By: Nazish Nehal, M. Tech (Biotechnology), University School of Biotechnology (USBT), Guru Gobind Singh Indraprastha University, New Delhi (INDIA)

1. Molecular basis of Plant resistance to
Abiotic stresses like High temperature
and Heavy metals

2. Introduction
• Higher plants are sessile and therefore cannot
escape abiotic stress factors
• This immobile nature requires more protection
and this enabled them cope with the different
stress factors
• Thus the plants have to alter their physiologies,
metabolic mechanisms, gene expressions and
developmental activities to cope with the stress
effects
• Gene products play a key role in the molecular
mechanisms of stress tolerance in plants.

5. Different types of Abiotic stress
•
•
•
•
•
•
•
Water stress
Salt stress
High temperature stress
Freezing stress
Photo-oxidative stress
Nutrient stress
Heavy metal stress

6. High Temperature stress
• High temperature stress in plants arises in response to
many factors such as :
 exposure of plants to high ambient temperatures
 exposure of germinating seeds to the soil which is
warmed by absorbed infrared radiation from the sun
 more plant transpiration followed by less water
absorption
 reduced transpiration capacity in certain plant organs
 forest fires
 natural gas blowouts, etc.

7. Effect of Heat stress
• Membranes self assemble from amphipathic lipids as a
result of hydrophobic interactions. Therefore, their
properties will depend critically on temperature.
• Membrane components suited to one temperature will
be inappropriate at other temperatures.
• Both membranes and proteins need to be flexible in
order to function in biological systems; this restricts
the temperature range over which they can be
biologically active.

8. Heat shock proteins
• Because of temperature sensitivity of the forces responsible for
protein folding , proteins are easily denatured by high temperature.
• Biological organisms have a suite of proteins that are made in
response to high temperature that appear to be designed to
prevent or reverse the effects of heat on protein denaturation
called heat shock proteins (HSPs) which are not present, or are
present in small quantities in unstressed organisms.
• These HSPs are involved in cellular repair, rescue, cleanup and/or
protection during the stress and from its recovery.
• The hsps are divided into families based on their molecular weight,
eg. Hsp 100 (with mol. Wt. between 100 to 104)
• The different hsp families appear to have different functions.

9. Different families of Hsp
•
•
•
•
Hsp 100
Hsp 90
Hsp 70
Hsp 60
• heat stress may affect membrane function and protein folding
• Two plant processes that are particularly sensitive to heat stress are
pollen development and photosynthesis.
• Recent evidence indicates that there are heat shock factors with
DNA binding domains that may be important components in the
transduction pathway between high temperature stress and gene
expression leading to accumulation of heat shock proteins

10. miRNA
• Wheat miRNAs showed differential expression in
response to heat stress by using Solexa highthroughput sequencing cloned the small RNAs from
wheat leaves treated by heat stress
• Among the 32 miRNA families detected in wheat, nine
conserved miRNAs were putatively heat responsive.
• For example:
 miR172 was significantly decreased, and miRNAs
(including
miR156, miR159, miR160, miR166, miR168, miR169,
miR393 and miR827) were upregulated under heat
stress

11. siRNA
• Abiotic stress responsiveness has also been observed in
a pool of T. aestivum small noncoding RNAs
• In wheat seedlings heat stress substantially change the
expression of four siRNAs:
 siRNA002061_0636_3054.1 is strongly downregulated by
heat
 siRNA 005047_0654_1904.1 is greatly downregulated by
heat
 siRNA080621_1340_0098.1 is downregulated by heat

12. Strategies for improving heat tolerance
• Improving heat tolerance has been attempted by various biotechnological
methods:
 Controlling the composition of membranes. For example, Arabidopisis
plants can be engineered to prevent them from making trienoic fatty
acids in their chloroplasts.
 Improving heat stress is engineering constitutive or over-expression of
Hsps. Hsp 100 family members and the smhsps have both been implicated
in thermotolerance of plants
 Perhaps more promise lies in overexpressing heat shock factors. These
are genes that control the expression of other genes and so changing the
expression of one heat shock factor will affect the expression of many
genes that through evolution, have come to be controlled by the same
control.

