Rhythmic lipid and gene expression responses to chilling in panicoid grasses.

Chilling stress threatens plant growth and development, particularly affecting membrane fluidity and cellular integrity. Understanding plant membrane responses to chilling stress is important for unraveling the molecular mechanisms of stress tolerance. Whereas core transcriptional responses to chilling stress and stress tolerance are conserved across species, the associated changes in membrane lipids appear to be less conserved, as which lipids are affected by chilling stress varies by species. Here, we investigated changes in gene expression and membrane lipids in response to chilling stress during one 24 hour cycle in chilling-tolerant foxtail millet (Setaria italica), and chilling-sensitive sorghum (Sorghum bicolor), and Urochloa (browntop signal grass, Urochloa fusca, lipids only), leveraging their evolutionary relatedness and differing levels of chilling-stress tolerance. We show that most chilling-induced lipid changes are conserved across the three species, while we observed distinct, time-specific responses in chilling-tolerant foxtail millet, indicating the presence of a finely orchestrated adaptive mechanism. We detected rhythmicity in lipid responses to chilling stress in the three grasses, which were also present in Arabidopsis (Arabidopsis thaliana), suggesting the conservation of rhythmic patterns across species and highlighting the importance of accounting for time of day. When integrating lipid datasets with gene expression profiles, we identified potential candidate genes that showed corresponding transcriptional changes in response to chilling stress, providing insights into the differences in regulatory mechanisms between chilling-sensitive sorghum and chilling-tolerant foxtail millet.

Identification of transcriptional networks controlling leaf sheath growth in Sorghum bicolor.

OBJECTIVES: The objective of this data set was to identify transcriptional networks that control elongation of seedling leaf sheaths in the C4 grass Sorghum bicolor. One motivation was that leaf sheaths are a primary constituent of stems in grass seedlings; therefore, genes that control growth of this organ are important contributors to successful transition from the seedling stage to the mature plant stage and, ultimately, crop success. Since diurnal rhythms contribute to regulation of signaling networks responsible for growth, a time course representing the late afternoon and early evening was anticipated to pinpoint important control genes for stem growth. Ultimately, the expected outcome was discovery of transcript networks that integrate internal and external signals to fine tune leaf sheath growth and, consequently, plant height. DATA DESCRIPTION: The data set is RNAseq profiling of upper leaf sheaths collected from wild type Sorghum bicolor (BTx623 line) plants at four-hour intervals from 12.5 h after dawn to 20 h after dawn. Global transcript levels in leaves were determined by deep sequencing of mRNA from four individual seedlings at each time point. This data set contains sequences representing the spectrum of mRNAs from individual genes. This data set enables detection of significant changes in gene-level expression caused by the progression of the day from late afternoon to the middle of the night. This data set is useful to identify gene expression networks regulating growth in the leaf sheath, an organ that is a major contributor to the sorghum seedling stem and defines seedling height.

Sorghum bicolor INDETERMINATE1 is a conserved primary regulator of flowering.

Introduction: A fundamental developmental switch for plants is transition from vegetative to floral growth, which integrates external and internal signals. INDETERMINATE1 (Id1) family proteins are zinc finger transcription factors that activate flowering in grasses regardless of photoperiod. Mutations in maize Id1 and rice Id1 (RID1) cause very late flowering. RID1 promotes expression of the flowering activator genes Early Heading Date1 (Ehd1) and Heading date 1 (Hd1), a rice homolog of CONSTANS (CO). Methods and results: Mapping of two recessive late flowering mutants from a pedigreed sorghum EMS mutant library identified two distinct mutations in the Sorghum bicolor Id1 (SbId1) homolog, mutant alleles named sbid1-1 and sbid1-2. The weaker sbid1-1 allele caused a 35 day delay in reaching boot stage in the field, but its effect was limited to 6 days under greenhouse conditions. The strong sbid1-2 allele delayed boot stage by more than 60 days in the field and under greenhouse conditions. When sbid1-1 and sbid1-2 were combined, the delayed flowering phenotype remained and resembled that of sbid1-2, confirming late flowering was due to loss of SbId1 function. Evaluation of major flowering time regulatory gene expression in sbid1-2 showed that SbId1 is needed for expression of floral activators, like SbCO and SbCN8, and repressors, like SbPRR37 and SbGhd7. Discussion: These results demonstrate a conserved role for SbId1 in promotion of flowering in sorghum, where it appears to be critical to allow expression of most major flowering regulatory genes.

