ROLE OF miRNAS IN PLANT DROUGHT
TOLERANCE
Faisal Qaseem
08-arid-876
PhD Scholar
Botany
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Drought
Mechanisms of drought tolerance
History of miRNAs
Formation of miRNA
Mechanism of action
miRNA responses to drought stress
Expression of mRNA in different plant species
miRNAs involved in auxin signaling
Drought
• Drought is one of the most common environmental
stresses affecting growth, development and yield of
plants
• Plant tolerance to drought is important for the
improvement of crop productivity
• Different defense strategies
– Escape the drought
– Drought avoidance (high tissue water potential)
– Enhanced water acquisition by deep root system
Mechanisms of drought tolerance
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Maintenance of turgor through osmotic adjustment
Increased cell elasticity and
Decreased cell size
Desiccation tolerance via protoplasmic tolerance.
Molecular Mechanism
Switching on of drought resistant genes
Transgenic plants overexpressing some droughtresponsive genes did not exhibit significant improvements
or had no improvement at all for drought Tolerance.
• This may reflect the fact that the plant drought stress
responses, tolerance mechanisms and genetic control of
tolerance are complex
• Expression of microRNAs (miRNAs) has been found
to be altered in plants during drought stress.
• Drought response mechanisms which can potentially
be targeted in development of new drought tolerant
crops
History of miRNAs
• miRNA were first discovered in the nematode
Caenorhabditis elegans in 1993 at which time they
were considered as small temporal RNAs
• In 2001, miRNAs were formally named and
recognized as a distinct class of RNAs with
regulatory functions
• Plant miRNAs were identified 10 years after animal
miRNAs
• 7385 mature miRNAs and 6150 precursor miRNAs
(pre-miRNAs) have been identified in 72 plant
species
• miRNAs are single-stranded noncoding RNAs sized
usually between 20 and 24 nucleotides (nt) that serve
as gene regulators in a wide range of organisms
Function of miRNA
• Different processes are affected by miRNA including
development of organs such as roots, stems, leaves
and flower parts
• miRNAs play key roles in plant responses to biotic
and abiotic stresses.
Mechanism of action
• miRNAs mediate the responses by
– modulating the amount of themselves,
– the amount of mRNA targets or
– the activity/mode of action of miRNA–protein
complexes.
• In turn, these changes modify the timing, location and
amount of proteins expressed from other genes upon
exposure to the stress.
• Plant miRNAs are derived by processing of their
RNA precursors.
• Such precursors are occasionally transcribed from an
intron or exon of a protein coding region, but most
precursors are transcribed from the intergenic regions
of genomes
• A few miRNAs can be generated independently of the
splicing pathway, but details of their maturation are
obscure
Formation of miRNA
• In Arabidopsis after transcription by Pol II or Pol III
enzyme into primary miRNA (primiRNA), the miRNA
gene is processed by Dicer-like (DCL) into a stem-loop
miRNA::miRNA* duplex.
• The miRNA::miRNA* duplex is processed by DCL1,
with assistance from the double-stranded RNA-binding
protein HYL1.
• The 3ʹ ends of miRNA duplexes are methylated by HEN1
and loaded onto AGO1.
• The miRNAs are then exported to the cytoplasm by a
HASTY protein and cleaved into mature miRNAs.
• Mature miRNAs are incorporated into the RNAinduced silencing complex (RISC), where the mature
single-stranded miRNA guides the RNA slicing
activity of AGO1 (Argonaute) to partially
complementary mRNA.
• In plants, miRNAs generally interact with their
targets
through
perfect
or
near-perfect
complementarity and lead to the cleavage of target
mRNA
• Loop-derived miRNAs were recently identified and shown to
be functional
• How these loop-derived miRNAs are generated has not yet
been elucidated.
