Coordination of the maize transcriptome by a conserved circadian clock
© Khan et al; licensee BioMed Central Ltd. 2010
Received: 20 November 2009
Accepted: 24 June 2010
Published: 24 June 2010
The plant circadian clock orchestrates 24-hour rhythms in internal physiological processes to coordinate these activities with daily and seasonal changes in the environment. The circadian clock has a profound impact on many aspects of plant growth and development, including biomass accumulation and flowering time. Despite recent advances in understanding the circadian system of the model plant Arabidopsis thaliana, the contribution of the circadian oscillator to important agronomic traits in Zea mays and other cereals remains poorly defined. To address this deficit, this study investigated the transcriptional landscape of the maize circadian system.
Since transcriptional regulation is a fundamental aspect of circadian systems, genes exhibiting circadian expression were identified in the sequenced maize inbred B73. Of the over 13,000 transcripts examined, approximately 10 percent displayed circadian expression patterns. The majority of cycling genes had peak expression at subjective dawn and dusk, similar to other plant circadian systems. The maize circadian clock organized co-regulation of genes participating in fundamental physiological processes, including photosynthesis, carbohydrate metabolism, cell wall biogenesis, and phytohormone biosynthesis pathways.
Circadian regulation of the maize genome was widespread and key genes in several major metabolic pathways had circadian expression waveforms. The maize circadian clock coordinated transcription to be coincident with oncoming day or night, which was consistent with the circadian oscillator acting to prepare the plant for these major recurring environmental changes. These findings highlighted the multiple processes in maize plants under circadian regulation and, as a result, provided insight into the important contribution this regulatory system makes to agronomic traits in maize and potentially other C4 plant species.
Plants match their physiology to daily and seasonal environmental changes through the circadian clock, an internal timekeeping mechanism that regulates a wide range of plant behavior. Overt circadian rhythms in plants include rhythmic leaf movements, stomatal conductance, and growth . Rhythms are maintained with a period of approximately 24 hours in the absence of environmental cues and over the normal range of ambient temperatures. The circadian system enables plants to anticipate and synchronize their physiology to the recurring environmental changes brought on by day and night. The consequence of proper clock and environment synchronization is optimized fitness [2, 3]. Beyond the daily control of plant biology, circadian rhythms also allow plants to track seasonal change according to day length, or photoperiod [4, 5]. The interplay of the circadian clock and photoperiod allows photoperiod sensitive plants to initiate floral development in accordance with the season [6, 7]. Thus, the circadian clock is an endogenous timer that maintains normal plant biology on both daily and seasonal timescales.
The circadian clock is best described in the model plant Arabidopsis thaliana. Arabidopsis mutants with impaired clock function show reduced fitness arising from mismatch between internal rhythms and external environmental conditions [8, 9]. In addition, many circadian clock mutants exhibit alterations in flowering time associated with defects in day length perception . At the molecular level, Arabidopsis circadian physiology requires the products of so-called core clock genes, whose mutation widely disrupts circadian physiology . The core clock proteins regulate expression of one another in a network of feedback loops . The myb-like transcription factors CCA1 and LHY directly control TOC1, PRR7, and PRR9 expression [13, 14]. In turn, these pseudoresponse regulators define the expression patterns of CCA1 and LHY. In addition, the TCP transcription factor CHE serves as a transcriptional regulator of CCA1 . Additionally, the clock-specific photoreceptor and F-box protein ZTL controls TOC1 function through 26S proteasome-mediated protein degradation of TOC1 at night [16, 17]. The core circadian oscillator also requires the activity of ELF3 and LUX in the evening [18, 19]. This regulatory network generates phase-specific 24-hour oscillations in each core clock gene, with the state of the overall system reflecting time of day.
Orthologs of circadian clock components in plants outside of Arabidopsis have been best characterized in rice  and Lemna . The rice genome encodes single orthologs of CCA1 and GI, but two potential orthologs of ZTL. In addition, rice possesses five unique PRR orthologs, including a clear ortholog of TOC1 . Overexpression of these rice orthologs in Arabidopsis modifies circadian rhythms, which is consistent with the function of these proteins being conserved between rice and Arabidopsis . In addition, rice TOC1 and PRR7 partially complement the corresponding Arabidopsis toc1 and prr7 mutants . Circadian clock related genes have also been described in two species of the monocot Lemna, including LHY, GI, ELF3, TOC1, and the other PRRs . Knockdown and overexpression of LHY, ELF3, and GI in Lemna suggest the topology of the Lemna circadian system is conserved with Arabidopsis and rice .