Unique and overlapping functions for the transcriptional regulators KANADI1 and ULTRAPETALA1 in Arabidopsis gynoecium and stamen gene regulation.

Plants generate their reproductive organs, the stamens and the carpels, de novo within the flowers that form when the plant reaches maturity. The carpels comprise the female reproductive organ, the gynoecium, a complex organ that develops along several axes of polarity and is crucial for plant reproduction, fruit formation, and seed dispersal. The epigenetic trithorax group (trxG) protein ULTRAPETALA1 (ULT1) and the GARP domain transcription factor KANADI1 (KAN1) act cooperatively to regulate Arabidopsis thaliana gynoecium patterning along the apical-basal polarity axis; however, the molecular pathways through which this patterning activity is achieved remain to be explored. In this study, we used transcriptomics to identify genome-wide ULT1 and KAN1 target genes during reproductive development. We discovered 278 genes in developing flowers that are regulated by ULT1, KAN1, or both factors together. Genes involved in developmental and reproductive processes are overrepresented among ULT1 and/or KAN1 target genes, along with genes involved in biotic or abiotic stress responses. Consistent with their function in regulating gynoecium patterning, a number of the downstream target genes are expressed in the developing gynoecium, including a unique subset restricted to the stigmatic tissue. Further, we also uncovered a number of KAN1- and ULT1-induced genes that are transcribed predominantly or exclusively in developing stamens. These findings reveal a potential cooperative role for ULT1 and KAN1 in male as well as female reproductive development that can be investigated with future genetic and molecular experiments.

Impact of the sickle mutant and temperature on the structure of transcripts and RNAs from Arabidopsis thaliana.

OBJECTIVES: The objective of this data set was to identify how interaction between temperature and the sickle-3 (sic-3) mutant alters the global messenger RNA (mRNA) content of Arabidopsis thaliana seedlings. The motivation was discovery of atypical mRNA splice variants in sic-3 that differed with seedling growth temperature. The expected outcome was identification of mRNA splice variants altered by sic-3, temperature, or the combination of temperature and genotype. DATA DESCRIPTION: The data set is RNAseq profiling of Arabidopsis (Col-0 ecotype) wild type and sic-3 seedlings under 16 °C or 28 °C. A comprehensive view of global mRNA sequences and their content was captured by deep sequencing of RNA pools made from sets of seedlings sampled every 4 h over 20 h. This data set contains sequences representing the spectrum of mRNA splice variants from individual genes, as well as from mRNA-related sequences like spliced introns. This data set enables detection of significant changes in gene-level expression and relative levels of mRNA splice variants caused by the different growth temperatures, the sic-3 mutation or both factors. This data set is useful to study production of mRNA splice variants and other mRNA-related RNAs in a range of plant species because Arabidopsis is a model plant.

72-h diurnal RNA-seq analysis of fully expanded third leaves from maize, sorghum, and foxtail millet at 3-h resolution.

OBJECTIVES: The purpose of this data set is to capture the complete diurnal (i.e., daily) transcriptome of fully expanded third leaves from the C4 panacoid grasses sorghum (Sorghum bicolor), maize (Zea mays), and foxtail millet (Setaria italica) with RNA-seq transcriptome profiling. These data are the cornerstone of a larger project that examined the conservation and divergence of gene expression networks within these crop plants. This data set focuses on temporal changes in gene expression to identify the network architecture responsible for daily regulation of plant growth and metabolic activities. The power of this data set is fine temporal resolution combined with continuous sampling over multiple days. DATA DESCRIPTION: The data set is 72 individual RNA-seq samples representing 24 time course samples each for sorghum, maize, and foxtail millet plants cultivated in a growth chamber under equal intervals of light and darkness. The 24 samples are separated by 3-h intervals so that the data set is a fine scale 72-h analysis of gene expression in the leaves of each plant type. FASTQ files from Illumina sequencing are available at the National Center for Biotechnology Information Sequence Read Archive.

A sorghum gigantea mutant attenuates florigen gene expression and delays flowering time.

