TRiP: Tracking Rhythms in Plants, an Automated Leaf Movement Analysis Program for Circadian Period Estimation

Background: A well characterized output of the circadian clock in plants is the daily rhythmic movement of leaves. This process has been used extensively in Arabidopsis to estimate circadian period in natural accessions as well as mutants with known defects in circadian clock function. Current methods for estimating circadian period by leaf movement involve manual steps throughout the analysis and are often limited to analyzing one leaf or cotyledon at a time. Methods: In this study, we describe the development of TRiP (Tracking Rhythms in Plants), a new method for estimating circadian period using a motion estimation algorithm that can be applied to whole plant images. To validate this new method, we apply TRiP to a Recombinant Inbred Line (RIL) population in Arabidopsis using our high-throughput imaging platform. We begin imaging at the cotyledon stage and image through the emergence of true leaves. TRiP successfully tracks the movement of cotyledons and leaves without the need to select individual leaves to be analyzed

The Importance of Ambient Temperature to Growth and the Induction of Flowering

Plant development is exquisitely sensitive to the environment. Light quantity, quality, and duration (photoperiod) have profound effects on vegetative morphology and flowering time. Recent studies have demonstrated that ambient temperature is a similarly potent stimulus influencing morphology and flowering. In Arabidopsis, ambient temperatures that are high, but not so high as to induce a heat stress response, confer morphological changes that resemble the shade avoidance syndrome. Similarly, these high but not stressful temperatures can accelerate flowering under short day conditions as effectively as exposure to long days. Photoperiodic flowering entails a series of external coincidences, in which environmental cycles of light and dark must coincide with an internal cycle in gene expression established by the endogenous circadian clock. It is evident that a similar model of external coincidence applies to the effects of elevated ambient temperature on both vegetative morphology and the vegetative to reproductive transition. Further study is imperative, because global warming is predicted to have major effects on the performance and distribution of wild species and strong adverse effects on crop yields. It is critical to understand temperature perception and response at a mechanistic level and to integrate this knowledge with our understanding of other environmental responses, including biotic and abiotic stresses, in order to improve crop production sufficiently to sustainably feed an expanding world population

Type II Protein Arginine Methyltransferase 5 (PRMT5) Is Required for Circadian Pperiod Determination in Arabidopsis Thaliana

Posttranslational modification is an important element in circadian clock function from cyanobacteria through plants and mammals. For example, a number of key clock components are phosphorylated and thereby marked for subsequent ubiquitination and degradation. Through forward genetic analysis we demonstrate that protein arginine methyltransferase 5 (PRMT5; At4g31120) is a critical determinant of circadian period in Arabidopsis. PRMT5 is coregulated with a set of 1,253 genes that shows alterations in phase of expression in response to entrainment to thermocycles versus photocycles in constant temperature. PRMT5 encodes a type II protein arginine methyltransferase that catalyzes the symmetric dimethylation of arginine residues (Rsme2). Rsme2 modification has been observed in many taxa, and targets include histones, components of the transcription complex, and components of the spliceosome. Neither arginine methylation nor PRMT5 has been implicated previously in circadian clock function, but the period lengthening associated with mutational disruption of prmt5 indicates that Rsme2 is a decoration important for the Arabidopsis clock and possibly for clocks in general

Crosstalk between the Circadian Clock and Innate Immunity in Arabidopsis

Plants are frequently challenged by various pathogens. The circadian clock, which is the internal time measuring machinery, has been implicated in regulating plant responses to biotic cues. To better understand the role of the circadian clock in defense control, we tested disease resistance with Arabidopsis mutants disrupted in CCA1 and LHY , two key components of the circadian clock. We found that consistent with their contributions to the circadian clock, cca1 and lhy mutants synergistically affect resistance to both bacterial and oomycete pathogens. Disrupting the circadian clock caused by overexpression of CCA1 or LHY also results in severe disease susceptibility. Thus, our data further demonstrate a direct role of the circadian clock mediated by CCA1 and LHY in defense regulation. We also found that CCA1 and LHY act independently of salicylic acid mediated defense but at least through the down- stream target gene GRP7 to regulate both stomata- dependent and -independent pathways. We further show that defense activation by bacterial infection and the treatment with the elicitor flg22 can also feed back to regulate clock activity. Together our study reveals for the first time reciprocal regulation of the circadian clock and plant innate immunity, significantly expanding our view of complex gene networks regulating plant defense responses and development