Genome-wide expression assays have revealed details behind circadian clock regulation of overt plant physiology. In Arabidopsis, the core oscillator genes are only a fraction of the genes showing cyclic expression in constant conditions. Whole genome transcriptional profiling demonstrates that the steady state transcripts of ~30% of Arabidopsis genes cycle every 24 hours in constant conditions [25, 26]. Clock controlled phasing of these cyclic genes throughout a day activates or represses metabolic and signal transduction pathways, thereby yielding macroscopic circadian rhythms in plant physiology. Genes involved in related pathways share timing of peak expression; for example, genes encoding enzymes in secondary metabolite biosynthesis, nutrient assimilation, and hormone signaling are co-regulated by the circadian clock [27–29].
Approximately half of the world's grass species employ C4 photosynthesis, including the food crops maize, sugarcane, and sorghum, as well as the potential biofuel crops switchgrass and Miscanthus. C4 plants capture CO2 as the 4-carbon compound oxaloacetate in specialized mesophyll cells and the newly captured carbon is then transported into adjacent bundle sheath cells to enter the Calvin cycle through the action of Rubisco. Primary carbon fixation in C3 plants like Arabidopsis occurs in mesophyll cells through Rubisco-mediated incorporation of CO2 into the 3-carbon compound 3-phosphoglycerate. Direct CO2 capture by Rubisco reduces the photosynthetic efficiency of C3 plants because photosynthetic rate is limited both by CO2 diffusion from the atmosphere and by photorespiration that increases at low CO2 concentrations and high temperatures. The physical partitioning of CO2 capture and the Calvin cycle in C4 plants improves photosynthetic efficiency under low CO2 concentrations and reduces photorespiration by Rubisco at high temperatures and low CO2 concentrations. As a consequence, C4 crop plants assimilate biomass more efficiently than C3 plants at the high temperatures typical of agricultural settings .
Previous studies have shown the circadian clock serves to coordinate expression of genes encoding the photosynthesis apparatus in plants that carry out C3 photosynthesis . The circadian system of C4 plants remains uncharacterized. Therefore, examination of the maize circadian system is fundamental to understanding the impact of circadian regulation on C4 photosynthesis, as well as the many other areas of maize metabolism where circadian rhythms are important. In this study, transcriptional profiling revealed the maize circadian transcriptome and this provided an initial characterization of the aspects of maize physiology under circadian clock influence.
Results and Discussion
Widespread circadian regulation of the maize transcriptome
To map the maize circadian transcriptome, mRNA levels in the aerial tissues of week-old maize B73 plants were determined by transcriptional profiling with the Affymetrix GeneChip® Maize Genome Array. The B73 inbred is widely used and is the source material for the complete maize genome sequence . Young plants were chosen as the experimental model because at this developmental stage all the tissues of the plant were easily sampled at once, unlike older maize plants that were too large to sample whole. Plants were exposed to 12 h light:12 h dark photocycles to set the circadian clock and then transferred to LL conditions for 48 additional hours. While in LL, aerial tissue was harvested every 4 hours and transcript levels in three pooled replicate samples were determined for the 13,339 maize genes represented on the microarray. The genes on this microarray account for approximately 41% of the entire maize genome, as the maize genome is predicted to have over 32,540 protein-encoding genes . Consequently, the genes with circadian expression identified here may represent nearly half of the number of predicted maize genes with rhythmic expression.
Comparing the genes called rhythmic by COSOPT and HAYSTACK revealed that the two methods had 210 genes in common (Figure 1A), a proportion similar to that described for Arabidopsis circadian expression datasets . COSOPT identified almost twice as many unique rhythmic transcripts as found by HAYSTACK. This outcome was unexpected, since HAYSTACK has the potential to match a larger diversity of waveforms  and HAYSTACK has been shown to identify a larger proportion of rhythmically expressed transcripts in Arabidopsis datasets, most notably those with expression patterns in the spike class . Not surprisingly, HAYSTACK appeared to favor maize genes showing the spike and box waveforms over other waveform classes, like cosine that COSOPT was designed to find (Figure 1B). Thus, under these standard cutoffs COSOPT was more sensitive than HAYSTACK in finding transcripts that match a cosine waveform. Furthermore, the cosine waveform appeared to be the dominant pattern of expression in maize seedlings under LL conditions.
