Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence.

Small RNAs (sRNAs) are essential for normal plant development and range in size classes of 21-24 nucleotides. The 22nt small interfering RNAs (siRNAs) and miRNAs are processed by Dicer-like 2 (DCL2) and DCL1 respectively and can initiate secondary siRNA production from the target transcript. 22nt siRNAs are under-represented due to competition between DCL2 and DCL4, while only a small number of 22nt miRNAs exist. Here we produce abundant 22nt siRNAs and other siRNA size classes using long hairpin RNA (hpRNA) transgenes. By introducing asymmetric bulges into the antisense strand of hpRNA, we shifted the dominant siRNA size class from 21nt of the traditional hpRNA to 22, 23 and 24nt of the asymmetric hpRNAs. The asymmetric hpRNAs effectively silenced a ß-glucuronidase (GUS) reporter transgene and the endogenous ethylene insensitive-2 (EIN2) and chalcone synthase (CHS) genes. Furthermore, plants containing the asymmetric hpRNA transgenes showed increased amounts of 21nt siRNAs downstream of the hpRNA target site compared to plants with the traditional hpRNA transgenes. This indicates that these asymmetric hpRNAs are more effective at inducing secondary siRNA production to amplify silencing signals. The 22nt asymmetric hpRNA constructs enhanced virus resistance in plants compared to the traditional hpRNA constructs.

Mutation in Polycomb repressive complex 2 gene OsFIE2 promotes asexual embryo formation in rice.

Prevention of autonomous division of the egg apparatus and central cell in a female gametophyte before fertilization ensures successful reproduction in flowering plants. Here we show that rice ovules of Polycomb repressive complex 2 (PRC2) Osfie1 and Osfie2 double mutants exhibit asexual embryo and autonomous endosperm formation at a high frequency, while ovules of single Osfie2 mutants display asexual pre-embryo-like structures at a lower frequency without fertilization. Earlier onset, higher penetrance and better development of asexual embryos in the double mutants compared with those in Osfie2 suggest that the autonomous endosperm facilitated asexual embryo development. Transcriptomic analysis showed that male genome-expressed OsBBM1 and OsWOX8/9 were activated in the asexual embryos. Similarly, the maternal alleles of the paternally expressed imprinted genes were activated in the autonomous endosperm, suggesting that the egg apparatus and central cell convergently adopt PRC2 to maintain the non-dividing state before fertilization, possibly through silencing of the maternal alleles of male genome-expressed genes.

Deregulation of ζ-carotene desaturase in Arabidopsis and tomato exposes a unique carotenoid-derived redundant regulation of floral meristem identity and function.

A level of redundancy and interplay among the transcriptional regulators of floral development safeguards a plant's reproductive success and ensures crop production. In the present study, an additional layer of complexity in the regulation of floral meristem (FM) identity and flower development is elucidated linking carotenoid biosynthesis and metabolism to the regulation of determinate flowering. The accumulation and subsequent cleavage of a diverse array of ζ-carotenes in the chloroplast biogenesis 5 (clb5) mutant of Arabidopsis results in the reprogramming of meristematic gene regulatory networks establishing FM identity mirroring that of the FM identity master regulator, APETALA1 (AP1). The immediate transition to floral development in clb5 requires long photoperiods in a GIGANTEA-independent manner, whereas AP1 is essential for the floral organ development of clb5. The elucidation of this link between carotenoid metabolism and floral development translates to tomato exposing a regulation of FM identity redundant to and initiated by AP1 and proposed to be dependent on the E class floral initiation and organ identity regulator, SEPALLATA3 (SEP3).

Contactless skin perfusion monitoring with video cameras: tracking pharmacological vasoconstriction and vasodilation using photoplethysmographic changes.