• Hypothetically, miRNA precursors could be processed into
three single-stranded short RNA molecules: the 50 -arm (5p),
the 30 -arm (3p) and the loop (loop-miR
Mechanism of action
• miRNA-directed DNA methylation
• This methylation occur at occurs at cytosine in all
sequence contexts
• This way of regulation is very similar to siRNA directed
DNA methylation
• siRNA is a class of double-stranded small RNAs of 21–24
base pairs in length, which plays important roles in the
RNA interference (RNAi) pathway.
canonical
miRNAs
(cmiRNAs)
are generated by DCL1 and specifically loaded into AGO1
clade proteins to form effector complexes to direct the
cleavage of their target mRNAs.
Long miRNAs (lmiRNAs) are processed by DCL3 and sorted into AGO4 clade proteins.
LmiRNAs bound to AGO4 proteins interact with nascent transcripts transcribed from
their own loci or target genes, thereby recruiting de novo cytosine methyltransferase
DRM2 to methylate the adjacent DNA.
• In contrast to siRNAs, miRNA directed DNA
methylation is affected by multiple factors
– Dicer member
– miRNA size
– AGO member
– Stability of the duplex miRNA
• In plants, miRNAs mainly function at the posttranscriptional gene silencing (PTGS) level and guide
the AGO protein to cleave the target mRNA between
positions 10 and 11
• Exact way of translational inhibition is still obscure
• During translation, miRNAs do not result in mRNA
cleavage, but arrest translation by blocking readthrough of the ribosome
• A recent study showed that miRNA-directed
translation inhibition occurs at the endoplasmic
reticulum (ER) and requires ALTERED MERISTEM
PROGRAM1 (AMP1)
• Homologues of AMP1 are present in animal
genomes, it is possible that the connection between
the ER and translation inhibition by miRNAs is
conserved across plants and animals.
• Taken together, it is suggested that miRNAs may
regulate the expression of their target genes via a
combination of the aforementioned mechanisms
miRNA responses to drought stress
• Drought stress has been revealed to alter expression of
many genes/metabolites
• Dehydrins
– Vacuolar acid invertase
– Glutathione S-transferase (GST)
– Abscisic acid (ABA)-inducible genes
• LEA (late embryo abundant)
• RAB (responsive to abscisic acid)
• COR (cold regulated)
• Rubisco (5-bisphosphate carboxylaseoxygenase)
• helicase, proline and carbohydrates
• miRNAs as gene regulators are expected to
participate in the regulation of these drought
responsive genes.
• Expression of miRNAs is themselves altered in
response to drought stress.
Expression of mRNA in different plant species
• Drought-responsive miRNAs have been reported in
many plant species such as
• Arabidopsis
• Cowpea
• Tobacco
• soya bean
• Phaseolus vulgaris
• In Arabidopsis, miR156, miR159, miR167, miR168,
miR171, miR172, miR319, miR393, miR394a,
miR395c, miR395e, miR396 and miR397 are upregulated
• while miR161, miR168a, miR168b, miR169,
miR171a and miR319c are down-regulated, under
drought stress
Role of some of miRNAs
• miR159
• MYB and TCP transcription factors
• ABA response, NaCl stress response, floral asymmetry
and leaf development
• miR171
• GRAS transcription factors
• response to abiotic stresses and floral development
• miR397
• Laccases
• lignin biosynthesis, ion absorption and stress response
• miR170
• SCL transcription factor
• radial patterning in roots, floral development and
shoot branching
• The up-regulated miRNAs were also shown to be
involved in different developmental stages suggesting that
the regulation of drought tolerance and development by
miRNAs is tightly linked, which probably undergoes via
the same mechanism.
• Expression level or drought responsiveness of a miRNA
is species dependent
• For example
• Drought up-regulates miR156 in Arabidopsis, Prunus
persica, barley, Panicum virgatum and Triticum
dicoccoides
• Down-regulates it in rice and maize
• In some plant species, members of the same families were
found to be differently expressed under drought stress
• For example
• Drought stress down- and up-regulates respective
members of the miR319 family in rice
• In fact, even the same miRNA in the same plant species
can show different responses to drought depending on the
exact conditions.
