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Regulation of Stomatal Development by Stomatal Lineage miRNAs

Regulation of Stomatal Development by Stomatal Lineage miRNAs
Jiali Zhu
DGIST Authors
Kwak, June M.Zhu, JialiKim, Yun Ju
Yun Ju Kim
Issued Date
Awarded Date
Plant biology
Stomata in the plant epidermis play a vital role for growth and survival by controlling gas exchange, transpiration, and immunity to pathogens. Plants modulate stomatal cell fate and patterning through key transcriptional factors and signaling cascades. miRNAs are known to contribute to functional and developmental plasticity in multicellular organisms, however, the extent to which miRNAs are involved in stomatal development remains elusive. Here, we reveal highly dynamic, development stage-specific miRNA expression profiles from stomatal lineage. We show that stomatal lineage miRNAs positively and negatively regulate stomatal formation and patterning to avoid clustered and paired stomata. Target prediction of stomatal lineage miRNAs suggests potential cellular processes implicated in stomatal development. We show that dysregulation of stomatal lineage miRNAs and their target mRNAs disclose unexpected genetic pathways modulating stomatal development. We reveal that miRNA399-regulated PHO2, involved in phosphate homeostasis, controls stomatal development. Our study demonstrates that miRNAs constitute a critical component in the regulatory mechanism controlling stomatal development.
Table Of Contents
Ⅰ Introduction 1
1.1 Organization of Theses 1
1.2 Plant stomata and development 2
1.2.1 The process of stomatal development 2
1.2.2 bHLH transcription factors in the control of cell fate determination 3
1.2.3 Cell patterning and signaling 5
1.2.4 Cell cycle regulators 7
1.2.5 Cell polarity 8
1.3 Environmental influences on stomatal development 9
1.3.1 Carbon dioxide 9
1.3.2 Temperature 12
1.3.3 Light 13
1.4 Epigenetic regulation in stomatal development 15
1.4.1 DNA demethylation 15
1.4.2 Histone modification 16
1.4.3 miRNA regulation in stomatal development 17
1.5 miRNAs 19
1.5.1 miRNAs biogenesis 19
1.5.2 Cell-type-specific miRNA profiling and challenging 20
1.5.3 Role of miRNAs in cell differentiation and cell pattering 21
1.6 Theses hypoTheses and specific aims 21
1.6.1 Theses hypoTheses 21
1.6.2 Specific aims 22
Ⅱ Stomatal lineage cell-type-specific miRNA profiling from AGO1-Associated miRNAs 22
2.1 Introduction 22
2.2 Experimental section 24
2.2.1 Plant materials and growth condition 24
2.2.2 Plasmid construction and plant transformation 24
2.2.3 Microscopy 24
2.2.4 RNA Immunoprecipitation and small RNA isolation 24
2.2.5 Western blot analysis 25
2.2.6 Small RNA library sequencing and data analysis 26
2.3 Results 26
Ⅲ Stomatal lineage miRNA dynamics 33
3.1 Identification of DE miRNAs 33
3.2 Results 34
Ⅳ Validation of Stage-Specific miRNAs 36
Ⅴ Developmental miRNAs Regulate Stomatal Development 38
5.1 Stomatal phenotype analysis 38
5.2 Results 38
Ⅵ Target mRNA and Genetic Pathway Predictions 41
6.1 Data analysis 41
6.1.1 Identification of potential target DEGs for DE miRNAs 41
6.1.2 Functional enrichment analysis of Gene Ontology biological processes (GOBPs) 42
6.1.3 Construction of miRNA-target mRNA regulatory network 43
6.2 Results 43
Ⅶ MiR399-mediated PHO2 Regulation Controls Stomatal Development 46
7.2 Experimental section 46
7.2.1. Confocal microscopy 46
7.2.2. Stomatal phenotype analysis 47
7.2.3. Total RNA extraction and RT-qPCR analysis 47
7.2.4. RNA ligase-mediated rapid amplification of 5’ cDNA ends 47
7.3 Results 48
Ⅷ Discussion 51
Ⅸ References 101
New Biology
Related Researcher
  • 곽준명 Kwak, June Myoung 뉴바이올로지학과
  • Research Interests Calcium Signaling; ABA;ROS Signaling; plant development; Development of Abscission; Culluar Features; Signaling network; Environmental Regulation
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Department of New Biology Theses Ph.D.


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