13. Heavy metal stress
• It
is
apparent
that
complex
network
of
transport, chelation, and sequestration processes has
evolved over time that functions in maintaining
concentrations of essential metal ions in different cellular
compartments within a narrow physiological range, thus
minimizing the damage caused by entry of non-essential
metal ions into the cytosol
• Important components of heavy metal homeostasis and
detoxification systems are:
 membrane-based heavy metal transporters
 intracellular metal chaperones for efficient distribution of
scarce essential metals, chelation and sequestration
processes
• Loss of any one of these critical processes will lead to

14. 4 main families of metal transport
proteins
• P-type ATPases; e.g.heavy-metal ATPases (P1B)
• Cation diffusion facilitators (CDF-transporters)
• ZRT-/IRT- like proteins (ZIP-transporters)
• Natural resistance associated Macrophage
proteins (Nramp-transporters); e.g. AtNramp

15. Iron-regulated zinc-regulated
transporters
• Arabidopsis IRT1 gene (first isolated transporter gene) is a major
transporter responsible for high affinity iron uptake from the soil
• Its abundance is controlled at both levels of transcript and protein
accumulation. Overexpression of IRT1 does not confer dominant
gain-of-function enhancement of metal uptake.
 Iron deficiency results in induction of IRT1 transcript accumulation
 Iron sufficiency results in reduction of IRT1 transcript levels.
• High levels of zinc and cadmium also contribute to reduction in
IRT1 transcript levels.
• ZIP1 and ZIP3 are expressed in roots in response to zinc deficiency,
thus suggesting roles in transport of zinc from the soil into the
plant.
• ZIP4 is induced in both shoots and roots of zinc-limited plants, thus
it may be involved in the transport of zinc either intracellularly or
between plant tissues.

17. Chelation
• Once metal ions enter the cell, they are bound by chelators and
chaperones.
 Chelators contribute to metal detoxification by buffering cytosolic
metal concentrations
 Chaperones specifically deliver metal ions to organelles and metalrequiring proteins.
• Among heavy metal- binding ligands in plant cells are:
 Phytochelatins (PCs) : Overexpression of the wheat PCS gene
TaPCS1 in Nicotiana glauca (shrub tobacco) also increased tolerance
to metals such as Pb and Cd.
 Metallothioneins (MTs): In Saccharomyces cerevisiae cup1∆ mutant
ABDE-1 (metal-sensitive) confirmed the functional nature of this
mcMT1 genein sequestering both essential (Cu, Zn) and nonessential metals (Cd, Pb, Cr).

18. Chaperones
• In recent years a family of soluble metal receptor
proteins, known as “metallochaperones”, that are active in
intercellular metal trafficking has emerged.
• For example:
 In prokaryotic cells and Arabidopsis thaliana, small
cytosolic proteins, designated as copper chaperones, have
been identified.
 A copper chaperone (CCH) and a responsive to antagonist 1
(RAN1), both identified in A. thaliana, were the first Cu
delivery systems identified in plant cells
 Orthologues of the three copper chaperones characterized
in yeast, ATX1, CCS and COX17, have been found in A.
thaliana

19. miRNA
• miRNAs are involved in plant responses to nutrient stress
• According
to
recent
studies
with
Arabidopsis, miR399, miR395, and miR398 are induced in
response to phosphate-, sulfate-, and copper-deprived
conditions, respectively.
• Phosphate homeostasis is partially controlled through
miR399, which targets a gene encoding a putative
ubiquitin- conjugating enzyme (UBC24).
• miR399 is upregulated in low phosphate-stressed plants
and is not induced by other common stresses, whereas its
target, UBC24 mRNA, was reduced primarily in roots of
plants exposed to low-phosphate stress

