Transgenic insertion of the cyanobacterial membrane protein ictB increases grain yield in Zea mays through increased photosynthesis and carbohydrate production.

The C4 crop maize (Zea mays) is the most widely grown cereal crop worldwide and is an essential feedstock for food and bioenergy. Improving maize yield is important to achieve food security and agricultural sustainability in the 21st century. One potential means to improve crop productivity is to enhance photosynthesis. ictB, a membrane protein that is highly conserved across cyanobacteria, has been shown to improve photosynthesis, and often biomass, when introduced into diverse C3 plant species. Here, ictB from Synechococcus sp. strain PCC 7942 was inserted into maize using Agrobacterium-mediated transformation. In three controlled-environment experiments, ictB insertion increased leaf starch and sucrose content by up to 25% relative to controls. Experimental field trials in four growing seasons, spanning the Midwestern United States (Summers 2018 & 2019) and Argentina (Winter 2018 & 2019), showed an average of 3.49% grain yield improvement, by as much as 5.4% in a given season and up to 9.4% at certain trial locations. A subset of field trial locations was used to test for modification of ear traits and ФPSII, a proxy for photosynthesis. Results suggested that yield gain in transgenics could be associated with increased ФPSII, and the production of longer, thinner ears with more kernels. ictB localized primarily to the microsome fraction of leaf bundle-sheath cells, but not to chloroplasts. Extramembrane domains of ictB interacted in vitro with proteins involved in photosynthesis and carbohydrate metabolism. To our knowledge, this is the first published evidence of ictB insertion into a species using C4 photosynthesis and the largest-scale demonstration of grain yield enhancement from ictB insertion in planta. Results show that ictB is a valuable yield gene in the economically important crop maize, and are an important proof of concept that transgenic manipulation of photosynthesis can be used to create economically viable crop improvement traits.

Temporal and spatial transcriptomic and microRNA dynamics of CAM photosynthesis in pineapple.

The altered carbon assimilation pathway of crassulacean acid metabolism (CAM) photosynthesis results in an up to 80% higher water-use efficiency than C3 photosynthesis in plants making it a potentially useful pathway for engineering crop plants with improved drought tolerance. Here we surveyed detailed temporal (diel time course) and spatial (across a leaf gradient) gene and microRNA (miRNA) expression patterns in the obligate CAM plant pineapple [Ananas comosus (L.) Merr.]. The high-resolution transcriptome atlas allowed us to distinguish between CAM-related and non-CAM gene copies. A differential gene co-expression network across green and white leaf diel datasets identified genes with circadian oscillation, CAM-related functions, and source-sink relations. Gene co-expression clusters containing CAM pathway genes are enriched with clock-associated cis-elements, suggesting circadian regulation of CAM. About 20% of pineapple microRNAs have diel expression patterns, with several that target key CAM-related genes. Expression and physiology data provide a model for CAM-specific carbohydrate flux and long-distance hexose transport. Together these resources provide a list of candidate genes for targeted engineering of CAM into C3 photosynthesis crop species.

Comparative Analysis of Vertebrate Diurnal/Circadian Transcriptomes.

From photosynthetic bacteria to mammals, the circadian clock evolved to track diurnal rhythms and enable organisms to anticipate daily recurring changes such as temperature and light. It orchestrates a broad spectrum of physiology such as the sleep/wake and eating/fasting cycles. While we have made tremendous advances in our understanding of the molecular details of the circadian clock mechanism and how it is synchronized with the environment, we still have rudimentary knowledge regarding its connection to help regulate diurnal physiology. One potential reason is the sheer size of the output network. Diurnal/circadian transcriptomic studies are reporting that around 10% of the expressed genome is rhythmically controlled. Zebrafish is an important model system for the study of the core circadian mechanism in vertebrate. As Zebrafish share more than 70% of its genes with human, it could also be an additional model in addition to rodent for exploring the diurnal/circadian output with potential for translational relevance. Here we performed comparative diurnal/circadian transcriptome analysis with established mouse liver and other tissue datasets. First, by combining liver tissue sampling in a 48h time series, transcription profiling using oligonucleotide arrays and bioinformatics analysis, we profiled rhythmic transcripts and identified 2609 rhythmic genes. The comparative analysis revealed interesting features of the output network regarding number of rhythmic genes, proportion of tissue specific genes and the extent of transcription factor family expression. Undoubtedly, the Zebrafish model system will help identify new vertebrate outputs and their regulators and provides leads for further characterization of the diurnal cis-regulatory network.

