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Advances in
eISSN: 2373-6402

Plants & Agriculture Research

Research Article Volume 8 Issue 6

Salt stress tolerance in plants: the role of miRNAs

Shaifaly Parmer, Birendra Prasad Shaw

Institute of Life Sciences, India

Correspondence: Birendra Prasad Shaw, Institute of Life Sciences, Nalco Square, Bhubaneswar-751023, Odisha, India, Tel 9437488362

Received: December 18, 2017 | Published: November 26, 2018

Citation: Parmer S, Shaw BP. Salt stress tolerance in plants: the role of miRNAs. Adv Plants Agric Res. 2018;8(6):411-415. DOI: 10.15406/apar.2018.08.00360

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Abstract

Soil salinity is a challenge for agriculture worldwide as crop plants are more sensitive to it compared to non-crops. Development of salinity tolerant crop plants through breeding approaches has been quite a task to the breeder so far because of quantitative nature of the trait. Hence, great emphasis has been given to understand the mechanism underlying salt tolerance in plant so that crop cultivars tolerant to salinity may be developed by biotechnological interventions. The studies on this have basically been directed into two paths; firstly, identification and functional characterization of the genes encoding the effector proteins, including those involved in maintenance of ion homeostasis, osmolytes accumulation and reactive oxygen scavenging and secondly, understanding the regulatory aspects of the effector genes. Accordingly this review describes the issues related to salinity in agriculture with emphasis on understanding on the current developments in regulatory aspects of salt tolerance. In past decade much emphasis has been given on understanding salt tolerance in terms of its regulation by miRNAs, the robust master regulator of the level of information to be carried out from gene to protein. The level of information that is currently available on salt-responsive miRNAs and their targets has enabled the researchers to look into their functional significance in salt tolerance by developing plants over expressing the concerned miRNAs. However, currently our understanding on the involvement on salt tolerance, or a biotic stress tolerance in general is far from complete, and hence requires further investigation, focusing particularly on salt responsiveness of miRNAs in halophytes and study of their expression pattern and targets across species.

Introduction

Salinity as an a biotic stress of global importance

Environmental disturbances adversely affect the normal physiological functioning of an organism.1 It can be the fluctuation of temperature, increase in salinity level, water deficit or sub mergance conditions, light-related variations, decrease in soil moisture content, etc. Being sessile, plants cope with the adverse environment by evolving stress tolerance mechanisms. Stress tolerance mainly involves biochemical, molecular and genetic mechanisms which are ultimately controlled by genes. The regulation of gene expression temporally and spatially in response to environmental cues is an important factor in plant survival and adaptability leading to development of an ecotype.2 A biotic stresses are the main cause of huge crop yield loss worldwide, and among these high salinity stress is a major threat to agriculture.   

Salt stress tolerance and miRNAs

NaCl is the most pervasive and highly abundant salt on the surface of earth. Increased salt levels leads to osmotic stress which subsequently leads to ion toxicity in the plants. This severely deteriorates the plant’s ability to take up nutrients and hence leads to nutrient stress and hampered shoot growth and organ development. The plants have evolved various interlinked mechanisms leading to osmotic tolerance and ion exclusion. Various genes and transcription factors get altered to great extents in order to complement the ill-effects of the stress.

Out of these important tolerance mechanisms, small RNAs establish themselves as huge players in post transcriptional regulation of gene expression. These sRNA are either positively regulated by stress, where they enhance the suppression of the genes serving as negative regulators of stress tolerance. Or negatively regulated where the target is positive regulator of stress causing more accumulation of gene product.3 Amongst these regulatory small RNAs, MicroRNAs (miRNAs) have generated considerable excitement recently.4

Tiny yet potent regulators, the miRNAs

The expression of a gene gets regulated at various levels by a large number of molecules. One such class of regulators involve tiny ~21 to 24nt long small-RNA molecules called the miRNAs, which regulate the expression of genes at the post-transcriptional and translational level. The biogenesis of miRNAs gets initiated with the MIR genes5 transcribing to primary-miRNAs (Figure 1). These pre-miRNAs fold up to characteristic stem-looped forms where one of the arms bears the mature miRNA and the opposite arm contains the miRNA* sequence, which get cleaved as a duplex.6 The ends of the duplex get ethylated following transportation of the duplex from the nucleus to the cytoplasm with the aid of HASTY protein.7 In the cytoplasm the two strands separate with the loading of mature miRNA strand on to the RISC,8 which is a multi-subunit assembly of AGO protein9,10 and the mature miRNA strand which possesses slicer/rib nuclease activity.

