MicroRNAomic profiling uncovers ZnO-NPs responsive gene regulatory networks in maize Sinchan Adhikari, Zahed Hossain Plant Stress and Molecular Biology Laboratory, Department of Botany, University of Kalyani, West Bengal, India
Normalized expression level Log2
Background: MicroRNAs (miRNAs) are endogenous non-coding small RNAs (20-24nt) that play critical roles in plant stress adaptation. Zinc (Zn) serves as an indispensible element in several plant metabolic processes, however excess Zn is found to be toxic. Multifaceted applications of Zn based nanoparticles (NPs) trigger indiscriminate release of Zn in arable land which in excess can reduce crop productivity including maize. Rationale: Despite substantial efforts paid towards assessment of phytotoxicity of ZnO-NPs, no meticulous study has been performed to decipher the miRNA-mediated underlying molecular mechanisms involved in stress response to ZnO-NPs. Strategy: A comparative small RNA transcriptome study on two sRNA libraries constructed from control and ZnO-NPs (800 ppm, <50 nm) challenged maize leaves was performed to unveil the roles of miRNAs mediated gene regulatory networks associated with ZnO-NPs stress adaptation. Expression of candidate miRNAs and their negative correlation with the corresponding target genes were validated by qRT-PCR. Results: Meager growth performance, adsorption of ZnO-NPs to root surface and accumulation of Zn in roots, root cell death, oxidative burst in leaves confirmed the stress response. Deep sequencing results revealed 2 up- and 73 down-regulated known miRNAs. Gene ontology (GO) annotations of their putative targets indicated that ZnO-NPs responsive miRNAs are potentially involved in key biological processes including photosynthesis (miR171b), redox homeostasis (miR166l), cell wall modification (miR156b.1), heavy metal transport and detoxification (miR444a), and hormone signaling (miR159f-3p). This comprehensive study provides a framework for understanding the plant adaptive response to ZnO-NPs stress. Our findings have recently been endorsed by chemosphere journal.
RNA extraction from leaves Small RNA library preparation Deep Sequencing and Bioinformatics Identification of differentially expressed known miRNAs Putative target prediction and GO annotations qRT-PCR validation of selected miRNAs and their targets
RESULTS A
CT
C
CT
B
NP
E
CT
NP
D
NP
G
CT
NP
NP
L
Control
20
*
Control
15 10 5
*
ZnO-NPs
* **
* *
*
0
Relative expression of target genes of the selected miRNAs using qRT-PCR. Symbol * and ** indicate significant difference at P<0.05 and P<0.01, respectively. Hypothetical model showing the putative roles of candidate miRNAs and their corresponding target genes in response to ZnO-NPs stress in maize
NP
Impact of ZnO-NPs (800 ppm) stress on maize seedlings. Complete drying of first leaf (B) and leaf tip yellowing of second leaf (D) under ZnO-NPs stress. NBT staining indicates superoxide deposits as blue spots (formazan) (E) and DAB staining indicate H2O2 deposits (F) on the ZnO-NPs stressed leaves. Evans blue staining indicates nonviable cells in ZnO-NPs stressed root tips (H). In situ localization of Zn using Zincon in maize roots where blue colouration indicatives of Zn-Zincon complex localization (J). Scanning electron micrographs showing adherence of ZnO-NPs on root surface (K). Venn diagram showing number distributions of known miRNAs between control and ZnO-NPs stressed libraries (L). CT: Control; NP: ZnO-NPs.
miR159f-3p
miR171b
miR166l Polyamine Oxidase 3
Scarecrow 6
GAMYB
Protochlorophyllide Oxidoreductase (POR)
H2O2 Oxidative Burst
Chlorophyll Biosynthesis
Programmed Cell Death (PCD)
Cell Proliferation in Vegetative Tissue
Restricted Plant Growth and Development
ZnO-NPs
miR156b.1 mir1432 mir156 mir159 mir160 mir162 mir164 mir165 mir166 mir167 mir168 mir171 mir172 mir2118 mir2275 mir319 mir3630 mir390 mir393 mir394 mir395 mir396 mir397 mir398 mir399 mir408 mir444 mir528 mir529 mir6173 mir6478 mir9773 mir827
50 40 30 20 10 0
Validation of expression pattern of selected miRNAs using stem-loop qRT-PCR and comparison with deep sequencing results. Bars indicate down-regulation of miRNAs in ZnO-NPs stressed leaves with respect to control.
K
J
H
-5
10µm
CT
CT
F
I
-3
Scarecrow 6
Morphological alterations In vivo detection of ROS using NBT and DAB staining Root cell viability assay using Evans blue staining In situ localization of Zn with Zincon dye Scanning electron microscopy study of root surfaces
-1
Thioredoxin CXXS1
Molecular Experiments
156b.1 miR159f-3p miR166h miR166l miR171b miR444a miR156b miR396
Polyamine oxidase 3
Physiological Experiments
Stem-loop qRT-PCR
Callose synthase 11
Tissue harvest after 6 days of stress treatment
1
ABC transporter G family member 29
ZnO-NPs treatment (800 ppm suspension, <50 nm in size)
Deep Sequencing
3
Cytochrome P450 superfamily
Control (Deionized water)
5
GAMYB
5 days old maize seedlings [Zea mays L.; Trivikram (TMMH-802)]
Relative expression
EXPERIMENTAL DESIGN
miR444a
Callose Synthase Restriction of Symplastic Movement of HMs (Zn)
Number of miRNA members in each known miRNA family of Z. mays
ABC Transporter G Family Member 29 Sequestration of HMs (Zn) in Vacuole
Plant Adaptation to ZnO-NPs Stress
SALIENT FINDINGS Toxicity of ZnO-NPs is characterized by cell death, bioaccumulation of Zn, reduction in chlorophyll content and oxidative bursts. ROS homeostasis, photosynthesis and heavy metal transport are the major biological processes influenced by the differentially expressed miRNAs. miR159f-3p mediated upregulation of GAMYB might contribute to meager growth performance of maize seedlings. Transcriptional up-regulation of SCARECROW 6 by miR171b might be responsible for negative regulation of chlorophyll biosynthesis. Down-regulation of miR166l and miR444a expression trigger severe oxidative burst and promote Zn detoxification, respectively. PUBLICATION: Adhikari et al. (2020) Chemosphere, 249: 126197. [DOI: 10.1016/j.chemosphere.2020.126197]