• Expression level of miR398a/b in Medicago truncatula
was increased under drought stress while in another study,
expression level of the same miRNA in the same plant
species decreased under drought stress
• Such differences may reflect different degrees of drought
stress and high sensitivity of some miRNAs to subtle
differences in growing conditions.
• It is possible that differential expression of the same
miRNA in the same plant species under drought
conditions is the result of different spatial–temporal
manner.
• It is likely that under drought conditions regulators of
miRNA genes change their expression, which in turn
leads to the change in expression of miRNAs and
ultimately that of miRNAs’ targets
Expression of miRNA between different tissues
• miR169 (encoded by many loci in plant spp.)
• In rice is induced more prominently in the roots than in
the shoots.
• Over expression of miR169c in tomato reduced stomatal
conductance and water loss compared to non-transgenic
tomato and hence enhanced drought tolerance
• These differences in outcomes of over expressing
miR169c in different plant species have been suggested to
be caused by different timing, duration and intensity of
the stress that was applied in the different studies
• The contribution of miR169 to drought tolerance or
intolerance
• depend on its promoter
• two dehydration- responsive elements (DREs) were
identified in the promoter of MIR169g
miRNAs involved in ABA response
• Abscisic acid (ABA), a key plant stress hormone, is
produced de novo under water-deficit conditions
– expression of stress-related genes
– initiation of stomatal closure
• Indications that miRNAs participate in the ABA response
were first provided by isolation of ABA-hypersensitive
mutants impaired in any of several key genes of the
miRNA biogenesis pathway, such as HYL1, DCL1,
HEN1, SE, and HASTY.
• The hyl1 mutant was shown to be hypersensitive to
ABA during Arabidopsis germination
• Mutant alleles for dcl1 and hen1 increased ABA
sensitivity during germination.
• It is possible that these mutants reveal a defect in a
particular miRNA that is rightfully involved in the
regulation of the ABA response process.
• miR159 was induced by ABA and drought treatments
in germinating Arabidopsis seeds
• In Arabidopsis, miR159a mediates the cleavage of
MYB33 and MYB101 transcripts
miRNAs involved in auxin signaling
• miR393 was commonly upregulated during drought
stress in Arabidopsis
• The target of miR393 encodes TIR1 (transport
inhibitor response 1), an auxin receptor in
Arabidopsis.
• The TIR1 enzyme is a positive regulator of auxin
signalling by promoting the degradation of Aux/ IAA
proteins through ubiquitination
• Increased levels of miR393 would downregulate
auxin signaling and may reduce plant growth under
drought stress.
• miR390 does not target a protein-coding mRNA but
rather triggers the production of tasiRNA
• regulates lateral root emergence and organ polarity
establishment by targeting transcription factors such
as ARF2, ARF3, and ARF4
• Auxin response factors (ARFs) are important
transcription factors involved in auxin signal
transduction by binding to specific cis-elements in the
upstream regions of auxin-inducible genes
• Several ARF gene family members have been
confirmed as target genes for miRNAs.
• ARF10, ARF16, and ARF17 are targeted by miR160,
while miR167 guides the regulation of ARF6 and
ARF8, which apparently negatively regulate free
indole acetic acid levels by controlling GH3-like gene
expression
miRNAs involved in osmotic adjustment
• Osmotic adjustment represents a general mechanism
to maintain cell turgor and to stabilize protein
structure during drought stress
• Plants and other organisms cope with drought stress
by producing and accumulating various
• Osmoprotectants
• Amino acids
• Sugars
• Sugar alcohols
• Proline has multifunctional role in defence
mechanisms. It acts as an osmolyte, a free radical
scavenger, and a stress-related signal
• Proline accumulation in plants is caused not only by
the activation of proline biosynthesis but also by the
inactivation of proline degradation.