GIGANTEA (GI) is a conserved plant-specific gene that modulates a range of environmental responses in multiple plant species, including playing a key role in photoperiodic regulation of flowering time. The C4 grass Sorghum bicolor is an important grain and subsistence crop, animal forage, and cellulosic biofuel feedstock that is tolerant of abiotic stresses and marginal soils. To understand sorghum flowering time regulatory networks, we characterized the sbgi-ems1 nonsense mutant allele of the sorghum GIGANTEA (SbGI) gene from a sequenced M4 EMS-mutagenized BTx623 population. sbgi-ems1 plants flowered later than wild type siblings under both long-day or short-day photoperiods. Delayed flowering in sbgi-ems1 plants accompanied an increase in node number, indicating an extended vegetative growth phase prior to flowering. sbgi-ems1 plants had reduced expression of floral activator genes SbCO and SbEHD1 and downstream FT-like florigen genes SbFT, SbCN8, and SbCN12. Therefore, SbGI plays a role in regulating SbCO and SbEHD1 expression that serves to accelerate flowering. SbGI protein physically interacts with the sorghum FLAVIN-BINDING, KELCH REPEAT, F-BOX1-like (SbFFL) protein, a conserved flowering-associated blue light photoreceptor, and the SbGI-SbFFL interaction is stimulated by blue light. This work demonstrates that SbGI is an activator of sorghum flowering time upstream of florigen genes under short- and long-day photoperiods, likely in association with the activity of the blue light photoreceptor SbFFL. SIGNIFICANCE STATEMENT: This study elucidates molecular details of flowering time networks for the adaptable C4 cereal crop Sorghum bicolor, including demonstration of a role for blue light sensing in sorghum GIGANTEA activity. This work validates the utility of a large publicly available sequenced EMS-mutagenized sorghum population to determine gene function.

Temporal Regulation of the Metabolome and Proteome in Photosynthetic and Photorespiratory Pathways Contributes to Maize Heterosis.

Heterosis or hybrid vigor is widespread in plants and animals. Although the molecular basis for heterosis has been extensively studied, metabolic and proteomic contributions to heterosis remain elusive. Here we report an integrative analysis of time-series metabolome and proteome data in maize (Zea mays) hybrids and their inbred parents. Many maize metabolites and proteins are diurnally regulated, and many of these show nonadditive abundance in the hybrids, including key enzymes and metabolites involved in carbon assimilation. Compared with robust trait heterosis, metabolic heterosis is relatively mild. Interestingly, most amino acids display negative mid-parent heterosis (MPH), i.e., having lower values than the average of the parents, while sugars, alcohols, and nucleoside metabolites show positive MPH. From the network perspective, metabolites in the photosynthetic pathway show positive MPH, whereas metabolites in the photorespiratory pathway show negative MPH, which corresponds to nonadditive protein abundance and enzyme activities of key enzymes in the respective pathways in the hybrids. Moreover, diurnally expressed proteins that are upregulated in the hybrids are enriched in photosynthesis-related gene-ontology terms. Hybrids may more effectively remove toxic metabolites generated during photorespiration, and thus maintain higher photosynthetic efficiency. These metabolic and proteomic resources provide unique insight into heterosis and its utilization for high yielding maize and other crop plants.

Interspecific analysis of diurnal gene regulation in panicoid grasses identifies known and novel regulatory motifs.

BACKGROUND: The circadian clock drives endogenous 24-h rhythms that allow organisms to adapt and prepare for predictable and repeated changes in their environment throughout the day-night (diurnal) cycle. Many components of the circadian clock in Arabidopsis thaliana have been functionally characterized, but comparatively little is known about circadian clocks in grass species including major crops like maize and sorghum. RESULTS: Comparative research based on protein homology and diurnal gene expression patterns suggests the function of some predicted clock components in grasses is conserved with their Arabidopsis counterparts, while others have diverged in function. Our analysis of diurnal gene expression in three panicoid grasses sorghum, maize, and foxtail millet revealed conserved and divergent evolution of expression for core circadian clock genes and for the overall transcriptome. We find that several classes of core circadian clock genes in these grasses differ in copy number compared to Arabidopsis, but mostly exhibit conservation of both protein sequence and diurnal expression pattern with the notable exception of maize paralogous genes. We predict conserved cis-regulatory motifs shared between maize, sorghum, and foxtail millet through identification of diurnal co-expression clusters for a subset of 27,196 orthologous syntenic genes. In this analysis, a Cochran-Mantel-Haenszel based method to control for background variation identified significant enrichment for both expected and novel 6-8 nucleotide motifs in the promoter regions of genes with shared diurnal regulation predicted to function in common physiological activities. CONCLUSIONS: This study illustrates the divergence and conservation of circadian clocks and diurnal regulatory networks across syntenic orthologous genes in panacoid grass species. Further, conserved local regulatory sequences contribute to the architecture of these diurnal regulatory networks that produce conserved patterns of diurnal gene expression.

