Expression and Promoter Analysis of Six Heat Stress-Inducible Genes in Rice

During the long evolutionary process, plant gradually formed a series of strategies and mechanisms to cope with stress environment such as drought, heat, cold, and high salinity. Six highly heat responsive genes were identified in rice by microarray data analysis. The qRT-PCR analysis confirmed that the expression of these six genes were highly heat inducible and moderately responded to salt stress, polyethylene glycol, and abscisic acid treatment, but little affected by cold treatment. Promoters of the three highly heat-inducible genes (OsHsfB2cp, PM19p, and Hsp90p) were used to drive GUS gene expression in rice. The results of the GUS gene expression, histochemical staining, and GUS activities in panicles and flag leaves of the transgenic rice plants confirmed high heat-induced GUS activities and moderate drought-induced activities. The three promoters exhibited similar high activity lever in rice leaf under heat, but OsHsfB2cp and PM19p showed much higher activities in panicles under heat stress. Our work confirmed that the OsHsfB2c and PM19 promoters were highly heat inducible and further characterization and reconstruction of cis-elements in their promoters could lead to the development of highly effective heat-inducible promoters for plant genetic engineering

Expression Profile in Rice Panicle: Insights into Heat Response Mechanism at Reproductive Stage

<div><p>Rice at reproductive stage is more sensitive to environmental changes, and little is known about the mechanism of heat response in rice panicle. Here, using rice microarray, we provided a time course gene expression profile of rice panicle at anther developmental stage 8 after 40°C treatment for 0 min, 20 min, 60 min, 2 h, 4 h, and 8 h. The identified differentially expressed genes were mainly involved in transcriptional regulation, transport, cellular homeostasis, and stress response. The predominant transcription factor gene families responsive to heat stress were Hsf, NAC, AP2/ERF, WRKY, MYB, and C₂H₂. KMC analysis discovered the time-dependent gene expression pattern under heat stress. The motif co-occurrence analysis on the promoters of genes from an early up-regulated cluster showed the important roles of GCC box, HSE, ABRE, and CE3 in response to heat stress. The regulation model central to ROS combined with transcriptome and ROS quantification data in rice panicle indicated the great importance to maintain ROS balance and the existence of wide cross-talk in heat response. The present study increased our understanding of the heat response in rice panicle and provided good candidate genes for crop improvement.</p> </div

Gene Ontology (GO) classification of the heat-responsive (HR) genes.

<p>The HR genes were designated as those have more than 3-fold expression changes compared with the control (untreated sample) in at least two of the five time points.</p

Regulation model central to ROS.

<p>The catalysis by Prx, SOD and NADPH oxidase (also called Rboh) lead to ROS accumulation (1, 2A,), and Rboh activity is controlled by the small GTPase (2B) that were positively regulated with PLD-derived PA (2C) and GAP proteins (2D), and negatively regulated by GDI2 (2E). PLD is a component of ABA signaling pathway to activate SAPK (3) and is the key point at which ethylene and ABA showed negative interplay (4). ABA can be diffused through ABC transporters (5). In ethylene signaling pathway, EIN3 activates ERF genes (6), and was degraded by the UPS (7). The UPS reglated plant growth and stress tolerance (8). ROS could be abolished by PrxR, AOX (9), CAT, APX (10) and P450 (11). The CAT and APX genes were trans-activated by ERF (12) and HSF (13). ROS activate calcium influx channel (14A), and stimulates Ca²⁺ release from internal Ca²⁺ stores (14B). Ca²⁺ stimulates CaM (14C), which activates HSF activity (14D). ROS stimulate HSF (15) and NAC (16). ROS exert negative regulation on auxin signaling pathway that is suppressed by SAUR and stimulated by nitrilase, respectively (17). Furthermore, ROS could be diffused through aquaporin (18). The HR genes in the present study were mapped on this model. The color bar represents the log2 transformed relative expression level on the time course of heat treatment (from left to right: 20 min, 60 min, 2 h, 4 h, and 8 h in sequence). The color scale is shown at the right. Blue: down-regulation, red: up-regulation.</p

Expression profiles of the genes in cellular response, secondary metabolism and the ubiquitin-proteasome system.

<p>MapMan was used to visualize the HR genes in the cellular response (A), secondary metabolism (B) and ubiquitin-proteasome system (C). Each BIN or subBIN is represented as a block with each transcript colored red for up-regulation or blue for down-regulation.</p

Significantly-regulated probes on the time course of heat stress.

<p>A, Venn diagram analysis on the differentially expressed probes at all time points identified 262 significantly regulated probes on the time course of heat stress. B, Expression pattern of the 262 significantly regulated probes using Hierachical Clustering (HCL) algorithm. The color scale (representing the average of normalized values) is shown at the right, and green bar indicates low expression, and red for high expression.</p

Quantitative Real-Time PCR verification of the genes expression level.

<p>Transcript levels were presented as relative values that were normalized with respect to the level of actin 1 gene. Error bars in the figures indicate standard deviation. Log2FC, log2 transformed fold change.</p

Detection and quantification of superoxide anion and hydrogen peroxide in rice panicle under heat shock.

<p>Note: ++ represent for the normal level of superoxide anion and hydrogen peroxide in rice panicle, +++ for the high accumulation, + for the low level.</p

GO analysis on the clusters from KMC.

<p>Functional distribution of the early-up- and early-down-regulated (A, B) plus late-up- and late-down-regulated (C, D) clusters showed that the genes are mainly involved in stress response, transcription factor, transporter, secondary metabolism, development as well as reproduction (p = 0.05).</p

Accumulation of superoxide anion and hydrogen peroxide in rice panicle under heat shock.

<p>Plants at the reproductive stage were treated at 40°C for 8 h, and the florets of young panicles were collected at 0 min (as a control), 20 min, 60 min, 2 h, 4 h and 8 h. Immediately following heat-treatment, the florets were treated with NBT solution to detect superoxide anion or DAB solution to detect hydrogen peroxide in the dark for 3 h and 24 h, respectively.</p