miRNA and stress regulation

1. “Small RNAs as Big Players in Plant Stress
Responses”
Title

2. Introduction
• Plant growth and productivity is adversely affected by frequent
exposure to a plethora of stress conditions
• All stress factors are a menace for plants and prevent them from
reaching their full genetic potential and limit the crop productivity
thereby threaten the sustainability of agricultural industry.
• Various genes are up regulated and down-regulated, to mitigate
the effect of stress and lead to adjustment of the cellular milieu
and plant tolerance.

3. The most practical definition of stress is “an adverse force or a
condition, which inhibits the normal functioning or well being of a
biological system such as plants” (Jones and Jones 1989).
Stress

4. • Abiotic stresses
• 1. Cold
• 2. Heat
• 3. Salinity
• 4. Drought
• 5. Radiations
• 6. Oxidative stress
• 7. Nutrient deprivation
• Biotic stresses
• 1. Pathogens
• 2. Insects
• 3. Hebivores
Various stress elicitors

5. Small RNA (sRNA)?
• A class RNAs with 20-30 nucleotides (nt) in length.
• They tend to target chromatin as well as transcripts, thus regulating
both genome and transcriptome.
• All known types of ncRNAs are recognized as small RNAs.
sRNAmRNA rRNA, tRNA
miRNAs*, siRNAs, piRNAs,
vsi RNAs

6. Classification of eukaryotic ncRNAs
Basis of
classification
Types
Origin, properties
and functions
1.DNA markers, playing roles in dosage compensation and
imprinting: Xist, roX, PAT-1
2.Gene regulators, affecting activity of genes: DISC2, RNAI,
RNA-OUT
3.Abiotic stress signals, synthesized/processed in response to
abiotic stress: gadd7/adapt15 adapt33, G90
4.Biotic stress signals, induced by biologically active
compound: His-1, CR20, GUT15
Predicted
functions
1.Cellular debris ncRNA: RNAs with no specific function
2.Housekeeping ncRNA: tRNA, rRNA, small nuclear RNA,
small nucleolar RNA, signal recognition particle RNA,
3.Regulatory ncRNA: miRNAs, siRNAs
Role in RNA
silencing
1. miRNA
2. siRNA
Yadav SK et al. 2011. Genomics Proteomics & Bioinformatics. 9: 183-199

7. Differences and similarities between sRNAs and mRNAs
Property Non coding RNAs Protein coding RNAs
Length 20-30 nt (processed small RNAs)
64-303 nt (plant precursors)
60-70 nt (animal precursors)
Polynucleotides
Location of synthesis Nucleus and cytoplasm Nucleus and cytoplasm
RNA polymerase required RNA polymerase II and IV RNA polymerase II
Protein synthesis No Yes
Binding to Argonaute protein Yes No
Expression pattern Mostly tissue- and developmental
stage-specific expression
Only few with tissue- and
developmental
stage -specific expression
Energy consumption Expressed without translation,
requiring less energy
Translation, requiring relatively
higher energy
Open reading frames Absent Present
Degradation rate Less stable More stable
Functions Transcriptional and post-
transcriptional gene silencing
Expression of genes
Yadav SK et al. 2011. Genomics Proteomics & Bioinformatics. 9: 183-199

8. • MicroRNAs (miRNAs):
• miRNAs are about 20–22(nt),
single-stranded RNAs processed by
the Dicer-like (DCl) family of
enzymes in plants.
• A capitalized "miR-" refers to the
mature form of the miRNA, while
the uncapitalized "mir-" refers to
the pre-miRNA and the pri-miRNA.
• Genes encoding miRNAs in plants
are annotated as MIR genes.
What are miRNA’s?
Bartel DP. 2004. Cell. 116:281–297

9. How miRNA was Discovered?
Victor Ambros & Rosalind Lee
Rhonda L Felnbaum
Victor Ambros and Gary Ruvkun won 2015’s
Breakthrough Prize in Life Sciences.

10. Gary Ruvkun
The first miRNA of plant originwas identified in 2002 in Arabidopsis thaliana (Llave et al.
2002; Park et al. 2002; Reinhart et al. 2002).