Grasses suppress shoot-borne roots to conserve water during drought.

Many important crops are members of the Poaceae family, which develop root systems characterized by a high degree of root initiation from the belowground basal nodes of the shoot, termed the crown. Although this postembryonic shoot-borne root system represents the major conduit for water uptake, little is known about the effect of water availability on its development. Here we demonstrate that in the model C4 grass Setaria viridis, the crown locally senses water availability and suppresses postemergence crown root growth under a water deficit. This response was observed in field and growth room environments and in all grass species tested. Luminescence-based imaging of root systems grown in soil-like media revealed a shift in root growth from crown-derived to primary root-derived branches, suggesting that primary root-dominated architecture can be induced in S. viridis under certain stress conditions. Crown roots of Zea mays and Setaria italica, domesticated relatives of teosinte and S. viridis, respectively, show reduced sensitivity to water deficit, suggesting that this response might have been influenced by human selection. Enhanced water status of maize mutants lacking crown roots suggests that under a water deficit, stronger suppression of crown roots actually may benefit crop productivity.

The genome of black raspberry (Rubus occidentalis).

Black raspberry (Rubus occidentalis) is an important specialty fruit crop in the US Pacific Northwest that can hybridize with the globally commercialized red raspberry (R. idaeus). Here we report a 243 Mb draft genome of black raspberry that will serve as a useful reference for the Rosaceae and Rubus fruit crops (raspberry, blackberry, and their hybrids). The black raspberry genome is largely collinear to the diploid woodland strawberry (Fragaria vesca) with a conserved karyotype and few notable structural rearrangements. Centromeric satellite repeats are widely dispersed across the black raspberry genome, in contrast to the tight association with the centromere observed in most plants. Among the 28 005 predicted protein-coding genes, we identified 290 very recent small-scale gene duplicates enriched for sugar metabolism, fruit development, and anthocyanin related genes which may be related to key agronomic traits during black raspberry domestication. This contrasts patterns of recent duplications in the wild woodland strawberry F. vesca, which show no patterns of enrichment, suggesting gene duplications contributed to domestication traits. Expression profiles from a fruit ripening series and roots exposed to Verticillium dahliae shed insight into fruit development and disease response, respectively. The resources presented here will expedite the development of improved black and red raspberry, blackberry and other Rubus cultivars.

The pineapple genome and the evolution of CAM photosynthesis.

Pineapple (Ananas comosus (L.) Merr.) is the most economically valuable crop possessing crassulacean acid metabolism (CAM), a photosynthetic carbon assimilation pathway with high water-use efficiency, and the second most important tropical fruit. We sequenced the genomes of pineapple varieties F153 and MD2 and a wild pineapple relative, Ananas bracteatus accession CB5. The pineapple genome has one fewer ancient whole-genome duplication event than sequenced grass genomes and a conserved karyotype with seven chromosomes from before the ρ duplication event. The pineapple lineage has transitioned from C3 photosynthesis to CAM, with CAM-related genes exhibiting a diel expression pattern in photosynthetic tissues. CAM pathway genes were enriched with cis-regulatory elements associated with the regulation of circadian clock genes, providing the first cis-regulatory link between CAM and circadian clock regulation. Pineapple CAM photosynthesis evolved by the reconfiguration of pathways in C3 plants, through the regulatory neofunctionalization of preexisting genes and not through the acquisition of neofunctionalized genes via whole-genome or tandem gene duplication.

Alternative splicing in plants: directing traffic at the crossroads of adaptation and environmental stress.

In recent years, high-throughput sequencing-based analysis of plant transcriptomes has suggested that up to ∼60% of plant gene loci encode alternatively spliced mature transcripts. These studies have also revealed that alternative splicing in plants can be regulated by cell type, developmental stage, the environment, and the circadian clock. Alternative splicing is coupled to RNA surveillance and processing mechanisms, including nonsense mediated decay. Recently, non-protein-coding transcripts have also been shown to undergo alternative splicing. These discoveries collectively describe a robust system of post-transcriptional regulatory feedback loops which influence RNA abundance. In this review, we summarize recent studies describing the specific roles alternative splicing and RNA surveillance play in plant adaptation to environmental stresses and the regulation of the circadian clock.