Figure 1 Biogenesis of miRNA and its action.

The miRNAs target their cognate mRNAs with perfect or near-perfect complementarily leading to cleavage, translational repression or sometimes de-adenylation. Therefore, the miRNAs negatively regulate the levels of target mRNA and consequently the proteins which are coded by them. They do not however, regulate the MIR genes from where they originate due to the sequence similarity rather the complementarily. There are a number of conserved miRNA families which are found to be involved in various stress responses and major developmental processes. However, the abundance of newer miRNA sequences is sticking due to their universal involvement, particularly in individual stress conditions or specific developmental stage. Till date 8675 mature miRNAs have been submitted in the miRbase database (version: 21) with Medicagotrunculata leading the chart with 756 reported mature miRNAs followed by 713 mature miRNAs in Oryza sativa. Recent studies on various plants have revealed important role of miRNAs in salt stress responses. Sunkar & Zhu11 for the first time created small RNA libraries and sequenced them from plants treated with various stress conditions such as salinity, cold, dehydration, and abscise acid (ABA). Cloning of small RNAs from stressed plants resulted in the identification of 15 novel miRNAs in addition to some siRNAs. The result showed that drought, cold and salinity stress strongly induces miR402 expression while other miRNAs such as miR319 is induced by either cold or other stresses.11 The various other important miRNAs involved in salinity stress, in different plant species are enlisted in Table 1.

Plant species

miRNA

miRNA Abundance

Target

References

Arabidopsis thaliana

miR156

Up-regulated

Squamosa promoter-binding protein-like11

12

miR158

Up-regulated

Pentatricopeptide repeat-containing protein(PPR)

miR159

Up-regulated

MYB and TCP transcription factors

miR165

Up-regulated

Class III HD-ZIP transcription factors

miR167

Up-regulated

Auxin response factor

miR168

Up-regulated

ARGONAUTE 1 (AGO1)

miR169

Up-regulated

CCAAT-binding transcription factor (CBF-B/NF-YA) family protein

miR171

Up-regulated

Scarecrow transcription factor

miR319

Up-regulated

TCP transcription factors

miR393

Up-regulated

F-box protein; bHLH (basic helix–loop–helix) transcription factor

miR394

Up-regulated

F-box family protein

miR396

Up-regulated

Growth regulating factor 2 transcription factor Rhodenase-like protein

miR397

Up-regulated

Laccases 2

miR398

Down regulated

InterPro domain Protein of unknown function DUF266,

Zea mays

miR156

Down regulated

SBP-domain protein

13

miR162

Up-regulated

 Dicer-like (DCL)

miR164

Down regulated

NAC domain protein NAC1

miR166

Down regulated

Homeo domain leucine Zipper protein (HD-ZIP)

miR167

Down regulated

Auxin response factor

miR168

Up-regulated

ARGONAUTE 1 (AGO1)

miR395

Up-regulated

ATP sulfurylase

miR396

Down regulated

Cytochrome oxidase subunit I

Populus trichocarpa

miR171

Down regulated

Scarecrow-like protein

26

miR482.2

Up-regulated

Disease resistance protein

miR530a

Down regulated

Zinc knuckle (CCHC-type) family protein,

miR1445

Down regulated

Dihydropyrimidinase

miR1446a-e

Down regulated

GCN5-related N-acetyltransferase (GNAT) Family protein,

miR1447

Up-regulated

Ankyrin repeat family protein,

miR1450

Up-regulated

Leucine-rich repeat transmembrane protein kinase

Populus tomentosa

miR319

Up-regulated

TCP transcription factor

27

miR393

Up-regulated

APETELA 2 (AP2)

miR394

Up-regulated

F-Box protein

miR395

Up-regulated

ATP sulfurylase

miR396

Down regulated

Growth regulating factor

miR398

Up-regulated

Copper superoxide dismutase

miR399

Up-regulated

E2-ubiquitin conjugating enzyme

miR408

Up-regulated

Plasma cell leukemia

miR1450

Down regulated

Ankyrin repeat L-RTMK

Solanum linnaeanum

miR156c

Down regulated

Squamosa promoter-binding protein

28

miR167a

Down-regulated

Annexin 1

miR397a

Up-regulated

Laccase

miR5300

Down-regulated

Nucleotide-binding site-leucine-rich repeat

Raphanus sativus

miR166g-3p

Down-regulated

S-adenosylmethionine-dependent methyltransferase

29

miR169b

Down-regulated

Nuclear transcription factor Y subunit A-3

miR172c

Down-regulated

AP2-like ethylene responsive transcription factor SNZ

Table 1 List of reported salt stress responsive miRNAs and their targets in plants