Objectives.Clinical assessment of skin perfusion informs prognosis in critically ill patients. Video camera monitoring could provide an objective, continuous method to monitor skin perfusion. In this prospective, interventional study of healthy volunteers, we tested whether video camera-derived photoplethysmography imaging and colour measurements could detect drug-induced skin perfusion changes.Approach.We monitored the lower limbs of 30 volunteers using video cameras while administering phenylephrine (a vasoconstrictor) and glyceryl trinitrate (a vasodilator). We report relative pixel intensity changes from baseline, as absolute values are sensitive to environmental factors. The primary outcome was the pre- to peak- infusion green channel amplitude change in the pulsatile PPGi waveform component. Secondary outcomes were pre-to-peak changes in the photoplethysmographic imaging waveform baseline, skin colour hue and skin colour saturation.Main results.The 30 participants had a median age of 29 years (IQR 25-34), sixteen (53%) were male. A 34.7% (p= 0.0001) mean decrease in the amplitude of the pulsatile photoplethysmographic imaging waveform occurred following phenylephrine infusion. A 30.7% (p= 0.000004) mean increase occurred following glyceryl trinitrate infusion. The photoplethysmographic imaging baseline decreased with phenylephrine by 2.1% (p= 0.000 02) and increased with glyceryl trinitrate by 0.5% (p= 0.026). Skin colour hue changed in opposite direction with phenylephrine (-0.0013,p= 0.0002) and glyceryl trinitrate (+0.0006,p= 0.019). Skin colour saturation decreased with phenylephrine by 0.0022 (p= 0.0002), with no significant change observed with glyceryl trinitrate (+0.0005,p= 0.21).Significance.Drug-induced vasoconstriction and vasodilation are associated with detectable changes in photoplethysmographic imaging waveform parameters and skin hue. Our findings suggest video cameras have great potential for continuous, contactless skin perfusion monitoring.

The key role of terminators on the expression and post-transcriptional gene silencing of transgenes.

Transgenes have become essential to modern biology, being an important tool in functional genomic studies and also in the development of biotechnological products. One of the major challenges in the generation of transgenic lines concerns the expression of transgenes, which, compared to endogenes, are particularly susceptible to silencing mediated by small RNAs (sRNAs). Several reasons have been put forward to explain why transgenes often trigger the production of sRNAs, such as the high level of expression induced by commonly used strong constitutive promoters, the lack of introns, and features resembling viral and other exogenous sequences. However, the relative contributions of the different genomic elements with respect to protecting genes from the silencing machinery and their molecular mechanisms remain unclear. Here, we present the results of a mutagenesis screen conceived to identify features involved in the protection of endogenes against becoming a template for the production of sRNAs. Interestingly, all of the recovered mutants had alterations in genes with proposed function in transcription termination, suggesting a central role of terminators in this process. Indeed, using a GFP reporter system, we show that, among different genetic elements tested, the terminator sequence had the greatest effect on transgene-derived sRNA accumulation and that a well-defined poly(A) site might be especially important. Finally, we describe an unexpected mechanism, where transgenes containing certain intron/terminator combinations lead to an increase in the production of sRNAs, which appears to interfere with splicing.

Resetting FLOWERING LOCUS C Expression After Vernalization Is Just Activation in the Early Embryo by a Different Name.

The reproductive success of many plants depends on their capacity to respond appropriately to their environment. One environmental cue that triggers flowering is the extended cold of winter, which promotes the transition from vegetative to reproductive growth in a response known as vernalization. In annual plants of the Brassicaceae, the floral repressor, FLOWERING LOCUS C (FLC), is downregulated by exposure to low temperatures. Repression is initiated during winter cold and then maintained as the temperature rises, allowing plants to complete their life cycle during spring and summer. The two stages of FLC repression, initiation and maintenance, are distinguished by different chromatin states at the FLC locus. Initiation involves the removal of active chromatin marks and the deposition of the repressive mark H3K27me3 over a few nucleosomes in the initiation zone, also known as the nucleation region. H3K27me3 then spreads to cover the entire locus, in a replication dependent manner, to maintain FLC repression. FLC is released from repression in the next generation, allowing progeny of a vernalized plant to respond to winter. Activation of FLC in this generation has been termed resetting to denote the restoration of the pre-vernalized state in the progeny of a vernalized plant. It has been assumed that resetting must differ from the activation of FLC expression in progeny of plants that have not experienced winter cold. Considering that there is now strong evidence indicating that chromatin undergoes major modifications during both male and female gametogenesis, it is time to challenge this assumption.

APETALA 2-like genes AP2L2 and Q specify lemma identity and axillary floral meristem development in wheat.

