We studied the regulatory and physiological implications of chilling stress (10°C) transcriptome of japonica rice based on integrative analysis of whole-genome expression profiles and promoter architectures. Our analysis has taken into consideration the hypothesis that oxidative signaling is central in integrating various components of the regulatory network. We addressed this hypothesis by dissecting the commonalities between a direct response to cold stimulus and responses to low temperature-independent mimic of oxidative signaling. From the systems-level approach employed in this study, several important themes emerged. First, oxidative signaling by H2
is at the center of the regulatory network, particularly in relation to the execution of 'early response
' mechanisms (initial 24 hours). H2
has long been recognized as a major trigger of stress response signaling and its important role in interfacing abiotic and biotic stress responses with growth and development has been established [67
]. Until now, direct links of the primary oxidative signals to chilling-associated transcriptional changes has not been demonstrated at the genome-wide scale. Our current results contribute important information that fills this knowledge gap.
About 60% of chilling stress induced genes are triggered by oxidative signals either as primary or secondary targets. Much of the oxidative-mediated changes in gene expression occurred during the initial 24 hours and the composition of the transcriptome has striking similarities to disease response mechanisms in terms of the activities of genes involved in redox regulation, responses to organic toxins and wounding, phenylpropanoid and indole alkaloid metabolism, and jasmonic acid, salicylic acid and ethylene signaling [39
is likely to be the primary signal closest to the original stimulus (chilling) providing the initial trigger for a cascade of molecular processes leading to an intricately interconnected gene expression circuitry [101
]. The timing and scope of oxidative mediated regulatory clusters justify their major contribution to immediate defenses, which are critical for a species like rice that can only endure transient exposure to milder cold stress.
Second, our current data revealed a number of oxidative-mediated early response expression clusters associated with specific classes of regulatory sequences such as as1/ocs/TGA-like, GCC-box/JAre-like and Myb2-box-like cis-elements. Several classes of TFs are likely to act on these clusters including bZIP Group-D (TGA), Group-S (ocs), and Group-I (GA-associated), ERF Group-I (subfamily A-6), Group-IV (subfamily A-2), Group-VI (subfamily B-5), Group-VII (subfamily B-2), Group-X (subfamily B-4), and R2R3-MYB [24
]. Known interactions of these TFs with such elements and their involvement in various oxidative mediated processes justify their presumed roles as regulators of early response transcriptome. Particularly interesting candidates for regulon engineering are the as1/ocs/TGA-like element-regulated clusters comprising the largest group of genes associated with oxidative defenses.
Third, we have identified regulatory clusters that appear to be independent of oxidative signals. Two of the well known regulons that function during cold acclimation at 4°C in Arabidopsis
were among those and they are the ABRE-like (bZIP-ABF) and CRT/DRE/rav1-like (CBF/DREB/RAP2/RAV1
) element-enriched clusters. The third cluster (GARE/pyrimidine-box-like element-enriched) which involves an R1-MYB appears to be a component of combinatorial control mechanism. ABRE-enriched clusters appear to be downstream to the oxidative-mediated clusters and this is consistent with the timing of ABA response, i.e., after 24 hours. Recent report estimated that 400 protein-coding genes of rice are regulated via an ABRE requiring mechanism [103
]. This number is close to the estimated number of genes that belong to the chilling induced ABRE-enriched clusters, which is much less than the estimated number of genes regulated by an oxidative mediated mechanism. This data provide additional support that oxidative-mediated mechanism is the primary route for activating early response genes.
The cold acclimation associated regulons controlled by DREB/CBF, RAP2
are induced maximally at 4°C in Arabidopsis
]. Apparently, orthologous TFs in rice are induced at 10°C, which probably induce a different set of target genes distinct from those that are activated in cold acclimating species. An interesting trend that we observed was that chilling upregulated clusters in rice have only moderate enrichment of DRE/CRT/rav1-like elements compared to other types of elements. The DRE/CRT/rav1-like elements associated clusters are also enriched with one or more other types of elements. A possible explanation is that at milder cold stress, DREB/CBF/RAP2/RAV1-
regulated genes account for a relatively smaller fraction of the transcriptome compared to the oxidative mediated components. It has also been suggested that the genomes of chilling insensitive (e.g., Arabidopsis
) and sensitive (e.g., rice) species might differ in terms of the distribution of DRE/CRT/rav1-like elements [42
Fourth, our data showed an important role of combinatorial control in chilling stress response gene expression. Combinatorial control has long been recognized and numerous examples are constantly being reported in relation to stress response [17
]. Interaction of several classes of TFs facilitates fine-tuned regulation. Examples of these are the possible roles of WRKY TFs as repressors of ABA-mediated gene expression, which allows fine-tuned regulation of oxidative mediated transcriptome in relation to ABA-mediated responses. Interaction of several TF classes also facilitates integration of various signals associated with stress, growth and development. GARE/pyrimidine-box-like elements associated with R1-MYB TFs were the most widely distributed elements among the chilling upregulated clusters. This class of elements occurred in various combinations with other classes. R1-MYB TFs are primarily involved in gibberellic acid regulated growth processes [79
]. R1-MYB factors appear to coordinate and fine-tune gene expression required for growth modulation and survival at sub-optimal temperature, which appear to be a key aspect of vigor enhancement and/or maintenance under stress conditions.
Finally, our results provide a global picture of biochemical and physiological consequences of the regulatory networks induced at 10°C. The obvious theme is the striking similarities of chilling-induced processes to those associated with defenses against pathogens and responses to wounding. The predominance of genes involved in disease response signaling was indicated by large number of LRR, NBS-LRR, NB-ARC types of receptor proteins, genes associated with hypersensitive response, wounding, herbivory, redox regulation, cellular detoxification, programmed cell death, and terpenoid metabolism most of which are linked to jasmonic acid, salicylic acid and ethylene signaling. Activities of these genes provide further evidence that oxidative signal was the origin of most of the early responses.
The agronomic significance of the current results is quite interesting. The data provide a global picture of how stress, growth and developmental responses are integrated by the interaction of at least four major classes of TF. The interesting relationship between TF expression and QTL for stress tolerance and seedling vigor might be the key for further understanding of the precise mechanisms that make japonica rice withstand chilling better than indica rice. Genes related to stress response, growth, development and energy partitioning are often enriched within the boundaries of QTL associated with vigor and yield. An emerging theme is that linkage blocks with certain allelic combinations of stress-related genes involved in responses to diseases, dehydration, temperature extremes, and light stress are important components of plant vigor and heterosis [18
]. Modulated growth, physiological sustenance and maintenance of vigor appear to be the downstream consequences of the transcriptional networks induced by chilling, providing a means to delay the occurrence of irreversible injuries. This hypothesis is consistent with the fact that many japonica cultivars are able to survive chilling for a much longer period of exposure than most indica cultivars.