MicroRNAs and new biotechnological tools for its modulation and improving stress tolerance in plants.

MicroRNAs (miRNAs) modulate the abundance and spatial-temporal accumulation of target mRNAs and indirectly regulate several plant processes. Transcriptional regulation of the genes encoding miRNAs (MIR genes) can be activated by numerous transcription factors, which themselves are regulated by other miRNAs. Fine-tuning of MIR genes or miRNAs is a powerful biotechnological strategy to improve tolerance to abiotic or biotic stresses in crops of economic importance. Current approaches for miRNA fine-tuning are based on the down- or up-regulation of MIR gene transcription and the use of genetic engineering tools to manipulate the final concentration of these miRNAs in the cytoplasm. Transgenesis, cisgenesis, intragenesis, artificial MIR genes, endogenous and artificial target mimicry, MIR genes editing using Meganucleases, ZNF proteins, TALENs and CRISPR/Cas9 or CRISPR/Cpf1, CRISPR/dCas9 or dCpf1, CRISPR13a, topical delivery of miRNAs and epigenetic memory have been successfully explored to MIR gene or miRNA modulation and improve agronomic traits in several model or crop plants. However, advantages and drawbacks of each of these new biotechnological tools (NBTs) are still not well understood. In this review, we provide a brief overview of the biogenesis and role of miRNAs in response to abiotic or biotic stresses, we present critically the main NBTs used for the manipulation of MIR genes and miRNAs, we show current efforts and findings with the MIR genes and miRNAs modulation in plants, and we summarize the advantages and drawbacks of these NBTs and provide some alternatives to overcome. Finally, challenges and future perspectives to miRNA modulating in important crops are also discussed.

Insights into soybean transcriptome reconfiguration under hypoxic stress: Functional, regulatory, structural, and compositional characterization.

Soybean (Glycine max) is one of the major crops worldwide and flooding stress affects the production and expansion of cultivated areas. Oxygen is essential for mitochondrial aerobic respiration to supply the energy demand of plant cells. Because oxygen diffusion in water is 10,000 times lower than in air, partial (hypoxic) or total (anoxic) oxygen deficiency is important component of flooding. Even when oxygen is externally available, oxygen deficiency frequently occurs in bulky, dense or metabolically active tissues such as phloem, meristems, seeds, and fruits. In this study, we analyzed conserved and divergent root transcriptional responses between flood-tolerant Embrapa 45 and flood-sensitive BR 4 soybean cultivars under hypoxic stress conditions with RNA-seq. To understand how soybean genes evolve and respond to hypoxia, stable and differentially expressed genes were characterized structurally and compositionally comparing its mechanistic relationship. Between cultivars, Embrapa 45 showed less up- and more down-regulated genes, and stronger induction of phosphoglucomutase (Glyma05g34790), unknown protein related to N-terminal protein myristoylation (Glyma06g03430), protein suppressor of phyA-105 (Glyma06g37080), and fibrillin (Glyma10g32620). RNA-seq and qRT-PCR analysis of non-symbiotic hemoglobin (Glyma11g12980) indicated divergence in gene structure between cultivars. Transcriptional changes for genes in amino acids and derivative metabolic process suggest involvement of amino acids metabolism in tRNA modifications, translation accuracy/efficiency, and endoplasmic reticulum stress in both cultivars under hypoxia. Gene groups differed in promoter TATA box, ABREs (ABA-responsive elements), and CRT/DREs (C-repeat/dehydration-responsive elements) frequency. Gene groups also differed in structure, composition, and codon usage, indicating biological significances. Additional data suggests that cis-acting ABRE elements can mediate gene expression independent of ABA in soybean roots under hypoxia.

SPF45-related splicing factor for phytochrome signaling promotes photomorphogenesis by regulating pre-mRNA splicing in Arabidopsis.

