manner throughout the progression
of leaf senescence. 2. The senescence associated
gene 113 (SAG113), that acts as a negative
regulator of ABA signaling. SAG113 is induced by
ABA in senescing leaves and is significantly
reduced in its expression in the ABA biosynthesis.
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Cytokinin

• Cytokinin as major senescence- delaying hormone
  but endogenous cytokinins levels
• declined during leaf senescence.
• The roots are the main sources of cytokinins for
  the leaves, a decrease
• in the level of cytokinin during leaf
  senescence must be due to
• 1. To an accelerated cytokinins metabolism in the
  leaves before their senescence.
• 2. To an accelerated transport from leaves to
  other structures
• 3. To a decrease in the supply of cytokinins by
  the roots

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Auxin

• The auxin level decreases with age of several
  species. IAA content in leaves (common beans,
  cotton) decreases with age.
• NAA retard senescence of leaf discs and bean
  pods. 2,4-D butyl ester retards the proteolysis
  of detached leaves of cherries (Prunus
  serrulata).
• Auxins are not generally found very potent in
  retarding leaf senescence.

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• Chloroindole auxins
• 4- chloroindole acetic acid (CIIAA) derivatives
  may be death hormone in Pisum, Vicia, Lens and
  Lathyrus.
• It induces very strong, almost irreversible
  ethylene production.
• It induces death of apical meristems in pea
  cuttings.
• Auxin in lanolin placed in deseeded bean pods
  induce leaf senescence.
• Five different chloroinodole auxin derinatives
  are present in large
• amount in mature pea seeds (mg/kg).
• Five different chloroinodle auxin derivatives are
  4-chloroindole acid, methyl esters of
  4-chloroindole acid, methyl esters of malonyl-4-
  chlorotryptophan, ethyl esters of
  malonyl-4-chlorotryptophan and monomethyl of
  4-chloro indole acetyle aspartic acid.

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Gibberllins

• Parallel decrease in content of gibberllins in
  senescence has been reported in Tropaeolum
  leaves. A similar decrease occurs in lettuce
  leaves under water stress. Endogenous gibberllin
  like components decline during the senescence of
  detached Nastursium and Dandelion leaves.

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Jasmonic acid

• Jasmonic acid derivatives may be death harmones
  have following effects,
• Jasmonic acid methyl ester is a senescence
  inducer in some assays
• it is stronger than ABA.
• Jasmonic acid widely distributed in plants
• Soybeans contain high conc. In vascular bundles
  of the pericarp.
• Jasmonic acid effects can often be alleviated or
  reversed by
• cytokinins.
• Jasmonic acid methyl ester induces ethylene
  formation

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Brassinosteroids
Brassinosteroids can be regarded as a new class
of plant horomones in addition to auxin, GA,
cytokinin, ABA, ethylene and jasmonic acid with
an independent role in the growth regulation of
plants. Recently it has been reported that
brassinosteroid exhibit delayed leaf senescence.
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Biochemical Changes
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Photosynthetic pigments
Colour changes are important criteria for a
visual evaluation of the advance of senescence,
especially in fruits. All photosynthetic
pigments, namely Chla, Chlb, carotenes and
xanthophylls are found to decline during leaf
senescence.
Chlorophyll
Breakdown of chlorophyll may be one of the
earliest symptoms of senescence. However,
chlorophyll decline is strongly retarded by
continuous illumination in the process regulated
by phytochrome. The chlorophyll a/b ratio has
been shown usually to decline with the advance of
senescence, probably as a result of the
nonsynchronous dismantling of lamellae and grana
thylakoides and the asymmetrical distribution of
photosystems between them.
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Leaf senescence is accompanied by changes of many
organelles. Chloroplasts of senescing leaves show
reduced volume, their shape is spherical and the
thylakoid system is reduced (Matile 1992). The
final developmental stage of chloroplasts
gerontoplasts shows an increase in the number
and diameter of plastoglobuli, a reduction of the
thylakoid system, a loosening of the stacking of
thylakoids and a swelling of intrathylakoid
spaces.
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Carotenoides
In higher plants shows a grater loss of
chlororphyll than carotenoides, which results in
yellow colouration of senescence
leaves. Chloroplasts produced during leaf
senescence (gerntoplasts) are characteristically
different from chloroplast of green mature
leaves, the transformation is expected to bring
changes in the compositon and distribution of
carotenoids. Parallel to a quantitative loss of
carotenoids, some of the remaining carotenoids in
gerontoplasts turn out to be qualitatively
different. Leaf senescence causes formation of
xanthophylls acyl esters. Senescence induced
qualitative alteration of xanthophylls may also
occur, leading to the appearance of other
carotenoid species. The possibility of senescence
induced formation of ß- carotene epoxide. The
epoxide is known to be a product of
photo-oxidative events associated with oxidation
of ß carotene.
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Nucleic acids
RNA Senescence is a process of overall decline
in metabolism, including RNA and protein
synthesis. The quantitative decline in RNA is
explained mainly due to the decrease in ribosomal
RNA (rRNA), which is the most abundant cellular
RNA in both the chloroplast and cytosol. This
decrease correlates with the decline in protein
synthesis. Variations in relative amounts of two
phenylalanyl transfer RNA (tRNA) have been also
detected during senescence. However other tRNA
levels do not change or even increase in
senescence tissues, although the variations could
be due to differential rates of degradation.
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tRNA synthase activities are grately reduced
during senescence, probably limiting the
translational capacity of senescing
chloroplasts. DNA The appearance of the nucleus
is maintained throughout senescence without major
changes, although a decrease in nuclear DNA has
been described at the final stages of senescence
in peanut leaves, tobacco and soyabean cotyledons.
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Enzymes