The spikelet is the basic unit of the grass inflorescence. In tetraploid (Triticum turgidum) and hexaploid wheat (Triticum aestivum), the spikelet is a short indeterminate branch with two proximal sterile bracts (glumes) followed by a variable number of florets, each including a bract (lemma) with an axillary flower. Varying levels of miR172 and/or its target gene Q (AP2L5) result in gradual transitions of glumes to lemmas, and vice versa. Here, we show that AP2L5 and its related paralog AP2L2 play critical and redundant roles in the specification of axillary floral meristems and lemma identity. AP2L2, also targeted by miR172, displayed similar expression profiles to AP2L5 during spikelet development. Loss-of-function mutants in both homeologs of AP2L2 (henceforth ap2l2) developed normal spikelets, but ap2l2 ap2l5 double mutants generated spikelets with multiple empty bracts before transitioning to florets. The coordinated nature of these changes suggest an early role of these genes in floret development. Moreover, the flowers of ap2l2 ap2l5 mutants showed organ defects in paleas and lodicules, including the homeotic conversion of lodicules into carpels. Mutations in the miR172 target site of AP2L2 were associated with reduced plant height, more compact spikes, promotion of lemma-like characters in glumes and smaller lodicules. Taken together, our results show that the balance in the expression of miR172 and AP2-like genes is crucial for the correct development of spikelets and florets, and that this balance has been altered during the process of wheat and barley (Hordeum vulgare) domestication. The manipulation of this regulatory module provides an opportunity to modify spikelet architecture and improve grain yield.

Automated method for detecting and reading seven-segment digits from images of blood glucose metres and blood pressure monitors.

There is an increasing need for fast and accurate transfer of readings from blood glucose metres and blood pressure monitors to a smartphone mHealth application, without a dependency on Bluetooth technology. Most of the medical devices recommended for home monitoring use a seven-segment display to show the recorded measurement to the patient. We aimed to achieve accurate detection and reading of the seven-segment digits displayed on these medical devices using an image taken in a realistic scenario by a smartphone camera. A synthetic dataset of seven-segment digits was developed in order to train and test a digit classifier. A dataset containing realistic images of blood glucose metres and blood pressure monitors using a variety of smartphone cameras was also created. The digit classifier was evaluated on a dataset of seven-segment digits manually extracted from the medical device images. These datasets along with the code for its development have been made public. The developed algorithm first preprocessed the input image using retinex with two bilateral filters and adaptive histogram equalisation. Subsequently, the digit segments were automatically located within the image by two techniques operating in parallel: Maximally Stable Extremal Regions (MSER) and connected components of a binarised image. A filtering and clustering algorithm was then designed to combine digit segments to form seven-segment digits. The resulting digits were classified using a Histogram of Orientated Gradients (HOG) feature set and a neural network trained on the synthetic digits. The model achieved 93% accuracy on digits found on the medical devices. The digit location algorithm achieved a F1 score of 0.87 and 0.80 on images of blood glucose metres and blood pressure monitors respectively. Very few assumptions were made of the locations of the digits on the devices so that the proposed algorithm can be easily implemented on new devices.

Zebularine treatment is associated with deletion of FT-B1 leading to an increase in spikelet number in bread wheat.

The number of rachis nodes (spikelets) on a wheat spike is a component of grain yield that correlates with flowering time. The genetic basis regulating flowering in cereals is well understood, but there are reports that flowering time can be modified at a high frequency by selective breeding, suggesting that it may be regulated by both epigenetic and genetic mechanisms. We investigated the role of DNA methylation in regulating spikelet number and flowering time by treating a semi-spring wheat with the demethylating agent, Zebularine. Three lines with a heritable increase in spikelet number were identified. The molecular basis for increased spikelet number was not determined in 2 lines, but the phenotype showed non-Mendelian inheritance, suggesting that it could have an epigenetic basis. In the remaining line, the increased spikelet phenotype behaved as a Mendelian recessive trait and late flowering was associated with a deletion encompassing the floral promoter, FT-B1. Deletion of FT-B1 delayed the transition to reproductive growth, extended the duration of spike development, and increased spikelet number under different temperature regimes and photoperiod. Transiently disrupting DNA methylation can generate novel flowering behaviour in wheat, but these changes may not be sufficiently stable for use in breeding programs.

Developmental responses of bread wheat to changes in ambient temperature following deletion of a locus that includes FLOWERING LOCUS T1.