Many studies have shown that one miRNA may respond differently to salt stress depending on the plant species. For example, salinity stress induces the expression of miR156 in Arabidopsis12 but decreases in maize.13 Similarly salinity treatment up-regulated the expression of miR396 in Arabidopsis12 and maize13 but down regulated in rice.14 The expression of miRNAs has also been studied in salt stress sensitive and salt tolerant plant varieties to investigate the importance of miRNAs in plant salt stress responses. Comparative analysis between salt sensitive maize genotypes Huangzao4 and salt tolerant maize genotype NC286 identified 98 miRNAs from 27 families, among these miR156, miR164, miR166, miR167 and miR396 family members were down-regulated, whereas miR162, miR168, miR395 and miR474 families were up-regulated in salt stressed maize roots.13 Microarray experiment on two cotton cultivars (salt tolerance SN-011 and salt sensitive LM-6) demonstrated that miR156, miR169, miR535 and miR827 were significantly up-regulated in LM-6, while miR167, miR397 and miR399 were down regulated in it.15 Differential expression of Ath-miR169b, Osa-miR1432, Hv-miRx5, Hv-miR166b/c was seen in well-watered and drought-induced varieties in Barley along with their putative targets.16 All these studies suggest that comprehensive analysis of miRNA response to salt stress in closely related genotypes with contrasting stress sensitivities would provide better insights into miRNA-guided gene regulation and such strategies can be used for improving the salt stress tolerance of crop plants.

In rice, three members of miR169 family-miR169g, miR169n and miR169o as well as miR393 were found to be upregulated during salinity stress17,18 which specifically cleaves the gene transcript of NF-YA transcription factor which is evolutionary conserved in a wide range of organisms from yeast to humans. In Arabidopsis, miR169 was also induced by salinity stress.19 A contrasting feature was observed between miR398 and its target genes CSD1 and CSD2 during salinity stress. Microarray analysis in Populus tremula demonstrated dynamic regulation of miR398 abundance during salt stress with an initial increase followed by decrease and then again increase.20 Such active regulation of miR398 was absent in Arabidopsis, in which the abundance of miR398 was decreased and its target genes (CSD1 and CSD1) abundance was increased during salt stress.21 The expression level of miR168 was found to be upregulated under salt stress in both Arabidopsis12 and maize.13 The miR168 regulates the expression of AGO1 through an auto-regulatory mechanism to maintain homeostasis of AGO1.10 Since, AGO1 is a key component of RISC and required for miRNA function, any variation in miR168 expression has potential influence on the function of other miRNAs.

In contrast to glycophytes, halophytes can grow well in high saline conditions and are well suited candidates to study salt adaptation mechanisms in plants. Hence, investigating salt responsive miRNAs from halophytes may help us in better understanding of molecular mechanisms of salt adaptation in plants. Although salt responsive miRNAs have been profiled in various glycophytes, they have been analyzed in only a few halophytes. Dassanayake et al.,22 2010 for the first time computationally predicted 12 conserved miRNA families using mangrove transcriptome database and proposed regulatory models for these miRNAs by comparing distribution and positions of miRNA targets in mangrove plants and Arabidopsis. In Avicennia marina (Red sea mangrove), total 193 conserved miRNAs and 23 novel miRNAs were identified by using small RNA deep sequencing.14 Further, experimental validation of predicted miRNA target genes of both novel (miR2.1, miR2.2, miR3.1, miR3.5, miR4, miR7, miR8, miR11 and miR12) and conserved (miR156, miR159,  miR160, miR166, miR170, miR390, miR397 and miR398) miRNAs using 5’-RACE PCR followed by sequencing revealed target cleavage occurs at an expected position (Position 10 to 11). Their results suggested that the expression profiles of salt-responsive miRNAs and their target genes in A. marina may help to elucidate the important role of miRNAs in mangroves, including their response to a biotic stress.23A recent study on Halostachys caspica, identified a total of 170 conserved miRNAs; among these miRNAs, 48 were significantly down regulated and 31 were significantly up-regulated by salinity stress.24 They also identified 102 novel miRNAs; among them, 13 miRNAs were significantly down regulated and 12 miRNAs were significantly up-regulated by salinity. Further investigation on miRNA-target interaction by GO and KEGG analysis suggested many miRNAs involved in stress-related pathway. The expression profiles of conserved and novel miRNAs during salinity stress and between the shoots and roots of Salicornia europaea, a salt marsh euhalophyte, revealed that they may play an important role in salt tolerance by regulating their downstream targets.25 Hence, all these studies of salt-responsive miRNAs in halophyte plants may elucidate salt tolerance mechanisms and could be used to improve salt tolerance in crops and other plants.

