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Plants typically orient their organs with respect to the Earth's gravity field by a dynamic process called gravitropism. To discover conserved genetic elements affecting seedling root gravitropism, we measured the process in a set of Zea mays (maize) recombinant inbred lines with machine vision and compared the results with those obtained in a similar study of Arabidopsis thaliana . Each of the several quantitative trait loci that we mapped in both species spanned many hundreds of genes, too many to test individually for causality. We reasoned that orthologous genes may be responsible for natural variation in monocot and dicot root gravitropism. If so, pairs of orthologous genes affecting gravitropism may be present within the maize and Arabidopsis QTL intervals. A reciprocal comparison of sequences within the QTL intervals identified seven pairs of such one-to-one orthologs. Analysis of knockout mutants demonstrated a role in gravitropism for four of the seven: CCT2 functions in phosphatidylcholine biosynthesis, ATG5 functions in membrane remodeling during autophagy, UGP2 produces the substrate for cellulose and callose polymer extension, and FAMA is a transcription factor. Automated phenotyping enabled this discovery of four naturally varying components of a conserved process (gravitropism) by making it feasible to conduct the same large-scale experiment in two species.

Background: Gene expression inheritance patterns in Arabidopsis hybrid plants were investigated for correlation with the presence of transposable elements (TEs) and small RNA profile., Results: The presence of TEs in a gene and the expression of small RNA matching a gene were both found to be associated with non-additive mRNA inheritance patterns in hybrids. Expression levels below mid-parent values in the hybrids were associated with low mRNA expression in parents, with the presence of small RNA from both strands, and with the presence of TEs. High-parent dominance of mRNA levels was found to be associated with high parental mRNA expression levels, the absence of TEs, and for some genes, with small RNA fragments that are predominantly from the sense strand. These small RNAs exhibit a broader size distribution than siRNA and reduced nucleotide end bias, which are consistent with an origin from degraded mRNA. Thus, increased as well as decreased gene expression in hybrids relative to the parental mean is associated with gene expression levels, TE presence and small RNA fragments with differing characteristics., Conclusions: The data presented here is consistent with a role for differential mRNA decay kinetics as one mechanism contributing to high-parent dominance in gene expression. Our evidence is also consistent with trans repression by siRNA and TEs as the cause of low-parent dominance.

Hybrids often display increased size and growth, and thus are widely cultivated in agriculture and horticulture. Recent discoveries demonstrating the important regulatory roles of small RNAs have greatly improved our understanding of many basic biological questions, and could illuminate the molecular basis for the enhanced growth and size of hybrid plants. We profiled small RNAs by deep sequencing to characterize the inheritance patterns of small RNA levels in reciprocal hybrids of two Arabidopsis thaliana accessions, Columbia and Landsberg erecta. We find 24-nt siRNAs predominate among those small RNAs that are differentially expressed between the parents. Following hybridization, the transposable element (TE)-derived siRNAs are often inherited in an additive manner, whereas siRNAs associated with protein-coding genes are often down-regulated in hybrids to the levels observed for the parent with lower relative siRNA levels. Among the protein-coding genes that exhibit this pattern, genes that function in pathogen defense, abiotic stress tolerance, and secondary metabolism are significantly enriched. Small RNA clusters from protein-coding genes where a TE is present within one kilobase show a different predominant inheritance pattern (additive) from those that do not (low-parent dominance). Thus, down-regulation in the form of low-parent dominance is likely the default pattern of inheritance for genic siRNA, and a different inheritance mechanism for TE siRNA is suggested.

Balancing selection is one mechanism that may explain why diversity is maintained in wild populations. However, relatively few examples of genes showing evidence of balancing selection have been identified, particularly in plants. In this issue, Reininga et al. (2009) present three Arabidopsis loci that show strong evidence of balancing selection. The loci, discovered using a genome-scanning approach, encode proteins with diverse predicted functions: starch synthesis and control of gene expression. These three genes were identified by scanning only a small fraction of the Arabidopsis genome, suggesting that balancing selection may be more prevalent than previously known.

Cytochrome P450 monooxygenases (P450s) play important roles in the synthesis of diverse secondary compounds in Arabidopsis (Arabidopsis thaliana). Comparison of four data sets analyzing seedlings harvested over a 2-d period of constant conditions after growth with varying photoperiods and thermocycles recorded a total of 98 P450 loci as circadian regulated for at least one of the four conditions. Here, we further describe the circadian-regulated pathways using, as reporters, individual P450 loci that are likely to be rate limiting in secondary metabolic pathways. Reverse transcription-polymerase chain reaction gel blot analyses have confirmed circadian regulation of P450s in phenylpropanoid, carotenoid, oxylipin, glucosinolate, and brassinosteroid biosyntheses and have shown that both P450 and non-P450 genes in the many branches of the phenylpropanoid pathway have similar circadian patterns of expression. In silico analyses of the subsets of coregulated promoters have identified overrepresented promoter elements in various biosynthetic pathway genes, including MYB and MYB4 elements that are significantly more abundant in promoters for the core and lignin sections of phenylpropanoid metabolism. Interactions with these elements important for circadian regulation do not involve the MYB transcription factor PAP1, as previously proposed, since the expression patterns of circadian-regulated P450s are the same in pap1-D mutant seedlings as in wild-type seedlings. Further analysis of circadian-regulated promoters in other biochemical pathways provides us with the opportunity to identify novel promoter motifs that might be important in P450 circadian regulation.