Light signals regulate plant growth and development by controlling a plethora of gene expression changes. Posttranscriptional regulation, especially pre-mRNA processing, is a key modulator of gene expression; however, the molecular mechanisms linking pre-mRNA processing and light signaling are not well understood. Here we report a protein related to the human splicing factor 45 (SPF45) named splicing factor for phytochrome signaling (SFPS), which directly interacts with the photoreceptor phytochrome B (phyB). In response to light, SFPS-RFP (red fluorescent protein) colocalizes with phyB-GFP in photobodies. sfps loss-of-function plants are hyposensitive to red, far-red, and blue light, and flower precociously. SFPS colocalizes with U2 small nuclear ribonucleoprotein-associated factors including U2AF65B, U2A', and U2AF35A in nuclear speckles, suggesting SFPS might be involved in the 3' splice site determination. SFPS regulates pre-mRNA splicing of a large number of genes, of which many are involved in regulating light signaling, photosynthesis, and the circadian clock under both dark and light conditions. In vivo RNA immunoprecipitation (RIP) assays revealed that SFPS associates with EARLY FLOWERING 3 (ELF3) mRNA, a critical link between light signaling and the circadian clock. Moreover, PHYTOCHROME INTERACTING FACTORS (PIFs) transcription factor genes act downstream of SFPS, as the quadruple pif mutant pifq suppresses defects of sfps mutants. Taken together, these data strongly suggest SFPS modulates light-regulated developmental processes by controlling pre-mRNA splicing of light signaling and circadian clock genes.

Functional Characterization of a Putative Glycine max ELF4 in Transgenic Arabidopsis and Its Role during Flowering Control.

Flowering is an important trait in major crops like soybean due to its direct relation to grain production. The circadian clock mediates the perception of seasonal changes in day length and temperature to modulate flowering time. The circadian clock gene EARLY FLOWERING 4 (ELF4) was identified in Arabidopsis thaliana and is believed to play a key role in the integration of photoperiod, circadian regulation, and flowering. The molecular circuitry that comprises the circadian clock and flowering control in soybeans is just beginning to be understood. To date, insufficient information regarding the soybean negative flowering regulators exist, and the biological function of the soybean ELF4 (GmELF4) remains unknown. Here, we investigate the ELF4 family members in soybean and functionally characterize a GmELF4 homologous gene. The constitutive overexpression of GmELF4 delayed flowering in Arabidopsis, showing the ELF4 functional conservation among plants as part of the flowering control machinery. We also show that GmELF4 alters the expression of Arabidopsis key flowering time genes (AtCO and AtFT), and this down-regulation is the likely cause of flowering delay phenotypes. Furthermore, we identified the GmELF4 network genes to infer the participation of GmELF4 in soybeans. The data generated in this study provide original insights for comprehending the role of the soybean circadian clock ELF4 gene as a negative flowering controller.

Accurate measurement of transgene copy number in crop plants using droplet digital PCR.

Genetic transformation is a powerful means for the improvement of crop plants, but requires labor- and resource-intensive methods. An efficient method for identifying single-copy transgene insertion events from a population of independent transgenic lines is desirable. Currently, transgene copy number is estimated by either Southern blot hybridization analyses or quantitative polymerase chain reaction (qPCR) experiments. Southern hybridization is a convincing and reliable method, but it also is expensive, time-consuming and often requires a large amount of genomic DNA and radioactively labeled probes. Alternatively, qPCR requires less DNA and is potentially simpler to perform, but its results can lack the accuracy and precision needed to confidently distinguish between one- and two-copy events in transgenic plants with large genomes. To address this need, we developed a droplet digital PCR-based method for transgene copy number measurement in an array of crops: rice, citrus, potato, maize, tomato and wheat. The method utilizes specific primers to amplify target transgenes, and endogenous reference genes in a single duplexed reaction containing thousands of droplets. Endpoint amplicon production in the droplets is detected and quantified using sequence-specific fluorescently labeled probes. The results demonstrate that this approach can generate confident copy number measurements in independent transgenic lines in these crop species. This method and the compendium of probes and primers will be a useful resource for the plant research community, enabling the simple and accurate determination of transgene copy number in these six important crop species.

The Arabidopsis sickle Mutant Exhibits Altered Circadian Clock Responses to Cool Temperatures and Temperature-Dependent Alternative Splicing.