Collectively, COSOPT and HAYSTACK indicated 1,444 transcripts, or ~10% of the expressed genes on the microarray showed rhythmic expression (Additional file 1 Table S1); therefore, a substantial part of the maize transcriptome is subject to regulation by the circadian clock. Genes in this collection included known maize circadian clock-regulated transcripts such as putative maize flowering time genes gi1A and conz1 , the well-established circadian clock-regulated gene cat3 , and several lhcb genes  (Additional file 2 Figure S1). Assuming a maize genome of 32,540 genes , the full circadian transcriptome of maize potentially represents a minimum of 3,254 genes. This degree of clock regulation is comparable to that observed in Arabidopsis with a partial genome array  and in other model systems like Drosophila melanogaster, Neurospora crassa, and Mus musculus .
Preferential expression of maize circadian gene expression at dawn and dusk
A fundamental role of the clock is to anticipate day and night; as a result, the majority of circadian gene expression in Arabidopsis, rice, and poplar is timed, or phased, to precede or coincide with these recurring environmental shifts [26, 28]. To determine whether the maize circadian transcriptome was similarly organized, the cycling maize transcripts were sorted into six phase bins based on expression waveform. The six phase bins were distributed in 4-hour intervals throughout the 24-hour subjective day: dawn (ZT0 hours), midday (ZT4), late day (ZT8), dusk (ZT12), midnight (ZT16), and early morning (ZT20). K-means clustering successfully placed all transcripts into one of these six phase bins (Additional file 3 Figure S2), with the exception of four outliers. As expected, rhythmically expressed transcripts were preferentially phased to the transitions into or out of subjective day (Figure 1C). This distribution was significantly different from that expected by chance, as assessed by χ2 testing (χ2 = 109.83, P(χ2) = 4.45 × 10-22, df = 5, α = .05). The single largest phase bin was ZT12 with 335 of cycling genes, followed by ZT0 encompassing 314 transcripts (Figure 1C). The early morning phase bin was also predominant, containing 249 genes with peak expression at ZT20. The remaining transcripts were distributed almost equally over the remaining phase bins. This analysis indicated the maize circadian oscillator parses gene expression in accordance with predictable environmental changes associated with day and night.
Phase-specific distribution of key physiological processes
The maize circadian system preferentially regulated expression of genes encoding components of energy and metabolism pathways
GO Slim Category
GO Slim term
generation of precursor metabolites and energy
carbohydrate metabolic process
2 Biological Process within phase group
cellular amino acid and derivative metabolic process
Coordinated circadian expression of key C4 photosynthesis genes
The maize proteins encoded by the transcripts represented on the array were given a functional annotation by matching amino acid sequences to likely orthologs in rice and Arabidopsis (Additional file 6 Table S3). The functional annotation grouped genes into a high confidence set and a low confidence set based on the criteria described in "Methods" and outlined in Additional file 4 Figure S3. Importantly, the high confidence genes represent those genes where orthologs were identified in both Arabidopsis and rice. The genes in this high confidence set were used to explore the influence of the circadian clock on maize physiology, in particular maize metabolic and signaling pathways.
The maize circadian clock orchestrated coordinated expression of genes involved in multiple key physiological processes
Maize protein ID
2Function Zmto At
C4 carbon dioxide fixation
C4 carbon dioxide fixation
C4 carbon dioxide fixation
Chlorophyll a/b Biosynthesis
Chlorophyll a/b Biosynthesis
Superoxide Radical Removal
Superoxide Radical Removal
Growth and Development
Blue light response
The state of the maize circadian system observed here is likely an incomplete representation of the network in field grown mature plants, since young maize seedlings are developing their C4 photosynthetic capacity. Burris and de Veau showed that while 9 day-old maize seedlings exhibited a 3 times higher rate of photorespiration than 3 month-old maize plants, consistent with limited C4 photosynthesis in seedlings compared to the mature plants, the rate of photorespiration in these maize seedlings was 5 to 7 times lower than mature wheat plants performing C3 photosynthesis . Their analysis demonstrates young maize seedlings have the capacity for C4 photosynthesis, albeit a modified version with a higher rate of photorespiration than mature plants. Therefore, 7 day-old maize seedlings represent a suitable model of C4 photosynthesis.