The bHLH transcription factor, Phytochrome Interacting Factor 3 (PIF3), interacts specifically with the photoactivated, Pfr, form of Arabidopsis phytochrome B (phyB). This interaction induces PIF3 phosphorylation and degradation in vivo and modulates phyB-mediated seedling deetiolation in response to red light. To identify missense mutations in the phyB N-terminal domain that disrupt this interaction, we developed a yeast reverse-hybrid screen. Fifteen individual mutations identified in this screen, or in previous genetic screens for Arabidopsis mutants showing reduced sensitivity to red light, were shown to also disrupt light-induced binding of phyB to PIF3 in in vitro co-immunoprecipitation assays. These phyB missense mutants fall into two general classes: Class I (eleven mutants) containing those defective in light signal perception, due to aberrant chromophore attachment or photoconversion, and Class II (four mutants) containing those normal in signal perception, but defective in the capacity to transduce this signal to PIF3. By generating a homology model for the three-dimensional structure of the Arabidopsis phyB chromophore-binding region, based on the crystal structure of Deinococcus radiodurans phytochrome, we predict that three of the four Class II mutated phyB residues are solvent exposed in a cleft between the presumptive PAS and GAF domains. This deduction suggests that these residues could be directly required for the physical interaction of phyB with PIF3. Because these three residues are also necessary for phyB-imposed inhibition of hypocotyl elongation in response to red light, they are functionally necessary for signal transfer from photoactivated phyB, not only to PIF3 and other related bHLH transcription factors tested here, but also to other downstream signaling components involved in regulating seedling deetiolation., Competing Interests: The authors have declared that no competing interests exist.

Plants are continuously exposed to a myriad of abiotic and biotic stresses. However, the molecular mechanisms by which these stress signals are perceived and transduced are poorly understood. To begin to identify primary stress signal transduction components, we have focused on genes that respond rapidly (within 5 min) to stress signals. Because it has been hypothesized that detection of physical stress is a mechanism common to mounting a response against a broad range of environmental stresses, we have utilized mechanical wounding as the stress stimulus and performed whole genome microarray analysis of Arabidopsis thaliana leaf tissue. This led to the identification of a number of rapid wound responsive (RWR) genes. Comparison of RWR genes with published abiotic and biotic stress microarray datasets demonstrates a large overlap across a wide range of environmental stresses. Interestingly, RWR genes also exhibit a striking level and pattern of circadian regulation, with induced and repressed genes displaying antiphasic rhythms. Using bioinformatic analysis, we identified a novel motif overrepresented in the promoters of RWR genes, herein designated as the Rapid Stress Response Element (RSRE). We demonstrate in transgenic plants that multimerized RSREs are sufficient to confer a rapid response to both biotic and abiotic stresses in vivo, thereby establishing the functional involvement of this motif in primary transcriptional stress responses. Collectively, our data provide evidence for a novel cis-element that is distributed across the promoters of an array of diverse stress-responsive genes, poised to respond immediately and coordinately to stress signals. This structure suggests that plants may have a transcriptional network resembling the general stress signaling pathway in yeast and that the RSRE element may provide the key to this coordinate regulation., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Background: Genetic control of gene transcription is a key component in genome evolution. To understand the transcriptional basis of natural variation, we have studied genome-wide variations in transcription and characterized the genetic variations in regulatory elements among Arabidopsis accessions., Results: Among five accessions (Col-0, C24, Ler, WS-2, and NO-0) 7,508 probe sets with no detectable genomic sequence variations were identified on the basis of the comparative genomic hybridization to the Arabidopsis GeneChip microarray, and used for accession-specific transcriptome analysis. Two-way ANOVA analysis has identified 60 genes whose mRNA levels differed in different accession backgrounds in an organ-dependent manner. Most of these genes were involved in stress responses and late stages of plant development, such as seed development. Correlation analysis of expression patterns of these 7,508 genes between pairs of accessions identified a group of 65 highly plastic genes with distinct expression patterns in each accession., Conclusion: Genes that show substantial genetic variation in mRNA level are those with functions in signal transduction, transcription and stress response, suggesting the existence of variations in the regulatory mechanisms for these genes among different accessions. This is in contrast to those genes with significant polymorphisms in the coding regions identified by genomic hybridization, which include genes encoding transposon-related proteins, kinases and disease-resistance proteins. While relatively fewer sequence variations were detected on average in the coding regions of these genes, a number of differences were identified from the upstream regions, several of which alter potential cis-regulatory elements. Our results suggest that nucleotide polymorphisms in regulatory elements of genes encoding controlling factors could be primary targets of natural selection and a driving force behind the evolution of Arabidopsis accessions.

