Effects of drought on gene expression in maize reproductive and leaf meristem tissue revealed by RNA-Seq

Abstract

Drought stress affects cereals especially during the reproductive stage. The maize (Zea mays) drought transcriptome was studied using RNA-Seq analysis to compare drought-treated and well-watered fertilized ovary and basal leaf meristem tissue. More drought-responsive genes responded in the ovary compared with the leaf meristem. Gene Ontology enrichment analysis revealed a massive decrease in transcript abundance of cell division and cell cycle genes in the drought-stressed ovary only. Among Gene Ontology categories related to carbohydrate metabolism, changes in starch and Suc metabolism-related genes occurred in the ovary, consistent with a decrease in starch levels, and in Suc transporter function, with no comparable changes occurring in the leaf meristem. Abscisic acid (ABA)-related processes responded positively, but only in the ovaries. Related responses suggested the operation of low glucose sensing in drought-stressed ovaries. The data are discussed in the context of the susceptibility of maize kernel to drought stress leading to embryo abortion and the relative robustness of dividing vegetative tissue taken at the same time from the same plant subjected to the same conditions. Our working hypothesis involves signaling events associated with increased ABA levels, decreased glucose levels, disruption of ABA/sugar signaling, activation of programmed cell death/senescence through repression of a phospholipase C-mediated signaling pathway, and arrest of the cell cycle in the stressed ovary at 1 d after pollination. Increased invertase levels in the stressed leaf meristem, on the other hand, resulted in that tissue maintaining hexose levels at an "unstressed" level, and at lower ABA levels, which was correlated with successful resistance to drought stress.

Figure 1.

RNA-Seq workflow showing different steps implemented from raw reads to GO functional enrichment. A, Eight libraries were constructed. MCC and MCD stand for maize ovary tissue, well watered and drought stress, respectively; MLC and MLD stand for maize basal leaf meristem, well watered and drought stress, respectively. Numbers 1 and 2 indicate the two biological replicates. B, The reads were mapped using Tophat onto the maize masked genome. C, Cufflinks was used for transcriptome reconstruction, resulting in four different assemblies corresponding to four samples in each tissue. D, Cuffmerge was used to merge the four assemblies and the reference annotations into a single consensus list of transcripts and categorize the assembled transcripts. E, Limma, a Bioconductor R package, was used to identify differentially expressed genes under drought in both the tissues. F, Parametric analysis of gene set enrichment was implemented to identify different biological process GO terms enriched.

Figure 2.

Relationship of the cumulative number of splice junctions of each type to the log of the number of supporting reads. All the splice junctions identified were classified as either annotated (present in the gene model) or novel (absent in the gene model).

Figure 3.

Heat map showing the different biological process GO terms enriched in maize reproductive and vegetative tissue under drought stress. The T statistic obtained from the Limma R package was used as a parameter for the parametric analysis of gene set enrichment analysis. A q value cutoff of 0.05 was used to select enriched GO terms in both the tissues. The heat map shows the Z score as obtained from parametric analysis of gene set enrichment for terms enriched in either ovary tissue or leaf meristem.

Figure 4.

Overview of differentially expressed transcripts involved in different metabolic processes under drought stress. A and B show drought-mediated expression changes in different metabolic processes in the ovary and leaf meristem, respectively. The images were obtained using MapMan, showing different functional categories that passed the cutoff (less than 0.05 q value and greater than 2-fold change) for differential expression.

Figure 5.

Effects of drought stress on the expression of genes associated with starch and Suc metabolism. Drought stress responses associated with starch (A) and Suc metabolism (B) in ovaries are shown, with a table showing gene names, putative functions, and fold change values. The values in red and blue indicate fold increase and decrease in expression in the drought-stressed tissue, respectively.

Figure 6.

RNA-Seq expression data in drought-stressed ovary tissue with associated cell cycle phases. Each box represents a gene product, or group of gene products, and indicates where they exert influence on the cell cycle. Cyclin (CYC) gene group boxes display a red background, and other cell cycle gene groups have a light blue background. Within each box, the expression data for drought-stressed ovary tissue is listed in the form annotation, maize gene identifier, and fold change. The values in red and blue indicate the fold decrease and increase in expression in the drought-stressed ovary tissue, respectively.

Figure 7.

qRT-PCR validation of differentially expressed genes in maize under drought. A, Correlation of fold change analyzed by RNA-Seq platform (x axis) with data obtained using real-time PCR (y axis). B, Expression analysis of maize vacuolar, cell wall, and neutral invertases assessed through qRT-PCR. Error bars represent se (n = 3). Asterisks indicate levels of significance of differential expression (t test: * P ≤ 0.05, ** P ≤ 0.01).

Figure 8.

Overview of drought stress effects on maize ovary tissue at 1 DAP. The core figure is drawn according to Systems Biology Graphical Notation Activity Flow specifications (Level 1 Version 1) using Virginia Tech’s Beacon software. Shaded colors group glyphs with similar processes or events. The relationships, or arcs, express the nature of the influence (positive, negative, or unknown) between the glyphs and summarize this paper’s findings along with support from the literature. References Zinselmeier et al. (1999), Andersen et al. (2002), and Ruan et al. (2010) support and provide metabolite measurements for the carbon metabolism relationships under drought perturbation. Reference Zhuang et al. (2007) supports the positive influence of drought on the trehalose/raffinose pathway. References Hanson and Smeekens (2009) and Bolouri-Moghaddam et al. (2010) substantiate the positive influence on low-sugar signaling. Reference Hong et al. (2010) and the review by Li et al. (2009) associate PLDα and PLDδ expression to ROS and drought stress. References Yu and Setter (2003) and Setter et al. (2011) document increases in ABA under drought stress.