FLOWERING LOCUS T (FT) is a central integrator of environmental signals that regulates the timing of vegetative to reproductive transition in flowering plants. In model plants, these environmental signals have been shown to include photoperiod, vernalization, and ambient temperature pathways, and in crop species, the integration of the ambient temperature pathway remains less well understood. In hexaploid wheat, at least 5 FT-like genes have been identified, each with a copy on the A, B, and D genomes. Here, we report the characterization of FT-B1 through analysis of FT-B1 null and overexpression genotypes under different ambient temperature conditions. This analysis has identified that the FT-B1 alleles perform differently under diverse environmental conditions; most notably, the FT-B1 null produces an increase in spikelet and tiller number when grown at lower temperature conditions. Additionally, absence of FT-B1 facilitates more rapid germination under both light and dark conditions. These results provide an opportunity to understand the FT-dependent pathways that underpin key responses of wheat development to changes in ambient temperature. This is particularly important for wheat, for which development and grain productivity are sensitive to changes in temperature.

What makes for sound science?

The inclusive threshold policy for publication in BMC journals including BMC Plant Biology means that editorial decisions are largely based on the soundness of the research presented rather than the novelty or potential impact of the work. Here we discuss what is required to ensure that research meets the requirement of scientific soundness. BMC Plant Biology and the other BCM-series journals ( https://www.biomedcentral.com/p/the-bmc-series-journals ) differ in policy from many other journals as they aim to provide a home for all publishable research. The inclusive threshold policy for publication means that editorial decisions are largely based on the soundness of the research presented rather than the novelty or potential impact of the work. The emphasis on scientific soundness ( http://blogs.biomedcentral.com/bmcseriesblog/2016/12/05/vital-importance-inclusive/ ) rather than novelty or impact is important because it means that manuscripts that may be judged to be of low impact due to the nature of the study as well as those reporting negative results or that largely replicate earlier studies, all of which can be difficult to publish elsewhere, are available to the research community. Here we discuss the importance of the soundness of research and provide some basic guidelines to assist authors to determine whether their research is appropriate for submission to BMC Plant Biology.Prior to a research article being sent out for review, the handling editor will first determine whether the research presented is scientifically valid. To be valid the research must address a question of biological significance using suitable methods and analyses, and must follow community-agreed standards relevant to the research field.

New alleles of the wheat domestication gene Q reveal multiple roles in growth and reproductive development.

The advantages of free threshing in wheat led to the selection of the domesticated Q allele, which is now present in almost all modern wheat varieties. Q and the pre-domestication allele, q, encode an AP2 transcription factor, with the domesticated allele conferring a free-threshing character and a subcompact (i.e. partially compact) inflorescence (spike). We demonstrate that mutations in the miR172 binding site of the Q gene are sufficient to increase transcript levels via a reduction in miRNA-dependent degradation, consistent with the conclusion that a single nucleotide polymorphism in the miRNA binding site of Q relative to q was essential in defining the modern Q allele. We describe novel gain- and loss-of-function alleles of Q and use these to define new roles for this gene in spike development. Q is required for the suppression of 'sham ramification', and increased Q expression can lead to the formation of ectopic florets and spikelets (specialized inflorescence branches that bear florets and grains), resulting in a deviation from the canonical spike and spikelet structures of domesticated wheat.

Mutants in the imprinted PICKLE RELATED 2 gene suppress seed abortion of fertilization independent seed class mutants and paternal excess interploidy crosses in Arabidopsis.

Endosperm cellularization is essential for embryo development and viable seed formation. Loss of function of the FERTILIZATION INDEPENDENT SEED (FIS) class Polycomb genes, which mediate trimethylation of histone H3 lysine27 (H3K27me3), as well as imbalanced contributions of parental genomes interrupt this process. The causes of the failure of cellularization are poorly understood. In this study we identified PICKLE RELATED 2 (PKR2) mutations which suppress seed abortion in fis1/mea by restoring endosperm cellularization. PKR2, a paternally expressed imprinted gene (PEG), encodes a CHD3 chromatin remodeler. PKR2 is specifically expressed in syncytial endosperm and its maternal copy is repressed by FIS1. Seed abortion in a paternal genome excess interploidy cross was also partly suppressed by pkr2. Simultaneous mutations in PKR2 and another PEG, ADMETOS (ADM), additively rescue the seed abortion in fis1 and in the interploidy cross, suggesting that PKR2 and ADM modulate endosperm cellularization independently and reproductive isolation between plants of different ploidy is established by imprinted genes. Genes upregulated in fis1 and downregulated in the presence of pkr2 are enriched in glycosyl-hydrolyzing activity, while genes downregulated in fis1 and upregulated in the presence of pkr2 are enriched with microtubule motor activity, consistent with the cellularization patterns in fis1 and the suppressor line. The antagonistic functions of FIS1 and PKR2 in modulating endosperm development are similar to those of PICKLE (PKL) and CURLY LEAF (CLF), which antagonistically regulate root meristem activity. Our results provide further insights into the function of imprinted genes in endosperm development and reproductive isolation.