20. Phosphate
• It is likely that an MYB transcription factor, PHOSPHATE
STARVATION RESPONSE 1 (PHR1), is involved in miR399
expression. PHR1 is expressed in response to phosphate starvation
and positively regulates a group of phosphate-responsive genes
by binding to GNATATNC cis-elements [78–80]. This cis-element
has been found upstream of all known miR399 genes in
Arabidopsis [76,78]. Further- more, phr1 mutants show a
significant decline in miR399 induction under phosphate stress
[76,78]. miR399 has been isolated from phloem, and its
abundance in phloem increases upon phosphate starvation
• miRNAs themselves can be subject to posttranscrip- tional
regulation, as revealed by the discovery of the INDUCED BY
PHOSPHATE STARVATION1 (IPS1) gene, which acts as a target mimic
to control miR399 action

21. Sulfur
• Sulfur (S) is one of the essential macronutrients and
is available in the form of sulfate in the soil.
• miR395 targets both ATP sulfurylases (APSs) and the
sulfate transporter AST68.
• Sulfate deprivation induces the expression of miR395
with a concomitant decrease in transcript levels of
APS1.
• The abundance of miR395 in the phloem increases for
Brassica plants deprived of S, and the increase was
much stronger in the phloem than in the root, stem,
or leaf tissue

22. Copper
• The plant micronutrient copper (Cu)
photosynthesis, oxidative responses, and
processes.
is essential for
other physiological
• miR398 is a key regulator of Cu homeostasis.
• When Cu is limiting, the level of miR398 increases, and this reduces
the allocation of Cu to CSDs (CSD1 and CSD2) and therefore makes
Cu available for other essential processes.
• In Brassica under Cu deprivation, miR398 is upregulated not only
in leaf, stem, and root tissue but also in phloem sap.
•
According to more recent research, Brassica phloem sap contains
a specific set of small RNAs that are distinct from those in leaves
and roots, and the phloem responds specifically to stress
• In higher plants, the Cu/Zn-SODs are replaced by Fe-SODs

23. Cadmium
•
Cadmium (Cd) is one of the most toxic metals in agricultural soils.
•
Recent studies identified a set of conserved and non-conserved miRNAs that are differentially
regulated in response to heavy metals in rice.
•
miRNAs help regulate plant responses to heavy metal stress in addition to other abiotic stresses. In
B. napus, expression of miRNAs shows different responses to sulfate deficiency and Cd exposure
•
miR160 was transcriptionally downregulated by sulphate deficiency and by Cd exposure
•
miR164b and miR394a,b,c in roots and stems were upregulated by sulfate deficiency
•
Similarly, treatment with Cd induced expression of miR164b and miR394a,b,c in all tissues except
that miR164b was downregulated in leaves.
•
miR156a and miR167b in roots and miR156a and miR167c in leaves were upregulated under
sulfate deficiency. In contrast, miR167a and miR168 in roots and miR167a, miR167b, and miR168
in leaves were downregulated.
•
Under Cd stress, most B. napus miRNAs are induced. Notably, miR156a, miR167a, and miR167c
in roots and miR167a and miR167c in leaves were strongly upregulated.
•
Rice miRNAs showed different patterns of expression in leaves and roots. miR601, miR602, and
miR603 in roots were upregulated while miR602 and miR606 in leaves and miR604 in roots were
downregulated by Cd exposure

24. Mercury and Aluminium
• Mercury (Hg) and aluminum (Al) regulate the expression of
miRNAs in M. truncatula.
• miR171, miR319, miR393, and
response to Hg, Cd, and Al
miR529 are upregulated in
• miR319 showed weak constitutive expression in leaves. It is
upregulated by Cd and Al but was not affected by Hg.
• Similarly, expression of miR393 was not affected by Al but
was slightly upregulated by Hg and Cd.
• In contrast, miR166 and miR398 are downregulated by Hg, Cd,
and Al exposure

25. Iron
• Expression of miRNAs also is affected by Fe deficiency,
in Arabidopsis
 miR169b, miR169c, miR172c, miR172d, miR173 and
miR394b in roots
 miR169c, miR172c, miR172d, miR173, miR394a and
miR394b in shoots
initially upregulated and then down- regulated during
the period of Fe deficiency.
• These results indicate that the cloned miRNAs
respond to Fe deficiency

26. Thank-you…
By:
Nazish Nehal,
M. Tech (Biotechnology),
University School of Biotechnology (USBT),
Guru Gobind Singh Indraprastha University,
New Delhi (INDIA)