11. Identification of Plant MicroRNA’s (miRNAs)
• Methods for identifying miRNAs are based on their
major characteristics :
• Small non-coding RNAs, with 20–22 nt for animals
and 20–24 nt for plants.
• A well-predicted stem loop hairpin structure.
• Many miRNAs are evolutionarily conserved.
• Usually, only the mature miRNAs are conserved in
plants instead of miRNA precursors that are usually
conserved in animals.

12. • Primary miRNA transcript (pri-miRNA)
• Precursor miRNA (pre-miRNA)
• miRNA/miRNA*
• Mature miRNA
• Dicer like enzyme (DCL1)
• HYPONASTIC LEAVES 1 (HYL 1)
• HUA ENHANCER (HEN 1)
• HASTY (HST) (Exportin-5 Homologue)
• Argonaute protein (AGO)
• RNA induced silencing complex (RISC)
Biogenesis of miRNA in plants
Terminology used

13. Biogenesis of miRNA
Rhoades et al., Annu. Rev. Plant Biol. 2006

15. The Actions of Small Silencing RNAs
• (A) Messenger RNA cleavage. Black arrowhead indicates site of cleavage.
• (B) Translational repression (H3K4me3)
• (C) Transcriptional silencing, thought to be specified by siRNAs.
Bartel DP. 2004. Cell. 116:281–297

16. sRNAs and abiotic stress
• Plants respond to these adverse conditions in various forms,
broadly categorized as:
• Physiological responses-involve various proteins, transcription
factors and metabolites, etc.,.
• Genetic responses-involve epigenetic changes, including RNA-
directed DNA methylation, histone and DNA modifications,
which play an important role in altering gene expression against
stress conditions.
• It has been reported that regulation of stress-related genes and
miRNAs are correlated (Sunkar and Zhu 2004).

17. miRNA-mediated gene regulation upon auxin
perception and signaling under normal growth
and stress condition. ( Source: Sunkar et al. 2012.
Trends in Plant Sciences.)
Guilfoyle T. 2007. Nature

18. miRNAs regulating stress caused by light
• Light acts as one of the essential elements and can be stress to plant like other kind
of stresses .
• UV-B fraction is considered as the harmful.
• miRNAs possess same array of proximal promoter motifs as the UV-B responsive
protein-encoding genes and the inferred expression of miRNAs is negatively
correlated with the expression of target genes.

19. Chung et al. 2016. Plant physiology

20. miRNAs regulating Drought stress
• Suppression of lateral root growth by drought stress allows redirection of
resources towards production of deeper roots, enabling more efficient
extraction of water from deep in the soil.
• Under drought stress, ABA is formed in the dehydrating roots, which inhibits
lateral root growth.
• miRNA393 is found to be strongly up-regulated by ABA .
• Drought stress also triggers ethylene production in higher plants, which in
turn enhances leaf senescence.
• miRNAs influence leaf senescence to avoid drought as it can reduce canopy
size and transpiration, and allow remobilization of water and nutrients to
organs more crucial for survival and reproduction of the plant.

21. • In this study, miR164 was proposed to be a regulator of leaf senescence in
Arabidopsis, based on the fact that EIN2 (ETHYLENE INSENSITIVE 2), an
ethylene signalling protein in Arabidopsis, negatively down-regulates miR164 in
older leaves. This results in increasing levels of its targets NAC1, ORE1 and
At5g61430. Accordingly, miR164 overexpression and/or lack of its target ORE1
activity resulted in enhanced leaf longevity.
Kim et al. 2009. Science

23. miRNAs regulating Oxidative Stress
• Abiotic stress causes generation of ROS and their accumulation to toxic
concentrations.
• ROS cause oxidative damage to membrane lipids, proteins and nucleic acids.
• Plants have developed sophisticated ROS detoxification system.
• In Arabidopsis, abiotic stress-inducible Cu-Zn SOD genes, namely CSD1
(encodes cytosolic SOD) and CSD2 (encodes chloroplastic SOD), have been
identified as targets of abiotic stress-down-regulated miR398.
.