Kicking against the PRCs - A Domesticated Transposase Antagonises Silencing Mediated by Polycomb Group Proteins and Is an Accessory Component of Polycomb Repressive Complex 2.

The Polycomb group (PcG) and trithorax group (trxG) genes play crucial roles in development by regulating expression of homeotic and other genes controlling cell fate. Both groups catalyse modifications of chromatin, particularly histone methylation, leading to epigenetic changes that affect gene activity. The trxG antagonizes the function of PcG genes by activating PcG target genes, and consequently trxG mutants suppress PcG mutant phenotypes. We previously identified the ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN1 (ALP1) gene as a genetic suppressor of mutants in the Arabidopsis PcG gene LIKE HETEROCHROMATIN PROTEIN1 (LHP1). Here, we show that ALP1 interacts genetically with several other PcG and trxG components and that it antagonizes PcG silencing. Transcriptional profiling reveals that when PcG activity is compromised numerous target genes are hyper-activated in seedlings and that in most cases this requires ALP1. Furthermore, when PcG activity is present ALP1 is needed for full activation of several floral homeotic genes that are repressed by the PcG. Strikingly, ALP1 does not encode a known chromatin protein but rather a protein related to PIF/Harbinger class transposases. Phylogenetic analysis indicates that ALP1 is broadly conserved in land plants and likely lost transposase activity and acquired a novel function during angiosperm evolution. Consistent with this, immunoprecipitation and mass spectrometry (IP-MS) show that ALP1 associates, in vivo, with core components of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a widely conserved PcG protein complex which functions as a H3K27me3 histone methyltransferase. Furthermore, in reciprocal pulldowns using the histone methyltransferase CURLY LEAF (CLF), we identify not only ALP1 and the core PRC2 components but also plant-specific accessory components including EMBRYONIC FLOWER 1 (EMF1), a transcriptional repressor previously associated with PRC1-like complexes. Taken together our data suggest that ALP1 inhibits PcG silencing by blocking the interaction of the core PRC2 with accessory components that promote its HMTase activity or its role in inhibiting transcription. ALP1 is the first example of a domesticated transposase acquiring a novel function as a PcG component. The antagonistic interaction of a modified transposase with the PcG machinery is novel and may have arisen as a means for the cognate transposon to evade host surveillance or for the host to exploit features of the transposition machinery beneficial for epigenetic regulation of gene activity.

Time-dependent stabilization of the +1 nucleosome is an early step in the transition to stable cold-induced repression of FLC.

In vernalized Arabidopsis, the extent of FLC repression and promotion of flowering are correlated with the length of winter (low temperature exposure), but how plants measure the duration of winter is unknown. Repression of FLC occurs in two phases: establishment and maintenance. This study investigates the early events in the transition between establishment and maintenance of repression. Initial repression was rapid but transient; within 24 h of being placed at low temperatures FLC transcription was reduced by 40% and repression was complete after 5 days in the cold. The extent to which repression was maintained depended on the length of the cold treatment. Occupancy of the +1 nucleosome in FLC chromatin increased in a time-dependent manner over a 4-week low temperature treatment concomitant with decreased histone acetylation and increased trimethylation of histone H3 lysine 27 (H3K27me3). Mutant analyses showed that increased nucleosome occupancy occurred independent of histone deacetylation and increased H3K27me3, suggesting that it is an early step in the switch between transient and stable repression. Both altered histone composition and deacetylation contributed to increased nucleosome occupancy. The time-dependency of the steps required for the switch between transient and stable repression suggests that the duration of winter is measured by the chromatin state at FLC. A chromatin-based switch is consistent with finding that each FLC allele in a cell undergoes this transition independently.

Direct links between the vernalization response and other key traits of cereal crops.

