Identification, Characterization, and Functional Validation of Drought-responsive MicroRNAs in Subtropical Maize Inbreds

MicroRNA-mediated gene regulation plays a crucial role in controlling drought tolerance. In the present investigation, 13 drought-associated miRNA families consisting of 65 members and regulating 42 unique target mRNAs were identified from drought-associated microarray expression data in maize and were subjected to structural and functional characterization. The largest number of members (14) was found in the zma-miR166 and zma-miR395 families, with several targets. However, zma-miR160, zma-miR390, zma-miR393, and zma-miR2275 each showed a single target. Twenty-three major drought-responsive cis-regulatory elements were found in the upstream regions of miRNAs. Many drought-related transcription factors, such as GAMYB, HD-Zip III, and NAC, were associated with the target mRNAs. Furthermore, two contrasting subtropical maize genotypes (tolerant: HKI-1532 and sensitive: V-372) were used to understand the miRNA-assisted regulation of target mRNA under drought stress. Approximately 35 and 31% of miRNAs were up-regulated in HKI-1532 and V-372, respectively. The up-regulation of target mRNAs was as high as 14.2% in HKI-1532 but was only 2.38% in V-372. The expression patterns of miRNA-target mRNA pairs were classified into four different types: Type I- up-regulation, Type II- down-regulation, Type III- neutral regulation, and Type IV- opposite regulation. HKI-1532 displayed 46 Type I, 13 Type II, and 23 Type III patterns, whereas V-372 had mostly Type IV interactions (151). A low level of negative regulations of miRNA associated with a higher level of mRNA activity in the tolerant genotype helped to maintain crucial biological functions such as ABA signaling, the auxin response pathway, the light-responsive pathway and endosperm expression under stress conditions, thereby leading to drought tolerance. Our study identified candidate miRNAs and mRNAs operating in important pathways under drought stress conditions, and these candidates will be useful in the development of drought-tolerant maize hybrids

Genomewide Expression and Functional Interactions of Genes under Drought Stress in Maize

A genomewide transcriptome assay of two subtropical genotypes of maize was used to observe the expression of genes at seedling stage of drought stress. The number of genes expressed differentially was greater in HKI1532 (a drought tolerant genotype) than in PC3 (a drought sensitive genotype), indicating primary differences at the transcriptional level in stress tolerance. The global coexpression networks of the two genotypes differed significantly with respect to the number of modules and the coexpression pattern within the modules. A total of 174 drought-responsive genes were selected from HKI1532, and their coexpression network revealed key correlations between different adaptive pathways, each cluster of the network representing a specific biological function. Transcription factors related to ABA-dependent stomatal closure, signalling, and phosphoprotein cascades work in concert to compensate for reduced photosynthesis. Under stress, water balance was maintained by coexpression of the genes involved in osmotic adjustments and transporter proteins. Metabolism was maintained by the coexpression of genes involved in cell wall modification and protein and lipid metabolism. The interaction of genes involved in crucial biological functions during stress was identified and the results will be useful in targeting important gene interactions to understand drought tolerance in greater detail

Combination of events and interactions during hypoxia, leading to programmed cell death (PCD).

<p>(<i>A</i>) No formation of aerenchyma at control stage in the absence of PCD and (<i>B</i>) formation of aerenchyma at severe stress stage due to PCD in the tolerant genotype (HKI 1105). (<i>C</i>) The differences in fold change of a few genes are represented with standard error in microarray and qRT-PCR assays.</p

Co-expression network of genes involved in response to moderate and severe waterlogging stress.

<p>The network comprises 1593 nodes and 178,449 edges in HKI 1105, and 1538 nodes and 367,958 edges in V 372. Each color represents a module. (<i>A</i>) and (<i>C</i>) represent overviews of the co-expression networks in HKI 1105 and V 372 genotypes, respectively. (<i>B</i>) and (<i>D</i>) show the seven modules each in the tolerant and susceptible genotypes, respectively, extracted from the corresponding networks. (<i>A</i>) and (<i>B</i>): black, module 1; green, 2; blue, 3; magenta, 4; red, 5; turquoise, 6; yellow, 7; (<i>C</i>) and (<i>D</i>): black, 1; blue, 2; green, 3; orange, 4; pink, 5; red, 6; yellow, 7.</p

Representation of DEGs during waterlogging stress in various pathways according to MapMan.

<p>Dark blue and dark red represent number of upregulated genes while light blue and light red represent number of downregulated genes in HKI 1105 (tolerant genotype) and V 372 (susceptible genotype), respectively.</p

Differentially expressed genes mapped for stress conditions.

<p>Co-expression networks of the HKI 1105 and V 372 genotypes in (<i>A</i>) and (<i>B</i>) respectively, color-coded according to the level or state of stress and the nature of regulation. Moderate stress: upregulation, turquoise; downregulation, blue; severe stress: upregulation, yellow; downregulation, red.</p

Functional clusters of genes identified in the tolerant genotype.

<p>Genes coding for (<i>A</i>) ethylene-responsive proteins, including AP2 domain-containing protein (GRMZM2G369472), ERF-like 1 (GRMZM2G053503), and EREBP 2 (GRMZM2G085964), and (<i>B</i>) Ca-binding proteins, including EF-HAND Ca-binding protein CCD1 (AC225718.2), calcineurin B-like protein 4 (GRMZM2G137751), calcium-dependent protein kinase isoform AK1 (GRMZM2G028086), and calmodulin (GRMZM2G062673) in HKI 1105.</p

An overview of the differentially expressed genes (DEGs) at <i>p</i> ≤ 0.001 and ≥ fivefold expression at moderate (M), severe (S) and recovery (R) stages of waterlogging stress.

<p>The total number of DEGs is shown in bold. The number of genes having cellular component, molecular function, and biological process GO terms is shown in italics. (<i>A</i>) and (<i>B</i>) represent genes up and downregulated, respectively in the tolerant genotype (HKI 1105), whereas (<i>C</i>) and (<i>D</i>) represent those in the susceptible genotype (V 372).</p

Stress tolerant gene clusters.

<p>Genes in the “response to stress” cluster in HKI 1105, shown (<i>A</i>) as part of the co-expression network. The nodes representing genes that play a crucial role in tolerance to waterlogging are shown magnified in (<i>B</i>). These include GRMZM2G078373: ASR protein, GRMZM2G168552: bundle sheath strand-specific gene 1, <i>PDC1,</i> GRMZM2G159285: <i>IAA13,</i> and <i>POR2.</i></p

Blast2GO-annotated DEGs grouped according to the MIPS functional catalogue.

<p>The gene expression pattern across moderate stress, severe stress and recovery is shown in the form of heat maps. Blue represents the percentage of upregulated genes in HKI 1105 (tolerant genotype) while red represents downregulated genes in V 372 (susceptible genotype).</p