• Proline degradation to glutamic acid in higher plants
is catalysed by proline dehydrogenase (PDH)
• Antisense suppression of PDH gene in Arabidopsis
led to an accumulation of proline
• miR474 is linked to the process of proline
degradation, because it targets the PDH gene in maize
• miR474 was upregulated during drought stress in
maize
• upregulation of miR474 decreased the expression
level of PDH, leading to the accumulation of proline
and thus an improvement in plant stress tolerance
under drought-prone conditions
miRNAs involved in antioxidant defense
• One of the inevitable consequences of drought stress is
enhanced reactive oxygen species (ROS) production in
the different cellular compartments
• ROS are potentially dangerous to plants
• Plants have evolved defence systems, consisting of
antioxidative enzymes and low-molecular-weight
antioxidants to scavenge ROS.
• Superoxide dismutase (SOD), catalase (CAT), peroxidase
(POD), ascorbate peroxidase (APX), and glutathione
reductase (GR) are important parts of the antioxidative
enzyme system
• miR528 was downregulated by drought in maize
seedlings
• POD is a predicted target of miR528
• RT-PCR analysis showed that the expression level of
POD was upregulated because of the downregulation
of miR528.
• Promote the removal of excessive H2O2 and alleviate
the injury caused by ROS
• miR398 targets two closely related Cu/Zn SODs
(CSD1 and CSD2) and cytochrome C oxidase subunit
V (COX5b).
• The CSD enzymes are involved in oxidative stress
detoxification
• COX5b functions in electron transport in the
mitochondrial respiratory pathway
• miR398 was downregulated under drought stress in
maize
• lead to increases in the activities of CSDs and
consequently oxidative stress tolerance.
miRNAs involved in photosynthesis and respiration
• Drought stress is known to inhibit photosynthetic
activity and photosynthetic electron transport capacity
• Photosynthetic activity is shown to be suppressed
after drought stress, whereas respiration is enhanced
by drought.
• The fixation of CO2 and the synthesis of starch are
important biochemical processes for plant growth
• miR397 is predicted to target a laccase gene which
was reported to reduce root growth under dehydration
in a knockout mutant
• Maintaining a reasonable rate of synthesis of carbonhydrogen compounds helps to protect against drought
stress in plants.
• The expression of miR397 was downregulated in
drought-stressed rice
• miR397 is predicted to target fructofuranosidase,
which takes part in starch and sucrose metabolism
• Change in miR397 expression plays a role in the
reductive carboxylate cycle (CO2 fixation) and
energy supply.
• miR398 plays a role in the regulation of respiration,
as it targets cytochrome C oxidase subunit V
(COX5b), which functions in electron transport in the
mitochondrial respiratory pathway
• miR398 was reported to be upregulated in T.
dicoccoides
• The increased level of miR398 led to the down
regulation of COX5b transcripts, indicating the
significance of miR398 in the regulation of
mitochondrial respiration under water deficit
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Conclusion
Changing climate, variable weather patterns and other
environmental stresses are a matter of concern for
agricultural crop production.
Drought tolerance is a complex trait involving a
number of gene regulatory networks that miRNAs
participate in.
The mechanisms of miRNAs involvement in stress
tolerance and their target regulatory networks are not
well understood.
This is partly due to the possibility of each
endogenous miRNA regulating multiple genes and
each gene being regulated by multiple miRNAs.
Future prospect
• Major challenge ahead will be to discover the miRNA
targets and how miRNAs function on the targets.
• Identification of miRNAs/ targets that influence
drought tolerance
• To characterize the cis-regulatory elements in the
miRNAs genes
• To determine the corresponding TFs and to describe
how the miRNAs are regulated by drought
References
• Ferdous, J., Hussain, S.S. and Shi, B.-J. (2015) Role
of microRNAs in plant drought tolerance. Plant
Biotechnology Journal.
• Y. Ding, Y. Tao and C. Zhu. 2013 Emerging roles of
microRNAs in the mediation of drought stress
response in plants. Journal of Experimental Botany.