The circadian clock allows plants to anticipate and respond to daily changes in ambient temperature. Mechanisms establishing the timing of circadian rhythms in Arabidopsis thaliana through temperature entrainment remain unclear. Also incompletely understood is the temperature compensation mechanism that maintains consistent period length within a range of ambient temperatures. A genetic screen for Arabidopsis mutants affecting temperature regulation of the PSEUDO-RESPONSE REGULATOR7 promoter yielded a novel allele of the SICKLE (SIC) gene. This mutant, sic-3, and the existing sic-1 mutant both exhibit low-amplitude or arrhythmic expression of core circadian clock genes under cool ambient temperature cycles, but not under light-dark entrainment. sic mutants also lengthen free running period in a manner consistent with impaired temperature compensation. sic mutant alleles accumulate LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1) splice variants, among other alternatively spliced transcripts, which is exacerbated by cool temperatures. The cca1-1 lhy-20 double mutant is epistatic to sic-3, indicating the LHY and CCA1 splice variants are needed for sic-3 circadian clock phenotypes. It is not expected that SIC is directly involved in the circadian clock mechanism; instead, SIC likely contributes to pre-mRNA metabolism, and the splice variants that accumulate in sic mutants likely affect the circadian clock response to cool ambient temperature.

Temporal Shift of Circadian-Mediated Gene Expression and Carbon Fixation Contributes to Biomass Heterosis in Maize Hybrids.

Heterosis has been widely used in agriculture, but the molecular mechanism for this remains largely elusive. In Arabidopsis hybrids and allopolyploids, increased photosynthetic and metabolic activities are linked to altered expression of circadian clock regulators, including CIRCADIAN CLOCK ASSOCIATED1 (CCA1). It is unknown whether a similar mechanism mediates heterosis in maize hybrids. Here we report that higher levels of carbon fixation and starch accumulation in the maize hybrids are associated with altered temporal gene expression. Two maize CCA1 homologs, ZmCCA1a and ZmCCA1b, are diurnally up-regulated in the hybrids. Expressing ZmCCA1 complements the cca1 mutant phenotype in Arabidopsis, and overexpressing ZmCCA1b disrupts circadian rhythms and biomass heterosis. Furthermore, overexpressing ZmCCA1b in maize reduced chlorophyll content and plant height. Reduced height stems from reduced node elongation but not total node number in both greenhouse and field conditions. Phenotypes are less severe in the field than in the greenhouse, suggesting that enhanced light and/or metabolic activities in the field can compensate for altered circadian regulation in growth vigor. Chromatin immunoprecipitation-sequencing (ChIP-seq) analysis reveals a temporal shift of ZmCCA1-binding targets to the early morning in the hybrids, suggesting that activation of morning-phased genes in the hybrids promotes photosynthesis and growth vigor. This temporal shift of ZmCCA1-binding targets correlated with nonadditive and additive gene expression in early and late stages of seedling development. These results could guide breeding better hybrid crops to meet the growing demand in food and bioenergy.

Transcriptome-Wide Identification of Reference Genes for Expression Analysis of Soybean Responses to Drought Stress along the Day.

The soybean transcriptome displays strong variation along the day in optimal growth conditions and also in response to adverse circumstances, like drought stress. However, no study conducted to date has presented suitable reference genes, with stable expression along the day, for relative gene expression quantification in combined studies on drought stress and diurnal oscillations. Recently, water deficit responses have been associated with circadian clock oscillations at the transcription level, revealing the existence of hitherto unknown processes and increasing the demand for studies on plant responses to drought stress and its oscillation during the day. We performed data mining from a transcriptome-wide background using microarrays and RNA-seq databases to select an unpublished set of candidate reference genes, specifically chosen for the normalization of gene expression in studies on soybean under both drought stress and diurnal oscillations. Experimental validation and stability analysis in soybean plants submitted to drought stress and sampled during a 24 h timecourse showed that four of these newer reference genes (FYVE, NUDIX, Golgin-84 and CYST) indeed exhibited greater expression stability than the conventionally used housekeeping genes (ELF1-ß and ß-actin) under these conditions. We also demonstrated the effect of using reference candidate genes with different stability values to normalize the relative expression data from a drought-inducible soybean gene (DREB5) evaluated in different periods of the day.

Daytime soybean transcriptome fluctuations during water deficit stress.