Crosstalk between the Circadian Clock and Innate Immunity in Arabidopsis

Plants are frequently challenged by various pathogens. The circadian clock, which is the internal time measuring machinery, has been implicated in regulating plant responses to biotic cues. To better understand the role of the circadian clock in defense control, we tested disease resistance with Arabidopsis mutants disrupted in CCA1 and LHY , two key components of the circadian clock. We found that consistent with their contributions to the circadian clock, cca1 and lhy mutants synergistically affect resistance to both bacterial and oomycete pathogens. Disrupting the circadian clock caused by overexpression of CCA1 or LHY also results in severe disease susceptibility. Thus, our data further demonstrate a direct role of the circadian clock mediated by CCA1 and LHY in defense regulation. We also found that CCA1 and LHY act independently of salicylic acid mediated defense but at least through the down- stream target gene GRP7 to regulate both stomata- dependent and -independent pathways. We further show that defense activation by bacterial infection and the treatment with the elicitor flg22 can also feed back to regulate clock activity. Together our study reveals for the first time reciprocal regulation of the circadian clock and plant innate immunity, significantly expanding our view of complex gene networks regulating plant defense responses and development

Variation in Arabidopsis Flowering Time Associated with Cis-Regulatory Variation in CONSTANS

The onset of flowering, the change from vegetative to reproductive development, is a major life history transition in flowering plants. Recent work suggests that mutations in cis-regulatory mutations should play critical roles in the evolution of this (as well as other) important adaptive traits, but thus far there has been little evidence that directly links regulatory mutations to evolutionary change at the species level. While several genes have previously been shown to affect natural variation in flowering time in Arabidopsis thaliana, most either show protein-coding changes and/or are found at low frequency (\u3c5%). Here we identify and characterize natural variation in the cis-regulatory sequence in the transcription factor CONSTANS that underlies flowering time diversity in Arabidopsis. Mutation in this regulatory motif evolved recently and has spread to high frequency in Arabidopsis natural accessions, suggesting a role for these cis-regulatory changes in adaptive variation of flowering time

Diurnal RNAPII-tethered chromatin interactions are associated with rhythmic gene expression in rice

Background: The daily cycling of plant physiological processes is speculated to arise from the coordinated rhythms of gene expression. However, the dynamics of diurnal 3D genome architecture and their potential functions underlying the rhythmic gene expression remain unclear. Results: Here, we reveal the genome-wide rhythmic occupancy of RNA polymerase II (RNAPII), which precedes mRNA accumulation by approximately 2 h. Rhythmic RNAPII binding dynamically correlates with RNAPII-mediated chromatin architecture remodeling at the genomic level of chromatin interactions, spatial clusters, and chromatin connectivity maps, which are associated with the circadian rhythm of gene expression. Rhythmically expressed genes within the same peak phases of expression are preferentially tethered by RNAPII for coordinated transcription. RNAPII-associated chromatin spatial clusters (CSCs) show high plasticity during the circadian cycle, and rhythmically expressed genes in the morning phase and non-rhythmically expressed genes in the evening phase tend to be enriched in RNAPII-associated CSCs to orchestrate expression. Core circadian clock genes are associated with RNAPII-mediated highly connected chromatin connectivity networks in the morning in contrast to the scattered, sporadic spatial chromatin connectivity in the evening; this indicates that they are transcribed within physical proximity to each other during the AM circadian window and are located in discrete “transcriptional factory” foci in the evening, linking chromatin architecture to coordinated transcription outputs. Conclusion: Our findings uncover fundamental diurnal genome folding principles in plants and reveal a distinct higher-order chromosome organization that is crucial for coordinating diurnal dynamics of transcriptional regulation