Transcription of the vernalization1 gene (VRN1) is induced by prolonged cold (vernalization) to trigger flowering of cereal crops, such as wheat and barley. VRN1 encodes a MADS box transcription factor that promotes flowering by regulating the expression of other genes. Here we use transcriptome sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) to identify direct targets of VRN1. Over 500 genomic regions were identified as potential VRN1-binding targets by ChIP-seq. VRN1 binds the promoter of flowering locus T-like 1, a promoter of flowering in vernalized plants. VRN1 also targets vernalization2 and ODDSOC2, repressors of flowering that are downregulated in vernalized plants. RNA-seq identified additional VRN1 targets that might play roles in triggering flowering. Other targets of VRN1 include genes that play central roles in low-temperature-induced freezing tolerance, spike architecture and hormone metabolism. This provides evidence for direct regulatory links between the vernalization response pathway and other important traits in cereal crops.

How is FLC repression initiated by cold?

Vernalization is the promotion of flowering in response to prolonged exposure to low temperatures. In Arabidopsis, FLOWERING LOCUS C (FLC), a suppressor of flowering, is repressed by low temperatures but the mechanism leading to the initial decrease in FLC transcription remains a mystery. No mutants that block the repression of FLC at low temperatures have been identified to date. If the failure to identify such a mutant is assumed to imply that no such mutant exists, then it follows that the first response to the drop in temperature is physical, not genetic. In this Opinion article we propose that the drop in temperature first causes a simple change in the topology of the chromatin polymer, which in turn initiates the repression of FLC transcription.

Ppd-1 is a key regulator of inflorescence architecture and paired spikelet development in wheat.

The domestication of cereal crops such as wheat, maize, rice and barley has included the modification of inflorescence architecture to improve grain yield and ease harvesting(1). Yield increases have often been achieved through modifying the number and arrangement of spikelets, which are specialized reproductive branches that form part of the inflorescence. Multiple genes that control spikelet development have been identified in maize, rice and barley(2-5). However, little is known about the genetic underpinnings of this process in wheat. Here, we describe a modified spikelet arrangement in wheat, termed paired spikelets. Combining comprehensive QTL and mutant analyses, we show that Photoperiod-1 (Ppd-1), a pseudo-response regulator gene that controls photoperiod-dependent floral induction, has a major inhibitory effect on paired spikelet formation by regulating the expression of FLOWERING LOCUS T (FT)(6,7). These findings show that modulated expression of the two important flowering genes, Ppd-1 and FT, can be used to form a wheat inflorescence with a more elaborate arrangement and increased number of grain producing spikelets.

Imprinting in rice: the role of DNA and histone methylation in modulating parent-of-origin specific expression and determining transcript start sites.

Over 200 imprinted genes in rice endosperm are known, but the mechanisms modulating their parental allele-specific expression are poorly understood. Here we use three imprinted genes, OsYUCCA11, yellow2-like and ubiquitin hydrolase, to show that differential DNA methylation and tri-methylation of histone H3 lysine 27 (H3K27me3 ) in the promoter and/or gene body influences allele-specific expression or the site of transcript initiation. Paternal expression of OsYUCCA11 required DNA methylation in the gene body whereas the gene body of the silenced maternal allele was hypomethylated and marked with H3K27me3 . These differential markings mirror those proposed to modulate paternal expression of two Arabidopsis genes, PHERES1 and a YUCCA homolog, indicating conservation of imprinting mechanisms. At yellow2-like, DNA hypomethylation in the upstream flanking region resulted in maternal transcripts that were longer than paternal transcripts; the maternal transcript initiation site was marked by DNA methylation in the paternal allele, and transcription initiated ~700 bp downstream. The paternal allele of an ubiquitin hydrolase gene exhibited gene body DNA methylation and produced full-length transcripts, while the maternal allele was hypomethylated in the 5' gene body and transcripts initiated from a downstream promoter. Inhibition of DNA methylation by 5-azacytidine or zebularine activated the long transcripts from yellow2-like and enhanced expression of the short transcripts from the ubiquitin hydrolase in seedlings, indicating that DNA methylation prevents transcript initiation from cryptic promoters. These observations suggest a paradigm whereby maternal genome hypomethylation is associated with the production of distinct transcripts, potentially diversifying the gene products from the two alleles.