Extensive Transcriptome Changes During Natural Onset and Release of Vegetative Bud Dormancy in Populus.

To survive winter, many perennial plants become endodormant, a state of suspended growth maintained even in favorable growing environments. To understand vegetative bud endodormancy, we collected paradormant, endodormant, and ecodormant axillary buds from Populus trees growing under natural conditions. Of 44,441 Populus gene models analyzed using NimbleGen microarrays, we found that 1,362 (3.1%) were differentially expressed among the three dormancy states, and 429 (1.0%) were differentially expressed during only one of the two dormancy transitions (FDR p-value < 0.05). Of all differentially expressed genes, 69% were down-regulated from paradormancy to endodormancy, which was expected given the lower metabolic activity associated with endodormancy. Dormancy transitions were accompanied by changes in genes associated with DNA methylation (via RNA-directed DNA methylation) and histone modifications (via Polycomb Repressive Complex 2), confirming and extending knowledge of chromatin modifications as major features of dormancy transitions. Among the chromatin-associated genes, two genes similar to SPT (SUPPRESSOR OF TY) were strongly up-regulated during endodormancy. Transcription factor genes and gene sets that were atypically up-regulated during endodormancy include a gene that seems to encode a trihelix transcription factor and genes associated with proteins involved in responses to ethylene, cold, and other abiotic stresses. These latter transcription factors include ETHYLENE INSENSITIVE 3 (EIN3), ETHYLENE-RESPONSIVE ELEMENT BINDING PROTEIN (EBP), ETHYLENE RESPONSE FACTOR (ERF), ZINC FINGER PROTEIN 10 (ZAT10), ZAT12, and WRKY DNA-binding domain proteins. Analyses of phytohormone-associated genes suggest important changes in responses to ethylene, auxin, and brassinosteroids occur during endodormancy. We found weaker evidence for changes in genes associated with salicylic acid and jasmonic acid, and little evidence for important changes in genes associated with gibberellins, abscisic acid, and cytokinin. We identified 315 upstream sequence motifs associated with eight patterns of gene expression, including novel motifs and motifs associated with the circadian clock and responses to photoperiod, cold, dehydration, and ABA. Analogies between flowering and endodormancy suggest important roles for genes similar to SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL), DORMANCY ASSOCIATED MADS-BOX (DAM), and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1).

Genome diversity in Brachypodium distachyon: deep sequencing of highly diverse inbred lines.

Brachypodium distachyon is small annual grass that has been adopted as a model for the grasses. Its small genome, high-quality reference genome, large germplasm collection, and selfing nature make it an excellent subject for studies of natural variation. We sequenced six divergent lines to identify a comprehensive set of polymorphisms and analyze their distribution and concordance with gene expression. Multiple methods and controls were utilized to identify polymorphisms and validate their quality. mRNA-Seq experiments under control and simulated drought-stress conditions, identified 300 genes with a genotype-dependent treatment response. We showed that large-scale sequence variants had extremely high concordance with altered expression of hundreds of genes, including many with genotype-dependent treatment responses. We generated a deep mRNA-Seq dataset for the most divergent line and created a de novo transcriptome assembly. This led to the discovery of >2400 previously unannotated transcripts and hundreds of genes not present in the reference genome. We built a public database for visualization and investigation of sequence variants among these widely used inbred lines.

Analysis of global gene expression in Brachypodium distachyon reveals extensive network plasticity in response to abiotic stress.

Brachypodium distachyon is a close relative of many important cereal crops. Abiotic stress tolerance has a significant impact on productivity of agriculturally important food and feedstock crops. Analysis of the transcriptome of Brachypodium after chilling, high-salinity, drought, and heat stresses revealed diverse differential expression of many transcripts. Weighted Gene Co-Expression Network Analysis revealed 22 distinct gene modules with specific profiles of expression under each stress. Promoter analysis implicated short DNA sequences directly upstream of module members in the regulation of 21 of 22 modules. Functional analysis of module members revealed enrichment in functional terms for 10 of 22 network modules. Analysis of condition-specific correlations between differentially expressed gene pairs revealed extensive plasticity in the expression relationships of gene pairs. Photosynthesis, cell cycle, and cell wall expression modules were down-regulated by all abiotic stresses. Modules which were up-regulated by each abiotic stress fell into diverse and unique gene ontology GO categories. This study provides genomics resources and improves our understanding of abiotic stress responses of Brachypodium.