Different Arabidopsis phytochrome (phy) family members (phyA through phyE) display differential photosensory and/or physiological functions in regulating growth and developmental responses to light signals. To identify the genes regulated by phyB in response to continuous monochromatic red light (Rc) during the induction of seedling de-etiolation, we have performed time-course, microarray-based expression profiling of wild type (WT) and phyB null mutants. Comparison of the observed expression patterns with those induced by continuous monochromatic far-red light (FRc; perceived exclusively by phyA) in WT and phyA null-mutant seedlings suggests early convergence of the FRc and Rc photosensory pathways to control a largely common transcriptional network. phyB mutant seedlings retain a surprisingly high level of responsiveness to Rc for the majority of Rc-regulated genes on the microarray, indicating that one or more other phys have a major role in regulating their expression. Combined with the robust visible morphogenic phenotype of the phyB mutant in Rc, these data suggest that different members of the phy family act in organ-specific fashion in regulating seedling de-etiolation. Specifically, phyB appears to be the dominant, if not exclusive, photoreceptor in regulating a minority population of genes involved in suppression of hypocotyl cell elongation in response to Rc signals. By contrast, this sensory function is apparently shared by one or more other phys in regulating the majority Rc-responsive gene set involved in other important facets of the de-etiolation process in the apical region, such as cotyledon cell expansion.

Several hundred Arabidopsis genes, transcriptionally regulated by phytochrome A (phyA), were previously identified using an oligonucleotide microarray. We have now identified, in silico, conserved sequence motifs in the promoters of these genes by comparing the promoter sequences to those of all the genes present on the microarray from which they were sampled. This was done using a Perl script (called Sift) that identifies over-represented motifs using an enumerative approach. The utility of Sift was verified by analysis of circadian-regulated promoters known to contain a biologically significant motif. Several elements were then identified in phyA-responsive promoters by their over-representation. Five previously undescribed motifs were detected in the promoters of phyA-induced genes. Four novel motifs were found in phyA-repressed promoters, plus a motif that strongly resembles the DE1 element. The G-box, CACGTG, was a prominent hit in both induced and repressed phyA-responsive promoters. Intriguingly, two distinct flanking consensus sequences were observed adjacent to the G-box core sequence: one predominating in phyA-induced promoters, the other in phyA-repressed promoters. Such different conserved flanking nucleotides around the core motif in these two sets of promoters may indicate that different members of the same family of DNA-binding proteins mediate phyA induction and repression. An increased abundance of G-box sequences was observed in the most rapidly phyA-responsive genes and in the promoters of phyA-regulated transcription factors, indicating that G-box-binding transcription factors are upstream components in a transcriptional cascade that mediates phyA-regulated development.

The Arabidopsis mutants far1 and fhy3 display a phenotype of reduced inhibition of hypocotyl elongation, which is specific to far-red light and therefore specific to the phytochrome A (phyA)-signaling pathway. We report that the proteins encoded by the FAR1 and FHY3 genes are both related to the transposases of type II MuDR family transposons. We demonstrate that the FAR1 protein is capable of activating transcription in Arabidopsis, indicating that it may define a type of transcriptional regulator. Using microarray expression analysis, we show that of 293 mRNAs twofold induced in wild-type Col-0 plants by continuous far-red light, 85% show reduced responsiveness in the fhy3 mutant. Notable alterations were observed in the responses of genes encoding certain transcription factors, proteins involved in cell wall extension, and proteins related to redox balance control. We also found genes, including some involved in transcriptional control, which showed altered transcriptional behavior in the dark-grown mutant plants. Taken together, our data suggest that FAR1 and FHY3 may function 'permissively' outside the signal transduction pathway of light-regulated development, yet be required for the expression of transcriptional regulatory components. An alternative possibility is that their role includes both light-signal transduction and transcriptional regulation of other genes not responsive to light. We propose that FAR1 and FHY3 control the expression of their target genes by a mechanism that has evolved directly from the way that an ancestral, MuDRA-like transposase bound to the TIRs of mobile elements.

