1. ROLE OF miRNAS IN PLANT DROUGHT TOLERANCE Faisal Qaseem 08-arid-876 PhD Scholar Botany

2. 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

3. 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

4. Mechanisms of drought tolerance 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 drought- responsive 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

5. 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

6. 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

7. 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

8. 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.

9. 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.

10. 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

11. 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 3 ʹ ends of miRNA duplexes are methylated by HEN1 and loaded onto AGO1.

12. The miRNAs are then exported to the cytoplasm by a HASTY protein and cleaved into mature miRNAs. Mature miRNAs are incorporated into the RNA- induced 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

13. 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

16. 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.

17. 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.

18. In contrast to siRNAs, miRNA directed DNA methylation is affected by multiple factors – Dicer member – miRNA size – AGO member – Stability of the duplex miRNA

19. In plants, miRNAs mainly function at the post- transcriptional 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 read- through of the ribosome

20. 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

21. 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

22. 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.

24. 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

25. In Arabidopsis, miR156, miR159, miR167, miR168, miR171, miR172, miR319, miR393, miR394a, miR395c, miR395e, miR396 and miR397 are up- regulated while miR161, miR168a, miR168b, miR169, miR171a and miR319c are down-regulated, under drought stress

26. 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

27. miR170 SCL transcription factor radial patterning in roots, floral development and shoot branching

28. 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

29. 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

30. 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

31. 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

32. 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

33. 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.

34. 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.

35. miR159 was induced by ABA and drought treatments in germinating Arabidopsis seeds In Arabidopsis, miR159a mediates the cleavage of MYB33 and MYB101 transcripts

36. 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

37. 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

38. 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.

39. 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

41. 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

42. 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)

43. 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

44. 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

45. 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

46. 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.

47. 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

48. Maintaining a reasonable rate of synthesis of carbon- hydrogen 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.

49. 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

50. 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.

51. 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

52. 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.