Sequencing and characterization of the anadromous steelhead (Oncorhynchus mykiss) transcriptome.

Identifying the traits that differ between hatchery and wild fish may allow for pragmatic changes to hatchery practice. To meet those ends, we sequenced, assembled, and characterized the anadromous steelhead (Oncorhynchus mykiss) transcriptome. Using the Illumina sequencing platform, we sequenced nearly 41million 76-mer reads representing 3.1 Gbp of steelhead transcriptome. Upon final assembly, this sequence data yielded 86,402 transcript scaffolds, of which, 66,530 (77%) displayed homology to proteins of the non-redundant NCBI database. Gene descriptions and gene ontology terms were used to annotate the transcriptome resulting in 4030 unique gene ontology (GO) annotations attributed to the assembled sequences. We also conducted a comparative analysis that identified homologous genes within four other fish species including zebrafish (Danio rerio), stickleback (Gasterosteus aculeatus), and two pufferfish species (Tetraodon nigroviridis and Takifugu rubripes). Comparing our steelhead reference assembly directly to the transcriptome for rainbow trout (the fresh water life-history variant of the same species) revealed that while the steelhead and rainbow trout transcriptomes are complementary, the steelhead data will be useful for investigating questions related to anadromous (ocean-going) fishes. These sequence data and web tools provide a useful set of resources for salmonid researchers and the broader genomics community (available at http://salmon.cgrb.oregonstate.edu).

Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon.

BACKGROUND: Lignin is a significant barrier in the conversion of plant biomass to bioethanol. Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the pathway of lignin monomer biosynthesis. Brown midrib mutants in Zea mays and Sorghum bicolor with impaired CAD or COMT activity have attracted considerable agronomic interest for their altered lignin composition and improved digestibility. Here, we identified and functionally characterized candidate genes encoding CAD and COMT enzymes in the grass model species Brachypodium distachyon with the aim of improving crops for efficient biofuel production. RESULTS: We developed transgenic plants overexpressing artificial microRNA designed to silence BdCAD1 or BdCOMT4. Both transgenes caused altered flowering time and increased stem count and weight. Downregulation of BdCAD1 caused a leaf brown midrib phenotype, the first time this phenotype has been observed in a C3 plant. While acetyl bromide soluble lignin measurements were equivalent in BdCAD1 downregulated and control plants, histochemical staining and thioacidolysis indicated a decrease in lignin syringyl units and reduced syringyl/guaiacyl ratio in the transgenic plants. BdCOMT4 downregulated plants exhibited a reduction in total lignin content and decreased Maule staining of syringyl units in stem. Ethanol yield by microbial fermentation was enhanced in amiR-cad1-8 plants. CONCLUSION: These results have elucidated two key genes in the lignin biosynthetic pathway in B. distachyon that, when perturbed, may result in greater stem biomass yield and bioconversion efficiency.

Detection and quantification of alternative splicing variants using RNA-seq.

Next-generation sequencing has enabled genome-wide studies of alternative pre-mRNA splicing, allowing for empirical determination, characterization, and quantification of the expressed RNAs in a sample in toto. As a result, RNA sequencing (RNA-seq) has shown tremendous power to drive biological discoveries. At the same time, RNA-seq has created novel challenges that necessitate the development of increasingly sophisticated computational approaches and bioinformatic tools. In addition to the analysis of massive datasets, these tools also need to facilitate questions and analytical approaches driven by such rich data. HTS and RNA-seq are still in a stage of very rapid evolution and are, therefore, only introduced in general terms. This chapter mainly focuses on the methods for discovery, detection, and quantification of alternatively spliced transcript variants.

Dynamic DNA cytosine methylation in the Populus trichocarpa genome: tissue-level variation and relationship to gene expression.