24. Source: Bej S and Basak J. 2014. American Journal of Plant Sciences. 5: 748-759.
Role of miR398 in Oxidative stress.

25. MicroRNAs Expression Under Salt Stress

26. Gao et al. 2016. Nature. 6: 19736

27. Role of miRNAs during Nutrient Depriviation
• Nutrient deficiency is caused by inadequate quantity of
mineral elements available in soil or the inability of plants to
absorb and use the minerals because of other factors.
• The nutrient acquisition and use depend on a precise
regulation of complex gene expression programs that govern
these mechanisms.
• miR399, miR395 and miR398 are induced in response to
phosphate-, sulfate- and Cu2+-deprived conditions,
respectively.

28. miRNAs regulating Sulfate Depriviation
• miR395 has been shown to be involved in the regulation of
sulfur homeostasis.
• Sulfur is taken up by plants in the form of inorganic sulfate.
Sulfur deficiency has resulted in some physiological changes
that suspended sulfate assimilation.
• miR395 has two potential targets:
• The first target is ATP sulfurylase (APS1, APS3 and APS4)
enzyme that catalyzes the first step of sulfur assimilation
pathway.
• The second target of miR395 is AST68, an Arabidopsis
sulphate transporter.

30. miRNAs regulating Phosphate Starvation
• Plants adapt to low Pi (Inorganic phosphate) soils in several ways; root
growth and architecture are altered to access a larger soil volume,
symbiotic association with mycorrhizal fungi, secretion of root exudates
(phosphatases, phosphodiesterases, organic acids), recycling of internal
Pi and alterations in metabolism.
• miR399 has two target genes belonging to different families: a
phosphate transporter and a putative ubiquitin conjugating enzyme
(UBS24) possibly involved in protein degradation .

31. Source: Bej S and Basak J. 2014. American Journal of Plant Sciences. 5: 748-759.

32. miRNAs regulating Copper Starvation
• Copper starvation induces miR398 expression, which further
suppresses the translation of CSD1 and CSD2 mRNA into Cu-
SOD proteins.
• CSD1 and CSD2 genes are induced under oxidative stress in
order to prevent superoxide radicals toxicity.
• Plants possessing CSD2 have demonstrated greater tolerance to
conditions inducing oxidative stress as compared to wild-type
plants. Reduction of Cu-SOD proteins also increases the
availability of copper for other biological processes.
• It suggests that miR398 is a part of regulatory network that
controls the copper ion concentration in plants.

33. ( Source: Sunkar R, Li FY and Jagadeeswaran G. 2012. Trends in Plant Sciences.)

34. miRNAs regulating cold and related stress conditions
• Cold stress i.e. chilling (below 20ºC) and freezing conditions
(below 0ºC) adversely affects plant growth and development.
• Chilling inhibits water uptake, while freezing induces cellular
dehydration, thus causing an osmotic stress and
hyperaccumulation of ROS as secondary effects.
• Plants are often acclimatized to cold conditions but certain
plants are sensitive to these chilling and freezing conditions.
• Post-transcriptional regulations are considered more critical for
cold tolerance.
• The up regulated cold-stress responsive miRNAs—miR393,
miR397b, miR402 and miR319c of Arabidopsis were first
reported by Sunkar and Zhu in 2004.

35. miRNAs regulating Mechanical Stress
• Various mechanical stresses involving wind, water, or any other
entity imposing physical forces upon the plant body have been
found to down- or up-regulate certain miRNAs.
• A comparative analysis of miRNA expression has been
performed in Populus trichocarpa subjected to mechanical stress
via bending the plant stem.
• The expression of miR156, miR162, miR164, miR475, miR480
and miR481 has been found to be down-regulated whereas
miR408 was up-regulated in the xylem tissue of mechanically
stressed plants as compared to the un-stressed control.
• Further studies on mutants over-expressing specific miRNAs or
miRNA-resistant target sequences would aid in determining the
role of these miRNAs.