MicroRNAs, a new target for improving plant tolerance to salt stress

Understanding miRNA-guided stress regulation followed by the use of this knowledge to engineer stress tolerant plants is unavoidable. Currently, many studies have shown that a biotic stresses induce aberrant expression of miRNAs, thus miRNAs could be used as a target for plant improvement, including enhanced tolerance to multiple stresses.30 Over expression of miRNA can be achieved by using an inducible promoter or constitutive promoter such as 35S or polyubiquitin promoters that is activated only under particular stress condition. To date, it has been shown in few reports that several miRNAs have been over expressed in multiple plant species and depending on their target genes they exhibited either higher tolerance or sensitivity to salt stress.  Most of the transgenic studies provide strong evidence for the use of miRNA-based technology for enhancing plant tolerance to various a biotic stresses particularly salt stress.

Conclusion

Research on understanding the mechanism of salt tolerance in plants is being carried out since decades. There is clear understanding of the probable underlying mechanisms, such as maintenance of cellular ion-homeostasis with regard to K+ and Na+, accumulation of osmolytes like proline and glycinebetaine for the maintenance of cellular osmotic potential, and effective removal of reactive oxygen species from cells and tissues. However, these processes require coherent participation of a number of genes, the expression of which is controlled by interplay of complex mechanisms involving signal transductions, cis- and trans- elements interaction, chromatin modifications, protein modification and localization, etc. So far it has not been possible to establish the linkage between various biochemical processes that enable the plant to achieve salt tolerance at the physiological level. In fact this is the major factor that has frustrated the researchers worldwide in their attempt to make a plant salt-sensitive plant salt tolerant. MiRNAs, the master regulator of gene expression is ray of hope in this regard, and that is why huge data on regulatory role of miRNAs in a biotic stress tolerance could be generated in a short stretch of time since their discovery. Although it is a long way for finally understanding the regulatory processes governing the salt tolerance, but identification of the salt-responsive miRNAs and their targets, and establishing the function of these targets is certainly going to yield promising results, which could be valuable contributions to the ongoing attempts of introducing the salt tolerance trait in the plant species of interest.

Acknowledgements

The authors are thankful to the Director, Institute of Life Sciences for providing the necessary facilities. SP is thankful to the Dept. of Biotechnology, New Delhi for fellowship

Conflicts of interest

Author declares that there is no conflicts of interest.