BACKGROUND: DNA cytosine methylation is an epigenetic modification that has been implicated in many biological processes. However, large-scale epigenomic studies have been applied to very few plant species, and variability in methylation among specialized tissues and its relationship to gene expression is poorly understood. RESULTS: We surveyed DNA methylation from seven distinct tissue types (vegetative bud, male inflorescence [catkin], female catkin, leaf, root, xylem, phloem) in the reference tree species black cottonwood (Populus trichocarpa). Using 5-methyl-cytosine DNA immunoprecipitation followed by Illumina sequencing (MeDIP-seq), we mapped a total of 129,360,151 36- or 32-mer reads to the P. trichocarpa reference genome. We validated MeDIP-seq results by bisulfite sequencing, and compared methylation and gene expression using published microarray data. Qualitative DNA methylation differences among tissues were obvious on a chromosome scale. Methylated genes had lower expression than unmethylated genes, but genes with methylation in transcribed regions ("gene body methylation") had even lower expression than genes with promoter methylation. Promoter methylation was more frequent than gene body methylation in all tissues except male catkins. Male catkins differed in demethylation of particular transposable element categories, in level of gene body methylation, and in expression range of genes with methylated transcribed regions. Tissue-specific gene expression patterns were correlated with both gene body and promoter methylation. CONCLUSIONS: We found striking differences among tissues in methylation, which were apparent at the chromosomal scale and when genes and transposable elements were examined. In contrast to other studies in plants, gene body methylation had a more repressive effect on transcription than promoter methylation.

Global profiling of rice and poplar transcriptomes highlights key conserved circadian-controlled pathways and cis-regulatory modules.

BACKGROUND: Circadian clocks provide an adaptive advantage through anticipation of daily and seasonal environmental changes. In plants, the central clock oscillator is regulated by several interlocking feedback loops. It was shown that a substantial proportion of the Arabidopsis genome cycles with phases of peak expression covering the entire day. Synchronized transcriptome cycling is driven through an extensive network of diurnal and clock-regulated transcription factors and their target cis-regulatory elements. Study of the cycling transcriptome in other plant species could thus help elucidate the similarities and differences and identify hubs of regulation common to monocot and dicot plants. METHODOLOGY/PRINCIPAL FINDINGS: Using a combination of oligonucleotide microarrays and data mining pipelines, we examined daily rhythms in gene expression in one monocotyledonous and one dicotyledonous plant, rice and poplar, respectively. Cycling transcriptomes were interrogated under different diurnal (driven) and circadian (free running) light and temperature conditions. Collectively, photocycles and thermocycles regulated about 60% of the expressed nuclear genes in rice and poplar. Depending on the condition tested, up to one third of oscillating Arabidopsis-poplar-rice orthologs were phased within three hours of each other suggesting a high degree of conservation in terms of rhythmic gene expression. We identified clusters of rhythmically co-expressed genes and searched their promoter sequences to identify phase-specific cis-elements, including elements that were conserved in the promoters of Arabidopsis, poplar, and rice. CONCLUSIONS/SIGNIFICANCE: Our results show that the cycling patterns of many circadian clock genes are highly conserved across poplar, rice, and Arabidopsis. The expression of many orthologous genes in key metabolic and regulatory pathways is diurnal and/or circadian regulated and phased to similar times of day. Our results confirm previous findings in Arabidopsis of three major classes of cis-regulatory modules within the plant circadian network: the morning (ME, GBOX), evening (EE, GATA), and midnight (PBX/TBX/SBX) modules. Identification of identical overrepresented motifs in the promoters of cycling genes from different species suggests that the core diurnal/circadian cis-regulatory network is deeply conserved between mono- and dicotyledonous species.

The genome of woodland strawberry (Fragaria vesca).

The woodland strawberry, Fragaria vesca (2n = 2x = 14), is a versatile experimental plant system. This diminutive herbaceous perennial has a small genome (240 Mb), is amenable to genetic transformation and shares substantial sequence identity with the cultivated strawberry (Fragaria × ananassa) and other economically important rosaceous plants. Here we report the draft F. vesca genome, which was sequenced to ×39 coverage using second-generation technology, assembled de novo and then anchored to the genetic linkage map into seven pseudochromosomes. This diploid strawberry sequence lacks the large genome duplications seen in other rosids. Gene prediction modeling identified 34,809 genes, with most being supported by transcriptome mapping. Genes critical to valuable horticultural traits including flavor, nutritional value and flowering time were identified. Macrosyntenic relationships between Fragaria and Prunus predict a hypothetical ancestral Rosaceae genome that had nine chromosomes. New phylogenetic analysis of 154 protein-coding genes suggests that assignment of Populus to Malvidae, rather than Fabidae, is warranted.