BACKGROUND: Since drought can seriously affect plant growth and development and little is known about how the oscillations of gene expression during the drought stress-acclimation response in soybean is affected, we applied Illumina technology to sequence 36 cDNA libraries synthesized from control and drought-stressed soybean plants to verify the dynamic changes in gene expression during a 24-h time course. Cycling variables were measured from the expression data to determine the putative circadian rhythm regulation of gene expression. RESULTS: We identified 4866 genes differentially expressed in soybean plants in response to water deficit. Of these genes, 3715 were differentially expressed during the light period, from which approximately 9.55% were observed in both light and darkness. We found 887 genes that were either up- or down-regulated in different periods of the day. Of 54,175 predicted soybean genes, 35.52% exhibited expression oscillations in a 24 h period. This number increased to 39.23% when plants were submitted to water deficit. Major differences in gene expression were observed in the control plants from late day (ZT16) until predawn (ZT20) periods, indicating that gene expression oscillates during the course of 24 h in normal development. Under water deficit, dissimilarity increased in all time-periods, indicating that the applied stress influenced gene expression. Such differences in plants under stress were primarily observed in ZT0 (early morning) to ZT8 (late day) and also from ZT4 to ZT12. Stress-related pathways were triggered in response to water deficit primarily during midday, when more genes were up-regulated compared to early morning. Additionally, genes known to be involved in secondary metabolism and hormone signaling were also expressed in the dark period. CONCLUSIONS: Gene expression networks can be dynamically shaped to acclimate plant metabolism under environmental stressful conditions. We have identified putative cycling genes that are expressed in soybean leaves under normal developmental conditions and genes whose expression oscillates under conditions of water deficit. These results suggest that time of day, as well as light and temperature oscillations that occur considerably affect the regulation of water deficit stress response in soybean plants.

Gibberellin driven growth in elf3 mutants requires PIF4 and PIF5.

The regulatory connections between the circadian clock and hormone signaling are essential to understand, as these two regulatory processes work together to time growth processes relative to predictable environmental events. Gibberellins (GAs) are phytohormones that control many growth processes throughout all stages of the plant life cycle, including germination and flowering. An increasing number of examples demonstrate that the circadian clock directly influences GA biosynthesis and signaling. EARLY FLOWERING 3 (ELF3) participates in a tripartite transcriptional complex known as the Evening Complex (EC). In this capacity, ELF3 is fundamental to core circadian clock activity, as well as time-of-day specific regulation of genes directly responsible for growth control, namely the PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and PIF5 genes. Here we show that the GA biosynthesis inhibitor paclobutrazol substantially reduces the long hypocotyl and petiole phenotypes of Arabidopsis elf3 mutants. In addition, loss of ELF3 activity causes upregulation of the key GA biosynthesis genes GA20ox1 and GA20ox2. Moreover, GA20ox1 and GA20ox2 expression depends strongly on the redundant activities of PIF4 and PIF5. These findings indicate that the defining growth phenotypes of elf3 mutants arise from altered GA biosynthesis due to misregulation of PIF4 and PIF5. These observations agree with recent work linking increased GA production with the elongated growth phenotypes of the barley elf3 mutant. Thus, the role of the EC in regulation of GA biosynthesis and signaling in eudicots is shared with monocots and, therefore, is a highly conserved mechanism for growth control.

Circadian Clock Genes Universally Control Key Agricultural Traits.

Circadian clocks are endogenous timers that enable plants to synchronize biological processes with daily and seasonal environmental conditions in order to allocate resources during the most beneficial times of day and year. The circadian clock regulates a number of central plant activities, including growth, development, and reproduction, primarily through controlling a substantial proportion of transcriptional activity and protein function. This review examines the roles that alleles of circadian clock genes have played in domestication and improvement of crop plants. The focus here is on three groups of circadian clock genes essential to clock function in Arabidopsis thaliana: PSEUDO-RESPONSE REGULATORs, GIGANTEA, and the evening complex genes early flowering 3, early flowering 4, and lux arrhythmo. homologous genes from each group underlie quantitative trait loci that have beneficial influences on key agricultural traits, especially flowering time but also yield, biomass, and biennial growth habit. Emerging insights into circadian clock regulation of other fundamental plant processes, including responses to abiotic and biotic stresses, are discussed to highlight promising avenues for further crop improvement.