References

  1. Munns R, Tester M. Mechanisms of salinity tolerance. Annu Rev Plant Biol. 2008;59:651‒681.
  2. Zhang Z, Wei L, Zou X, et al. Submergence-responsive MicroRNAs are potentially involved in the regulation of morphological and metabolic adaptations in maize root cells. Ann bot. 2008;102(4):509‒519.
  3. Sunkar R, Chinnusamy V, Zhu J, et al. Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends Plant Sci. 2007;12(7):301‒309.
  4. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116(2):281‒297.
  5. He L, Hannon GJ. MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet. 2004;5(7):522‒531.
  6. Lee Y, Kim M, HanJ, et al. MicroRNA genes are transcribed by RNA polymerase II. EMBO J. 2004;23(20):4051‒4060.
  7. Huntzinger E, Izaurralde E. Gene silencing by microRNAs: contributions of translational repression and mRNA decay. Nat Rev Genet. 2001;12(2):99–110.
  8. Baumberger N, Baulcombe DC. ArabidopsisARGONAUTE1 is an RNA Slicerthat selectively recruits microRNAs and short interfering RNAs. Proc Natl Acad Sci U S A. 2005;102(33):11928‒11933.
  9. Meister G. Argonaute proteins: functional insights and emerging roles. Nature reviews genetics. 2013;14:447–459.
  10. Vaucheret H, Vazquez F, Crete P, et al. The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes Dev. 2004;18(10):1187‒1197.
  11. Sunkar R, Zhu JK. Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant cell. 2004;16(8):2001‒2019.
  12. Liu HH, Tian X, Li YJ, et al. Microarray-based analysis of stress-regulated microRNAs in Arabidopsis thaliana. RNA. 2008;14(5):836‒843.
  13. Ding D, Zhang L, Wang H, et al. Differential expression of miRNAs in response to salt stress in maize roots. Ann Bot. 2009;103(1):29‒38.
  14. Zhou L, Liu Y, Liu Z, et al. Genome-wide identification and analysis of drought-responsive microRNAs in Oryza sativa. J Exp Bot. 2010;61(15):4157‒4168.
  15. Yin Z, Li Y, Han X, et al. Genome-wide profiling of miRNAs and other small non-coding RNAs in the Verticillium dahliae-inoculated cotton roots. PloS one. 2012;7(4):e35765.
  16. Ferdous J, Sanchez-Ferrero JC, Langridge P, et al. Differential expression of microRNAs and potential targets under drought stress in barley. Plant Cell Environ. 2017;40(1):11–24.
  17. Gao P, Bai X, Yang L, et al. osa-MIR393: a salinity- and alkaline stress-related microRNA gene. Mol Bio Rep. 2011;38(1):237‒242.
  18. Zhao B, Ge L, Liang R, et al. Members of miR-169 family are induced by high salinity and transiently inhibit the NF-YA transcription factor. BMC Mol Bio. 2009;10:29.
  19. Xu MY, Zhang L, Li WW, et al. Stress-induced early flowering is mediated by miR169 in Arabidopsis thaliana. J Exp Bot. 2014;65(1):89‒101.
  20. Jia X, Ren L, Chen QJ, et al. UV-B-responsive microRNAs in Populus tremula. J Plant Physiol. 2009;166(18):2046‒2057.
  21. Jagadeeswaran G, Saini A, Sunkar R. Biotic and abiotic stress down-regulate miR398 expression in Arabidopsis. Planta. 2009;229(4):1009‒1014.
  22. Dassanayake M, Haas JS, Bohnert HJ, et al. Comparative transcriptomics for mangrove species: an expanding resource. Funct Integr Genomics. 2010;10(4):523‒532.
  23. Khraiwesh B, Pugalenthi G, Fedoroff NV. Identification and analysis of red sea mangrove (Avicennia marina) microRNAs by high-throughput sequencing and their association with stress responses. PloS one. 2013;8(4):e60774.
  24. Yang R, Zeng Y, Yi X, et al. Small RNA deep sequencing reveals the important role of microRNAs in the halophyte Halostachys caspica. Plant Biotechnol J. 2015;13(3):395‒408.
  25. Feng J, Wang J, Fan P, et al. High-throughput deep sequencing reveals that microRNAs play important roles in salt tolerance of euhalophyte Salicornia europaea. BMC Plant Bio. 2015;15:63.
  26. Lu XY, Huang XL. Plant miRNAs and abiotic stress responses. Biochem Biophys Res Commun. 2008;368(3):458‒462.
  27. Ren Y, Chen L, Zhang Y, et al. Identification and characterization of salt-responsive microRNAs in Populus tomentosa by high-throughput sequencing. Biochimie. 2013;95(4):743‒750.
  28. Zhuang Y, Zhou XH, Liu J. Conserved miRNAs and their response to salt stress in wild eggplant Solanum linnaeanum roots. Int J Mol Sci. 2014;15:839‒849.
  29. Sun X, Xu L, Wang Y, et al. Identification of novel and salt-responsive miRNAs to explore miRNA-mediated regulatory network of salt stress response in radish (Raphanus sativus L.). BMC genomics. 2015;16:197.
  30. Zhang B, Wang Q. MicroRNA-based biotechnology for plant improvement. J Cell Physiol. 2015;230:1‒15.
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