• There is loss of Rubisco activity during
  senescence.
• Decrease in the level of mRNA responsible for the
  synthesis of the small subunit of
• Rubisco during dark-induced senescence
  (mustard cotyledons).
• The activity of protease increases, which
  increases degradation of proteins.
• There is an increase in peroxidase, which takes
  part in degradation of lipid and
• bleaching of carotenoids.

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Proteins/ amino acid

• Leaf senescence is characterized by a progressive
  decrease of total protein content (Brady 1988).
  The patterns of protein loss are characteristic
  and independent of the cause of senescence. Many
  specific proteins are degraded while others
  remain intact.

• Reduced synthesis and enhanced proteolysis are
  responsible for protein loss observed during
  senescence.

Increased protein breakdown may result from
different mechanisms de novo synthesis of
proteolytic enzymes, activation of pre-existing
proteases, decompartmentalization of proteases
and their substrates for degradation
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In green organs, chloroplast proteins are
principle targets of degradation during early
phase of senescence. The loss of chlorophyll
correlates with degradation of chlorophyll
carrying thylakoidal proteins, whose lysis is
strongly retarded by continuous illumination.
However, stromal proteins rapidly disappear under
the same conditions, indicating that breakdown of
the membrane and soluble proteins is differently
regulated by light.
The most abundant soluble protein in
chloroplasts, rubisco (ribulose 1,5-bisphosphate
carboxlyase/oxygenase) represents more than 50
of the chloroplast nitrogen and about 25 of that
of the whole cell. The rubisco is known to be
degraded extensively and selectively at early
stages of senescence in many plants.
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The specific proteolysis of this enzyme makes up
to 85 of the soluble protein lost in senescing
barley leaves and more than 90 of the nitrogen
mobilized from leaves before abscission in apple
trees. During senescence amino acids are
accumulated in detached leaves and with time
there is an increase utilization of these amino
acids are respiratory substrates, while the
carbon skeletons are used in respiratory
metabolism. The nitrogen components appear to be
accumulated in the amino residues of glutamine
and aspartate. In early senescence glutamine
formation predominates while in the later stages
of senescence aspartate biosynthesis
predominates.
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Petal senescence in D. chinensis has been found
to be accompanied by increased protein
degradation with a concomitant increase in the
protease activity.
Stages of flower development and senescence in
Dianthus chinensis L
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Loss of lipids and membranes
The outstanding change, which occurs in the cells
of senescence plant, is the deterioration of
membranes. Changes in cells membrane composition
and membrane permeability occur during
senescence. Many studies have been conducted on
changes in the ultra structure of leaves and
cotyledons during senescence.
In photosynthetic tissue, the chloroplast
thylakoids are typically the first membrane to
signs of deterioration. As deterioration of
thylakoid continues, the ER, mitochondria and
tonoplast show evidence of deterioration. Such
changes also occur in non photosynthetic tissues
like cotyledons, roots, xylem and phloem during
senescence.
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• In photosynthetic tissue, lipids associated with
  the chloroplast are the first lipids to decline,
  while the non-chloroplast lipids decline later.
  In plasma, microsomal and chloroplast membranes,
  it has been reported that there are 400 increase
  in the sterol phospholipid ratio as senescence
  progresses.

• Changes in the fatty acid content have also been
  reported in many tissues undergoing senescence.
  Degradation of lipid and the subsequent action of
  lipoxygenase on fatty acids play an important
  role in destruction of the various membranes of
  the cell. The activity of superoxide dismutase
  decreases during senescence.

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• Many studies reveal that membranes in mature
  tissue are in liquid crystalline state, while in
  senescence tissue the membranes or portion,
  thereof, are in crystalline gel phase at
  physiological temperature. Therefore during
  senescence the membrane lipid progressively
  becomes crystalline.

• Senescence process increases the permeability of
  the membrane and so contributes to loss of
  compartmentation. It is very likely that the
  increase in the permeability is at least partial
  due to the occurance of the crystalline gel phase
  in membrane lipids. It also helps in
  translocation of materials from cotyledons to
  other tissue.

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Petal senescence has been shown to be genetically
programmed and involves degradation of proteins,
lipids, carbohydrates and nucleic acid. Flower
petals are ideal tissues for cell death studies
as they are short lived, the tissue is relatively
homogenous and chemical manipulation can be
applied without substantial wounding. Petal
senescence has been found to be accompanied by an
increase in the activity of catabolic enzymes,
ion leakage and nuclear fragmentation. This is
all directed towards mobilization of nutrients
from petals to other parts of the plant such as
developing ovary.
a
b
a) Stages of flower development and senescence in
Helleborus orientalis. Note that during
senescence the sepals turn green. b) Increase in
the pistil dimension during flower development
and senescence
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Thank You.