BackgroundThe transcriptional regulatory network involved in low temperature response leading to acclimation has been established in Arabidopsis. In japonica rice, which can only withstand transient exposure to milder cold stress (10°C), an oxidative-mediated network has been proposed to play a key role in configuring early responses and short-term defenses. The components, hierarchical organization and physiological consequences of this network were further dissected by a systems-level approach.ResultsRegulatory clusters responding directly to oxidative signals were prominent during the initial 6 to 12 hours at 10°C. Early events mirrored a typical oxidative response based on striking similarities of the transcriptome to disease, elicitor and wounding induced processes. Targets of oxidative-mediated mechanisms are likely regulated by several classes of bZIP factors acting on as1/ocs/TGA-like element enriched clusters, ERF factors acting on GCC-box/JAre-like element enriched clusters and R2R3-MYB factors acting on MYB2-like element enriched clusters.Temporal induction of several H2O2-induced bZIP, ERF and MYB genes coincided with the transient H2O2 spikes within the initial 6 to 12 hours. Oxidative-independent responses involve DREB/CBF, RAP2 and RAV1 factors acting on DRE/CRT/rav1-like enriched clusters and bZIP factors acting on ABRE-like enriched clusters. Oxidative-mediated clusters were activated earlier than ABA-mediated clusters.ConclusionGenome-wide, physiological and whole-plant level analyses established a holistic view of chilling stress response mechanism of japonica rice. Early response regulatory network triggered by oxidative signals is critical for prolonged survival under sub-optimal temperature. Integration of stress and developmental responses leads to modulated growth and vigor maintenance contributing to a delay of plastic injuries.

The Arabidopsis (Arabidopsis thaliana) gene that encodes the probable ortholog of the 30-kD subunit of the mammalian cleavage and polyadenylation specificity factor (CPSF) is a complex one, encoding small (approximately 28 kD) and large (approximately 68 kD) polypeptides. The small polypeptide (AtCPSF30) corresponds to CPSF30 and is the focus of this study. Recombinant AtCPSF30 was purified from Escherichia coli and found to possess RNA-binding activity. Mutational analysis indicated that an evolutionarily conserved central core of AtCPSF30 is involved in RNA binding, but that RNA binding also requires a short sequence adjacent to the N terminus of the central core. AtCPSF30 was found to bind calmodulin, and calmodulin inhibited the RNA-binding activity of the protein in a calcium-dependent manner. Mutational analysis showed that a small part of the protein, again adjacent to the N terminus of the conserved core, is responsible for calmodulin binding; point mutations in this region abolished both binding to and inhibition of RNA binding by calmodulin. Interestingly, AtCPSF30 was capable of self-interactions. This property also mapped to the central conserved core of the protein. However, calmodulin had no discernible effect on the self-association. These results show that the central portion of AtCPSF30 is involved in a number of important functions, and they raise interesting possibilities for both the interplay between splicing and polyadenylation and the regulation of these processes by stimuli that act through calmodulin.

BackgroundPlants respond to many unfavorable environmental conditions via signaling mediated by altered levels of various reactive oxygen species (ROS). To gain additional insight into oxidative signaling responses, Arabidopsis mutants that exhibited tolerance to oxidative stress were isolated. We describe herein the isolation and characterization of one such mutant, oxt6.Methodology/Principal FindingsThe oxt6 mutation is due to the disruption of a complex gene (At1g30460) that encodes the Arabidopsis ortholog of the 30-kD subunit of the cleavage and polyadenylation specificity factor (CPSF30) as well as a larger, related 65-kD protein. Expression of mRNAs encoding Arabidopsis CPSF30 alone was able to restore wild-type growth and stress susceptibility to the oxt6 mutant. Transcriptional profiling and single gene expression studies show elevated constitutive expression of a subset of genes that encode proteins containing thioredoxin- and glutaredoxin- related domains in the oxt6 mutant, suggesting that stress can be ameliorated by these gene classes. Bulk poly(A) tail length was not seemingly affected in the oxt6 mutant, but poly(A) site selection was different, indicating a subtle effect on polyadenylation in the mutant.Conclusions/SignificanceThese results implicate the Arabidopsis CPSF30 protein in the posttranscriptional control of the responses of plants to stress, and in particular to the expression of a set of genes that suffices to confer tolerance to oxidative stress.

Background: Plants respond to low temperature through an intricately coordinated transcriptional network. The CBF/DREB-regulated network of genes has been shown to play a prominent role in freeze-tolerance of Arabidopsis through the process of cold acclimation (CA). Recent evidence also showed that the CBF/DREB regulon is not unique to CA but evolutionarily conserved between chilling-insensitive (temperate) and chilling-sensitive (warm-season) plants. In this study, the wide contrast in chilling sensitivity between indica and japonica rice was used as model to identify other regulatory clusters by integrative analysis of promoter architecture (ab initio) and gene expression profiles.