Circadian expression was evident for genes encoding enzymes involved in biosynthesis of carotenoids including PSY, BCH, and ZEO (also known as ABA DEFICIENT 1 in Arabidopsis) (Table 2, Figure 3). Carotenoids serve as photoprotective pigments and are also important structural components of light harvesting complexes. PSY catalyzes the first and rate-limiting step in the production of carotenoids and both BCH and ZEO lie downstream of PSY [44, 45]. Expression of these genes was phased to either early morning (ZT20) or midday (ZT4), clearly showing the maize circadian system orchestrates expression of upstream and downstream enzymes for carotenoid production to coincide with the time when these compounds are needed for photosynthesis and non-photochemical quenching. PSY is also circadian regulated in Arabidopsis , with a phase matching that found here for maize (Table 2), which again suggests strong conservation of the regulatory networks underlying the maize transcriptome. Protection from ROS produced by photosynthesis is provided by the action of catalase and superoxide dismutase enzymes. Genes encoding maize CAT3 and an ortholog of MSD1 exhibited circadian expression in maize seedlings (Table 2). Consistent with their role in scavenging ROS arising from photosynthesis, expression of the cat3 and msd1 genes reached peak levels during late day (ZT8) when ROS is more likely to accumulate (Figure 3). Overall, these findings show the circadian clock organizes maize C4 photosynthesis to ensure the plant makes maximal use of light energy available during the day.
Organization of carbohydrate metabolism gene expression by the maize circadian clock
The maize clock also regulates genes contributing to carbohydrate metabolism and carbon flux (Figure 3, Table 1). For example, the circadian clock controlled expression of two PFK encoding genes and peak expression for each occurred around dawn (Figure 3). Note that the annotation matched two different maize transcripts to the same Arabidopsis gene (PFK3), which explains the different phase values shown in Table 2. PFK enzymes mediate the central regulatory step of glycolysis. Two glycolysis enzymes of the FBAase class, which reversibly convert fructose bisphosphate to triose phosphate, were clock regulated such that one peaked in the morning (FBAase2) and another expressed in the evening (FBAase1). As in energy generation, the clock regulates genes involved in energy storage. AGPase is the major regulatory enzyme in starch biosynthesis, where it converts glucose 6-phosphate to ADP-glucose, the substrate for starch synthase . The transcript encoding AGPL, a subunit of AGPase, reached peak levels near dawn at ZT20 (Figure 3). Early morning accumulation of AGPase may be an anticipatory strategy to prepare the system for midday when photosynthate is in excess and starch biosynthesis commences. Similarly, two ss transcripts encoding starch synthases were maximally expressed early in the day (ZT0 and ZT4) like their Arabidopsis orthologs (Table 2). Thus, the maize circadian clock anticipated the need for carbon metabolism components and up-regulated expression of these genes so that enzymes would be present at the time when photosynthate would be available for energy production and storage. Together, the coordinated regulation of carbohydrate metabolism and C4 photosynthesis indicates that the circadian clock in maize organizes gene expression to ensure efficient and maximal energy production, use, and storage throughout the day.
Co-regulation of cell wall synthesis genes by the maize circadian clock
Cell walls are a major constituent of plant biomass, and their enlargement exhibits a biological rhythm [47, 48]. Consistent with rhythmic growth, the transcripts of several enzymes involved in cell wall biosynthesis were found to be circadian-regulated in maize (Table 2, Figure 3). CCR and CAD catalyze two of the final steps in the conversion of phenylpropanoid into monolignins to achieve wall hardening . Transcripts encoding both these enzymes were found to have cyclic expression peaking at dawn (Figure 3). The maize CAD is orthologous to Arabidopsis CAD4. Arabidopsis CAD4 is a class II CAD enzyme that acts on sinapaldehyde instead of coniferaldehyde ; therefore, the substrate for the maize CAD is likely to be sinapaldehyde (Figure 3). 4-CL acts early in phenylpropanoid synthesis and a maize gene encoding an ortholog of Arabidopsis 4-CL3 is rhythmically expressed with peak expression at ZT16, instead of ZT0 like the ccr and cad transcripts. Since the Arabidopsis 4-CL3 enzyme participates in flavonoid biosynthesis instead of contributing to lignin production , this maize 4-CL is likely not involved in lignin biosynthesis but flavonoid biosynthesis instead. The similar expression waveforms of ccr and cad in maize and Arabidopsis suggests the timing of this aspect of lignin biosynthesis is conserved (Table 2) . Several cellulose synthase genes shared nearly the same dawn phasing as the ccr and cad transcripts (Figure 3). Similarly, transcripts for two endoglucanases and a cellulase cycled at dawn in the maize dataset (Figure 3). Circadian dawn expression of cell wall-related enzymes correlates with the time of maximal plant growth rate reported for Arabidopsis [49, 51]; therefore, the maize circadian clock seems to regulate daily cell wall construction so that it coincides with growth trigged by phytohormone signaling.