Plants typically orient their organs with respect to the Earth’s gravity field by a dynamic process called gravitropism. To discover conserved genetic elements affecting seedling root gravitropism, we measured the process in a set of Zea mays (maize) recombinant inbred lines with machine vision and compared the results with those obtained in a similar study of Arabidopsis thaliana. Each of the several quantitative trait loci that we mapped in both species spanned many hundreds of genes, too many to test individually for causality. We reasoned that orthologous genes may be responsible for natural variation in monocot and dicot root gravitropism. If so, pairs of orthologous genes affecting gravitropism may be present within the maize and Arabidopsis QTL intervals. A reciprocal comparison of sequences within the QTL intervals identified seven pairs of such one-to-one orthologs. Analysis of knockout mutants demonstrated a role in gravitropism for four of the seven: CCT2 functions in phosphatidylcholine biosynthesis, ATG5 functions in membrane remodeling during autophagy, UGP2 produces the substrate for cellulose and callose polymer extension, and FAMA is a transcription factor. Automated phenotyping enabled this discovery of four naturally varying components of a conserved process (gravitropism) by making it feasible to conduct the same large-scale experiment in two species. [ABSTRACT FROM AUTHOR]

Background: Gene expression inheritance patterns in Arabidopsis hybrid plants were investigated for correlation with the presence of transposable elements (TEs) and small RNA profile. Results: The presence of TEs in a gene and the expression of small RNA matching a gene were both found to be associated with non-additive mRNA inheritance patterns in hybrids. Expression levels below mid-parent values in the hybrids were associated with low mRNA expression in parents, with the presence of small RNA from both strands, and with the presence of TEs. High-parent dominance of mRNA levels was found to be associated with high parental mRNA expression levels, the absence of TEs, and for some genes, with small RNA fragments that are predominantly from the sense strand. These small RNAs exhibit a broader size distribution than siRNA and reduced nucleotide end bias, which are consistent with an origin from degraded mRNA. Thus, increased as well as decreased gene expression in hybrids relative to the parental mean is associated with gene expression levels, TE presence and small RNA fragments with differing characteristics. Conclusions: The data presented here is consistent with a role for differential mRNA decay kinetics as one mechanism contributing to high-parent dominance in gene expression. Our evidence is also consistent with trans repression by siRNA and TEs as the cause of low-parent dominance. [ABSTRACT FROM AUTHOR]

We have developed an approach to facilitate the use of model organism microarrays in related, nonmodel organisms. We demonstrate the method by using Arabidopsis oligonucleotide microarrays to analyze gene expression in Brassica. Probes with low hybridization signals to Brassica genomic DNA were excluded from the transcriptional analysis of an Arabidopsis microarray at the software level, forming a virtual Brassica microarray. We then performed an experiment on transcriptional responses during seed germination in Brassica using 1/886 homologous probesets of the virtual array where Brassica mRNA hybridization was detected. We subjected seed to hydration or priming (a step to improve germination vigor) and subsequent heating and drying treatments (steps to prolong shelf life of dried seed). Exploration of the microarray results indicated two likely expression patterns shared by many genes. One class of transcripts was strongly, globally, and irreversibly downregulated by priming, while other transcripts were induced, hut often reversibly. Legacy seed-storage protein messages were in the first class, and many protein synthesis components and some resource mobilization enzymes were in the second. We were able to validate our results by confirming transcriptional responses using reverse transcription polymerase chain reaction (RT—PCR). [ABSTRACT FROM AUTHOR]

The Arabidopsis mutants far1 and fhy3 display a phenotype of reduced inhibition of hypocotyl elongation, which is specific to far-red light and therefore specific to the phytochrome A (phyA)-signaling pathway. We report that the proteins encoded by the FAR1 and FHY3 genes are both related to the transposases of type II MuDR family transposons. We demonstrate that the FAR1 protein is capable of activating transcription in Arabidopsis, indicating that it may define a type of transcriptional regulator. Using microarray expression analysis, we show that of 293 mRNAs twofold induced in wild-type Col-0 plants by continuous far-red light, 85% show reduced responsiveness in the fhy3 mutant. Notable alterations were observed in the responses of genes encoding certain transcription factors, proteins involved in cell wall extension, and proteins related to redox balance control. We also found genes, including some involved in transcriptional control, which showed altered transcriptional behavior in the dark-grown mutant plants. Taken together, our data suggest that FAR1 and FHY3 may function 'permissively' outside the signal transduction pathway of light-regulated development, yet be required for the expression of transcriptional regulatory components. An alternative possibility is that their role includes both light-signal transduction and transcriptional regulation of other genes not responsive to light. We propose that FAR1 and FHY3 control the expression of their target genes by a mechanism that has evolved directly from the way that an ancestral, MuDRA-like transposase bound to the TIRs of mobile elements. [ABSTRACT FROM AUTHOR]