Transcription factors in light and circadian clock signaling networks revealed by genomewide mapping of direct targets for neurospora white collar complex.

Light signaling pathways and circadian clocks are inextricably linked and have profound effects on behavior in most organisms. Here, we used chromatin immunoprecipitation (ChIP) sequencing to uncover direct targets of the Neurospora crassa circadian regulator White Collar Complex (WCC). The WCC is a blue-light receptor and the key transcription factor of the circadian oscillator. It controls a transcriptional network that regulates ∼20% of all genes, generating daily rhythms and responses to light. We found that in response to light, WCC binds to hundreds of genomic regions, including the promoters of previously identified clock- and light-regulated genes. We show that WCC directly controls the expression of 24 transcription factor genes, including the clock-controlled adv-1 gene, which controls a circadian output pathway required for daily rhythms in development. Our findings provide links between the key circadian activator and effectors in downstream regulatory pathways.

Supersplat--spliced RNA-seq alignment.

MOTIVATION: High-throughput sequencing technologies have recently made deep interrogation of expressed transcript sequences practical, both economically and temporally. Identification of intron/exon boundaries is an essential part of genome annotation, yet remains a challenge. Here, we present supersplat, a method for unbiased splice-junction discovery through empirical RNA-seq data. RESULTS: Using a genomic reference and RNA-seq high-throughput sequencing datasets, supersplat empirically identifies potential splice junctions at a rate of approximately 11.4 million reads per hour. We further benchmark the performance of the algorithm by mapping Illumina RNA-seq reads to identify introns in the genome of the reference dicot plant Arabidopsis thaliana and we demonstrate the utility of supersplat for de novo empirical annotation of splice junctions using the reference monocot plant Brachypodium distachyon. AVAILABILITY: Implemented in C++, supersplat source code and binaries are freely available on the web at http://mocklerlab-tools.cgrb.oregonstate.edu/.

Genome-wide mapping of alternative splicing in Arabidopsis thaliana.

Alternative splicing can enhance transcriptome plasticity and proteome diversity. In plants, alternative splicing can be manifested at different developmental stages, and is frequently associated with specific tissue types or environmental conditions such as abiotic stress. We mapped the Arabidopsis transcriptome at single-base resolution using the Illumina platform for ultrahigh-throughput RNA sequencing (RNA-seq). Deep transcriptome sequencing confirmed a majority of annotated introns and identified thousands of novel alternatively spliced mRNA isoforms. Our analysis suggests that at least approximately 42% of intron-containing genes in Arabidopsis are alternatively spliced; this is significantly higher than previous estimates based on cDNA/expressed sequence tag sequencing. Random validation confirmed that novel splice isoforms empirically predicted by RNA-seq can be detected in vivo. Novel introns detected by RNA-seq were substantially enriched in nonconsensus terminal dinucleotide splice signals. Alternative isoforms with premature termination codons (PTCs) comprised the majority of alternatively spliced transcripts. Using an example of an essential circadian clock gene, we show that intron retention can generate relatively abundant PTC(+) isoforms and that this specific event is highly conserved among diverse plant species. Alternatively spliced PTC(+) isoforms can be potentially targeted for degradation by the nonsense mediated mRNA decay (NMD) surveillance machinery or regulate the level of functional transcripts by the mechanism of regulated unproductive splicing and translation (RUST). We demonstrate that the relative ratios of the PTC(+) and reference isoforms for several key regulatory genes can be considerably shifted under abiotic stress treatments. Taken together, our results suggest that like in animals, NMD and RUST may be widespread in plants and may play important roles in regulating gene expression.

Cis-regulatory elements in plant cell signaling.

Plant cell signaling pathways are in part dependent on transcriptional regulatory networks comprising circuits of transcription factors (TFs) and regulatory DNA elements that control the expression of target genes. Here, we describe experimental and bioinformatic approaches for identifying potential cis-regulatory elements. We also discuss recent integrative genomics studies aimed at elucidating the functions of cis-regulatory elements in aspects of plant biology, including the circadian clock, interactions with the environment, stress responses, and regulation of growth and development by phytohormones. Finally, we discuss emerging technologies and approaches that offer great potential for accelerating the discovery and functional characterization of cis-elements and interacting TFs--which will help realize the promise of systems biology.