Circadian clock regulation of maize GA, ethylene, and ABA biosynthesis genes
Several recent studies have shown a fundamental role of the Arabidopsis circadian clock is to indirectly control growth and development through transcriptional regulation of genes involved in phytohormone biosynthesis and response [25, 27, 29]. Placement of the maize cycling genes in the context of metabolic pathways revealed that the maize circadian clock also regulates genes involved in the synthesis of phytohormones fundamental to plant growth (Table 2).
The plant stress hormone ABA antagonizes the growth promoting effects of GA and ethylene  and, like GAs and ethylene, ABA biosynthesis genes are part of a transcriptional regulatory network that exerts daily control over plant growth . The carotenoid biosynthesis pathway supplies precursors for the biosynthesis of ABA . As noted above, the transcript encoding PSY, the enzyme governing the first committed step in carotenoid synthesis, was under circadian control with peak expression in the early morning (Table 2, Figure 4A). Several genes downstream of PSY that are involved in ABA synthesis also showed dawn expression (Table 2, Figure 4A), which is consistent with ABA synthesis taking place in the early morning. Comparable circadian regulation of PSY and genes encoding downstream components of this pathway has been noted in Arabidopsis . The conserved circadian regulation of maize carotenoid biosynthesis genes observed here suggests that synthesis of ABA in maize leaves is under circadian control, which likely impacts the key role ABA plays in antagonizing cell growth and in stimulating stomatal closure . The latter role of ABA is associated higher-water use efficiencies in crop plants, since closure of stomata reduces water loss. Therefore, the maize circadian system is likely a critically important, but under appreciated, contributor to maize productivity in the field.
Rhythmic expression of putative maize flowering time, circadian clock, and phototropism genes
Several orthologs of Arabidopsis circadian clock and flowering time genes were present in the maize cycling gene set; however, the array had limited overall representation of genes in these categories. Included in the rhythmic collection was the gene encoding CONZ1, which the annotation matched to Arabidopsis COL1 (Table 2, Figure 4B). CONZ1 is a strong candidate for the functional ortholog of Arabidopsis CONSTANS and rice Heading date 1 . CONSTANS and Heading date 1 serve in control of flowering time to regulate the expression of the FT class of proteins. FT proteins act as florigen molecules that promote floral development at the shoot apical meristem . gi1A and gi1B are paralogous flowering time and circadian clock genes identified in the same study as conz1 . A probe set for gi1A was on the array and this transcript exhibited a waveform and phase similar to that of Arabidopsis GI expression (Figure 4B, Table 2) . Orthologs of Arabidopsis LDL1 and FCA were two other potential flowering time transcripts with rhythmic expression in the maize dataset (Figure 4B). LDL1 and FCA are known to act in the Arabidopsis autonomous flowering pathway . LDL1 is involved in modification of chromatin and, therefore, is not likely to be strictly involved in flowering time . Similarly, FCA plays a role in RNA-mediated silencing through DNA methylation . Circadian clock components present on the array included orthologs of Arabidopsis PRR7 and LUX (Table 2, Figure 4B). Expression of maize prr7 and lux exhibited the same phasing as their Arabidopsis orthologs (Table 2), indicating the conserved nature of the core maize circadian clock. Finally, the maize phot1 transcript, encoding a blue light photoreceptor of the phototropin family likely to be involved in phototropism, was robustly rhythmic with the identical phase as its Arabidopsis counterpart (Figure 4B). This restricted sampling of flowering time, circadian clock, and phototropism genes suggests that maize relies on conserved signaling networks for these key processes.