The R2R3-type OsMyb4 transcription factor of rice has been shown to play a role in the regulation of osmotic adjustment in heterologous overexpression studies. However, the exact composition and organization of its underlying transcriptional network has not been established to be a robust tool for stress tolerance enhancement by regulon engineering. OsMyb4 network was dissected based on commonalities between the global chilling stress transcriptome and the transcriptome configured by OsMyb4 overexpression. OsMyb4 controls a hierarchical network comprised of several regulatory sub-clusters associated with cellular defense and rescue, metabolism and development. It regulates target genes either directly or indirectly through intermediary MYB, ERF, bZIP, NAC, ARF and CCAAT-HAP transcription factors. Regulatory sub-clusters have different combinations of MYB-like, GCC-box-like, ERD1-box-like, ABRE-like, G-box-like, as1/ocs/TGA-like, AuxRE-like, gibberellic acid response element (GARE)-like and JAre-like cis-elements. Colddependent network activity enhanced cellular antioxidant capacity through radical scavenging mechanisms and increased activities of phenylpropanoid and isoprenoid metabolic processes involving various abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), ethylene and reactive oxygen species (ROS) responsive genes. OsMyb4 network is independent of drought response element binding protein/C-repeat binding factor (DREB/CBF) and its sub-regulons operate with possible co-regulators including nuclear factor-Y. Because of its upstream position in the network hierarchy, OsMyb4 functions quantitatively and pleiotrophically. Supra-optimal expression causes misexpression of alternative targets with costly trade-offs to panicle development.

Zoysiagrass (Zoysia japonica Steud.) is commonly found in temperate climate regions and widely used for lawns, in part, owing to its uniform green color. However, some zoysiagrass cultivars accumulate red to purple pigments in their spike and stolon tissues, thereby decreasing the aesthetic value. Here we analyzed the anthocyanin contents of two zoysiagrass cultivars ‘Anyang-jungji’ (AJ) and ‘Greenzoa’ (GZ) that produce spikes and stolons with purple and green colors, respectively, and revealed that cyanidin and petunidin were primarily accumulated in the pigmented tissues. In parallel, we performed a de novo transcriptome assembly and identified differentially expressed genes between the two cultivars. We found that two anthocyanin biosynthesis genes encoding anthocyanidin synthase (ANS) and dihydroflavonol 4-reductase (DFR) were preferentially upregulated in the purple AJ spike upon pigmentation. Both ANS and DFR genes were also highly expressed in other zoysiagrass cultivars with purple spikes and stolons, but their expression levels were significantly low in the cultivars with green tissues. We observed that recombinant ZjDFR1 and ZjANS1 proteins successfully catalyze the conversions of dihydroflavonols into leucoanthocyanidins and leucoanthocyanidins into anthocyanidins, respectively. These findings strongly suggest that upregulation of ANS and DFR is responsible for tissue-specific anthocyanin biosynthesis and differential pigmentation in zoysiagrass. The present study also demonstrates the feasibility of a de novo transcriptome analysis to identify the key genes associated with specific traits, even in the absence of reference genome information.

Plants perceive and respond to environmental stresses with complex mechanisms that are often associated with the activation of antioxidant defenses. A genetic screen aimed at isolating oxidative stress-tolerant lines of Arabidopsis thaliana has identified oxt1, a line that exhibits improved tolerance to oxidative stress and elevated temperature but displays no apparent deleterious growth effects under non-stress conditions. Oxt1 harbors a mutation that arises from the altered expression of a gene encoding adenine phosphoribosyltransferase (APT1), an enzyme that converts adenine to adenosine monophosphate (AMP), indicating a link between purine metabolism, whole-plant growth responses, and stress acclimation. The oxt1 mutation results in decreased APT1 expression that leads to reduced enzymatic activity. Correspondingly, oxt1 plants possess elevated levels of adenine. Decreased APT enzyme activity directly correlates with stress resistance in transgenic lines that ectopically express APT1. The metabolic alteration in oxt1 plants also alters the expression of several antioxidant defense genes and the response of these genes to oxidative challenge. Finally, it is shown that manipulation of adenine levels can induce stress tolerance to wild-type plants. Collectively, these results show that alterations in cellular adenine levels can trigger stress tolerance and improve growth, leading to increases in plant biomass. The results also suggest that adenine might play a part in the signals that modulate responses to abiotic stress and plant growth.

Plants produce an immense number of natural products and undifferentiated cells from various plant tissues have long been considered an ideal source for their synthesis. However, undifferentiated plant cells often either lose their biosynthetic capacity over time or exhibit immediate repression of the required pathways once dedifferentiated. In this study, freshly prepared callus tissue was employed to further investigate the regulation of a natural product pathway in undifferentiated tobacco cells. Putrescine N-methyltransferase (PMT) is a pathway-specific enzyme required in nicotinic alkaloid production in Nicotiana species. Callus derived from transgenic Nicotiana tabacum plants harboring PMT promoter-GUS fusions were used to study factors that influence PMT expression. Under normal callus growth conditions in the presence of light and auxin, PMT promoter activity was strongly repressed. Conversely, dark conditions and the absence of auxin were found to upregulate PMT promoter activity, with light being dominant to the repressive effects of auxin. Since reactive oxygen species (ROS) are known by-products of photosynthesis and have been implicated in signaling, their involvement was investigated in transgenic callus by treatment with the ROS scavenger, dimethylthiourea, or catalase. Under highly repressive conditions for alkaloid synthesis, including normal culture conditions in the light, both ROS scavengers resulted in significant induction of PMT promoter activity. Moreover, treatment of callus with catalase resulted in the upregulation of PMT promoter activity and alkaloid accumulation in this tissue. These results suggest that ROS impact the regulation of the alkaloid pathway in undifferentiated cells and have implications for regulation of the pathway in other plant tissues.