36. Lu et al. 2005. The plant cell

37. miRNAs regulating Hypoxia
• Hypoxia is a stressful state caused by loss of oxygen.
• Plants possess various hypoxia responsive transcription factors
that possibly trigger anaerobic responsive genes.
• Licausi et al. (2011) analyzed around 1,900 transcription factors
and 180 miRNA primary transcripts in stressed Arabidopsis and
reported that simultaneous interaction of various transcription
factors possibly regulates the hypoxia induced genes, although
one miRNA (miR391) showed minor activity.

38. miRNA and Biotic stress
• Pathogenic bacteria, fungi, viruses, insect pests and nematodes
cause severe damage to plants.
• miRNA-guided posttranscriptional regulation plays a crucial role
in the plant defense against pathogens through targeting transport
inhibitor response 1 (TIR1), an auxin receptor.
• On the other hand, to counteract the host encoded
miRNAs/siRNAs-mediated silencing, pathogens (viruses) have
evolved silencing suppressor proteins such as P1-HcPro, P15,
P19, P25 and P38 proteins, which inhibit diverse steps of the
PTGS/RNAi pathway.

39. Model for auxin crosstalk in plant immune defense pathways
(Naseem et al. 2015. Journal of Experimental Botany.

40. Navarro et al. 2006. Science.

41. Stress–miRNA networks responsive miRNAs in Arabidopsis
( Source: Khraiwesh B, Zhu KJ, Zhu J. 2012. Biochimica et Biophysica Acta 1819: 137–148.)

42. The potential miRNA identified in biotic stress
conditions

43. The potential miRNA identified in different abiotic stress
conditions.
Source: Bej S and Basak J. 2014. American Journal of Plant Sciences. 5: 748-759.

44. Applications of miRNA
Strategies for developing miRNA/target mimics (TM) based genetically modified
technology
Gupta, Curr. Sci. 2015

45. Trait improved microRNA Target (mRNA) Plant Reference
Bacterial resistance miR167 TIR1 Arabidopsis Navarro et al.
2006
Cauliflower mosaic
virus (CaMV)
resistance
miR393 2b protein of
CaMV
Tomato Qu et al. 2006
Tomato leaf curl
New Delhi Virus
(ToCNDV)
resistance
amiR171 AV1/AV2 Tomato Vu et al. 2013
Turnip yellow
mosaic virus
(TYMV) and Turnip
mosaic virus
(TuMV) reistance
amiR-AV1-1 P69 of TYMV
and HC-Pro of
TuMV
Arabidopsis Niu et al. 2006
Drought tolerance miR169 NFYA5 Arabidopsis Li et al. 2008
miR169 GmNFYA3 Soybean and
Arabidopsis
Ni et al. 2013
Cold tolerance miR319 PCF5/PCF8 Rice Yang et al. 2013
miRNA in improvement of crops

47. Conclusion and Future prospects
•Plant stress (biotic and abiotic) tolerance is a complex traits and could
be improve thorough understanding of the transcriptional,
posttranscriptional and post-translational changes during stress.
•Post-transcriptional gene regulation mediated by miRNAs is
particularly interesting, because they have the ability to regulate
several protein coding genes belonging to related gene families or
genes potentially implicated in the same pathway.
•miRNAs could be serve as master regulators, as an altered miRNA in
response to stress can silence more than one genes simultaneously.
•Identification of stress-regulated small RNAs as well as elucidating
their functional significance will likely to broaden our understanding
of the post-transcriptional gene regulations involving small RNAs
during plant stress responses.

48. Conclusion and Future prospects
•Plant stress (biotic and abiotic) tolerance is a complex traits and
could be improve thorough understanding of the transcriptional,
posttranscriptional and post-translational changes during stress.
•Post-transcriptional gene regulation mediated by miRNAs is
particularly interesting, because they have the ability to regulate
several protein coding genes belonging to related gene families or
genes potentially implicated in the same pathway.
•miRNAs can serve as master regulators, as an altered miRNA in
response to stress can silence more than one genes simultaneously.
•Identification of stress-regulated small RNAs as well as elucidating
their functional significance will likely to broaden our understanding
of the post-transcriptional gene regulations involving small RNAs
during plant stress responses.