Identification of maize genes under circadian clock regulation and the predicted contribution of these genes to metabolism, growth, and development indicates the maize circadian clock plays an important role in coordinating the overall physiology of this C4 crop plant. In general, circadian regulation of the enzymes mediating C4 photosynthesis was predictable based on the Arabidopsis C3 model and without any apparent large-scale changes to accommodate the specialized C4 anatomy and enzymology. Recent investigation of the global effects of the circadian system on Arabidopsis physiology demonstrated that rhythms are critical to plant fitness and optimal growth . A more complete appreciation of the maize circadian clock will reveal where the circadian system impacts maize growth and development, as well as highlight novel approaches for optimization of crop production through targeted modification of the circadian system.
Plant materials and growth conditions
Seeds of inbred B73 were obtained from the Maize Genetics Cooperation Center. B73 is widely used and is the source material for the complete maize genome sequence . Seedlings were germinated and grown in 57 × 30 × 6 cm flats filled with Scotts Supersoil (scotts.com) inside a Conviron (conviron.com) growth chamber at constant 26°C and constant 70% humidity. High intensity cool white fluorescent bulbs provided illumination at a fluence rate of 200 μmoles m-2 s-1. Seedlings were grown for seven days under 12 hours of light followed by 12 hours of dark before being transferred to LL for two days at the same temperature and humidity.
RNA sample preparation and array hybridization
Tissue harvest began following transfer of seedlings to LL on the 8th day. The entire aerial portion (corresponding to all tissue above the prop roots) of five seedlings was harvested beginning at dawn and every 4 hours thereafter for the next 48 hours. Samples were immediately frozen in liquid nitrogen. Three separate experimental replicates were collected in this way. Tissue was ground to a fine powder under liquid nitrogen and total RNA isolated by Trizol extraction (Invitrogen; invitrogen.com) followed by Qiagen RNeasy columns and treatment with RNase-free DNase I (Qiagen; qiagen.com). cRNA was generated from total RNA of three pooled replicate samples with the GeneChip® One-Cycle Target Labeling kit according to the manufacturer's recommendations (Affymetrix, affymetrix.com). The University of California, Berkeley Functional Genomics Laboratory  hybridized samples to Affymetrix GeneChip® Maize Genome Arrays and scanned the washed arrays as suggested by manufacturer. Present, Marginal, and Absent calls for each probe set were determined with MAS5 analysis  and these are presented in Additional file 7 Table S4. Probe sets called "Absent" at more than nine time points were removed from the downstream analysis . Raw hybridization intensities were normalized across all twelve arrays using RMAExpress in Perfect Match mode [64, 65]. The raw microarray data is available online as experiment ZM28 at the Plant Expression Database .
Identification of maize transcripts with circadian expression waveforms
Rhythmic expression waveforms within the normalized dataset were identified by analysis with the COSOPT curve fitting algorithm and the pattern matching function HAYSTACK. The COSOPT algorithm is a cosine waveform fitting method that identifies transcripts with circadian expression by first performing an arithmetic linear-regression detrend of the normalized time series data for each transcript followed by testing detrended data for a fit to 101 cosine test models . An approximate goodness of fit value produced by COSOPT analysis is MMC-β, where lower values correspond to a better fit to an ideal cosine curve. Based on previous studies in Arabidopsis [25, 26, 28, 33] and our empirical tests, a MMC-β threshold of ≤ 0.05 was chosen to call a given probe set circadian. HAYSTACK is a model-based, pattern-matching tool that identifies rhythmic expression patterns by aligning time-series microarray data to a set of discrete diurnal and circadian models . HAYSTACK conducts a least square linear regression for each gene against all the models and calculates the best-fit model, phase-of-expression, and p-value for each gene. HAYSTACK was run on the normalized maize dataset with the following parameters: fold cutoff = 2, p-value cutoff = 0.05, background cutoff = 50, and PCC = 0.7 with the 552 model set described in Michael et al. 2008 . In general, goodness of fit between the model and experimental traces is revealed by the PCC value. We empirically determined that a PCC ≥ 0.7 produced good matches between model and experimental data. This cutoff was less stringent than that applied in previous studies investigating Arabidopsis circadian expression [26, 29].
Phase clustering of circadian waveforms
Probe sets with similar expression waveforms were grouped together by K-means clustering. The RMAExpress normalized values for probe sets with MMC-β ≤ 0.05 and those with PCC ≥ 0.7 were converted to normalized values with the following calculation: value = [(expression at single time point) - mean(all time points)]/[standard deviation(all time points)]. These values were used to fit the waveforms to six clusters representing each ZT phase with the K-means clustering algorithm built into the TIGR Multiexpression Viewer tool . Clustering ran for a total of 50 iterations and was considered complete when probe sets were no longer exchanged between clusters. In two independent trials, no greater than 7 iterations were required to build the clusters.
Matching of probe sets to maize genes, assignment of GO Slim terms to genes, and functional annotation
Additional file 4 Figure S3 outlines the annotation scheme used to match probe sets to genes in the maize genome, identify GO Slim terms for maize genes, and functionally annotate maize genes. Transcripts represented on the Affymetrix GeneChip® Maize array were mapped to maize transcripts with a custom Perl script that used the DNA sequences provided by Affymetrix and BLASTn to query the ZmB73 4.53a filtered CDS sequences . After the maize gene ID was identified for each probe set, GO Slim terms and associated PlantCyc pathways  were extracted from the Maize Genome Consortium website . Maize gene IDs for each gene were used with a local implementation of BLASTp to identify likely rice orthologs in the Michigan State University Rice Pseudomolecule and Genome Annotation release 6.1 and likely Arabidopsis orthologs in TAIR9_pep_20090619 sequences. The output of these queries were separated into high and low confidence lists (Additional file 6 Table S3). Criteria for the high confidence list were amino acid identity of 40% or greater, Highest Scoring Segment Pair (HSP) of 50 amino acids or greater, and a match to putative orthologs in both Arabidopsis and rice. The low confidence list contains the remaining genes that encode proteins not meeting these three criteria. Putative functions of matched orthologs were derived from Kyoto Encyclopedia of Genes and Genomes . Phase of expression for Arabidopsis orthologs shown in Table 2 was determined with the DIURNAL tool from the "LL12" dataset and matches between model and data considered significant at PCC values ≥ 0.7 . Overrepresentation of GO Slim terms was calculated with hypergeometric distribution followed by a modified Bonfferoni correction for multiple testing as implemented in GeneMerge . GeneMerge produced an e-score comparable to a P-value that was used to determine significance of overrepresentation. GO Slim terms for the entire array were the population set and the indicated collection of cycling genes served as the study set. Overrepresented GO Slim terms were considered those with e-scores < 0.05.
List of Abbreviations
ABSCISIC ACID 8-HYDROXYLASE
ADP-GLUCOSE PYROPHOSPHORYLASE LARGE SUBUNIT
CINNAMYL ALCOHOL DEHYDROGENASE
CIRCADIAN CLOCK ASSOCIATED 1
CCA1 HIKING EXPEDITION
CONSTANS OF ZEA MAYS1
EARLY FLOWERING 3
GA REQUIRING 1
ENT-KAURENOIC ACID HYDROXYLASE 2
KETOSE-BISPHOSPHATE ALDOLASE CLASS II
LYSINE-SPECIFIC DEMETHYLASE 1
LIGHT HARVESTING COMPLEX B
LATE ELONGATED HYPOCOTYL
- MALIC E:
Multiple Measures Corrected-β
MANGANESE SUPEROXIDE DISMUTASE1
METHIONINE OVER-ACCUMULATOR 3
Pearson Correlation Coefficient
phospho enol pyruvate
PHOSPHO ENOL PYRUVATE CARBOXYLASE
PYRUVATE; ORTHOPHOSPHATE DIKINASE
RUBISCO SMALL SUBUNIT
reactive oxygen species
RIBOSE PHOSPHATE ISOMERASE
RIBULOSE BISPHOSPHATE CARBOXYLASE OXYGENASE
TIMING OF CAB EXPRESSION 1
TRIOSE PHOSPHATE ISOMERASE
Uridine diphosphate glucose
We thank Dr. Hector Candela-Anton, James Schnable, and Imran N. Amirani for writing perl scripts, as well as Dr. Robin Buell for help and advice on annotation with rice sequences. We also thank Bryan Thines for critical reading of the manuscript. This work is supported by United States Department of Agriculture grant CRIS 5335-21000-026-00D to FGH.
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