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Water scarcity is a serious agricultural problem causing significant losses to crop yield and product quality. The development of technologies to mitigate the damage caused by drought stress is essential for ensuring a sustainable food supply for the increasing global population. We herein report that the exogenous application of ethanol, an inexpensive and environmentally friendly chemical, significantly enhances drought tolerance in Arabidopsis thaliana, rice and wheat. The transcriptomic analyses of ethanol-treated plants revealed the upregulation of genes related to sucrose and starch metabolism, phenylpropanoids and glucosinolate biosynthesis, while metabolomic analysis showed an increased accumulation of sugars, glucosinolates and drought-tolerance-related amino acids. The phenotyping analysis indicated that drought-induced water loss was delayed in the ethanol-treated plants. Furthermore, ethanol treatment induced stomatal closure, resulting in decreased transpiration rate and increased leaf water contents under drought stress conditions. The ethanol treatment did not enhance drought tolerance in the mutant of ABI1, a negative regulator of abscisic acid (ABA) signaling in Arabidopsis, indicating that ABA signaling contributes to ethanol-mediated drought tolerance. The nuclear magnetic resonance analysis using 13C-labeled ethanol indicated that gluconeogenesis is involved in the accumulation of sugars. The ethanol treatment did not enhance the drought tolerance in the aldehyde dehydrogenase (aldh) triple mutant (aldh2b4/aldh2b7/aldh2c4). These results show that ABA signaling and acetic acid biosynthesis are involved in ethanol-mediated drought tolerance and that chemical priming through ethanol application regulates sugar accumulation and gluconeogenesis, leading to enhanced drought tolerance and sustained plant growth. These findings highlight a new survival strategy for increasing crop production under water-limited conditions.

Conferring drought resistant traits to crops is one of the major aims of current breeding programs in response to global climate changes. We previously showed that exogenous application of acetic acid to roots of various plants could induce increased survivability under subsequent drought stress conditions, but details of the metabolism of exogenously applied acetic acid, and the nature of signals induced by its application, have not been unveiled. In this study, we show that rice rapidly induces jasmonate signaling upon application of acetic acid, resulting in physiological changes similar to those seen under drought. The major metabolite of the exogenously applied acetic acid in xylem sap was determined as glutamine—a common and abundant component of xylem sap—indicating that acetic acid is not the direct agent inducing the observed physiological responses in shoots. Expression of drought-responsive genes in shoot under subsequent drought conditions was attenuated by acetic acid treatment. These data suggest that acetic acid activates root-to-shoot jasmonate signals that partially overlap with those induced by drought, thereby conferring an acclimated state on shoots prior to subsequent drought.

Histone modification is an important epigenetic mechanism in eukaryotes. Histone acetyltransferase and deacetylase regulate histone acetylation levels antagonistically, leading to dynamic control of chromatin structure. One of the histone deacetylases, HDA6, is involved in gene silencing in the heterochromatin regions, chromocenter formation, and metabolic adaptation under drought stress. Although HDA6 plays an important role in chromatin control and response to drought stress, its intracellular localization has not been observed in detail. In this paper, we generated transformants expressing HDA6-GFP in the model plant, Arabidopsis thaliana, and the crops, rice, and cassava. We observed the localization of the fusion protein and showed that HDA6-GFP was expressed in the whole root and localized at the nucleus in Arabidopsis, rice, and cassava. Remarkably, HDA6-GFP clearly formed speckles that were actively colocalized with chromocenters in Arabidopsis root meristem. In contrast, such speckles were unlikely to be formed in rice or cassava. Because AtHDA6 directly binds to the acetate synthesis genes, which function in drought tolerance, we performed live imaging analyses to examine the cellular dynamics of pH in roots and the subnuclear dynamics of AtHDA6 responding to acetic acid treatment. The number of HDA6 speckles increased during drought stress, suggesting a role in contributing to drought stress tolerance.

Rice seedlings (Oryza sativa) that have died from drought cannot be rescued by watering afterward, but pre-treatment with exogenous acetic acid enabled the plants to produce shoots again after being watered (hereinafter referred to as "drought resilience"). To elucidate the metabolism of acetic acid, we treated rice plants with 13C-labeled acetic acid and traced 13C-labeled metabolites using LC-MS and 13C-NMR techniques. The LC-MS and 13C-NMR spectral data of the root extracts indicated that the acetic acid treatment was absorbed into the plants and then was metabolized to gamma-aminobutyric acid (GABA) by glutamic acid decarboxylase (GAD). GABA accumulation in the roots took place in advance of that in the shoots, and the survival rate against drought stress increased in proportion to the amount of GABA accumulated in the shoots. Therefore, GABA accumulation in shoots may be a key step in drought resilience induced by the acetic acid treatment.

Leaf color is an important indicator when evaluating plant growth and responses to biotic/abiotic stress. Acquisition of images by digital cameras allows analysis and long-term storage of the acquired images. However, under field conditions, where light intensity can fluctuate and other factors (shade, reflection, and background, etc.) vary, stable and reproducible measurement and quantification of leaf color are hard to achieve. Digital scanners provide fixed conditions for obtaining image data, allowing stable and reliable comparison among samples, but require detached plant materials to capture images, and the destructive processes involved often induce deformation of plant materials (curled leaves and faded colors, etc.). In this study, by using a lightweight digital scanner connected to a mobile computer, we obtained digital image data from intact plant leaves grown in natural-light greenhouses without detaching the targets. We took images of soybean leaves infected by Xanthomonas campestris pv. glycines, and distinctively quantified two disease symptoms (brown lesions and yellow halos) using freely available image processing software. The image data were amenable to quantitative and statistical analyses, allowing precise and objective evaluation of disease resistance.

Nature Plants 3, 17097 (2017); published 26 June 2017; corrected 17 July 2017. The Supplementary Information files originally published with this Letter were not the intended final versions. The Supplementary Information PDF and Supplementary Tables 3, 5 and 6 have now been updated to fix a number of cosmetic problems.

Production of rice (Oryza sativa)—the staple food of over half the world’s population, especially those living in poverty—must continue to increase to meet the rising demand. The availability of a wide variety of natural rice resources has enabled highly efficient breeding approaches that have successfully improved productivity as well as biotic and abiotic stress tolerances. However, recent changes in global climate tendencies are imposing additional pressures on rice production, with the need for varieties showing unprecedented characteristics to counter adverse environments calling for innovative responses from breeders and researchers. Recent developments in epigenetic research in Arabidopsis thaliana have provided a plethora of data on epigenetic regulation in gene expression and development, paving the way to crop improvement via epigenetic manipulation. At ~400 Mb, the rice genome is the smallest among cereal crops and is relatively tractable with current molecular genetics techniques. This chapter begins by comparing characteristics of the rice genome and epigenome with those of Arabidopsis, before presenting some examples of epigenetic regulation in plants, with the emphasis on agriculturally important traits including abiotic stress responses. Most molecular studies on epigenetic modifications affecting plant phenotypes have been done in Arabidopsis, but examples of epigenetic regulation of agriculturally important traits in rice are accumulating rapidly. Current problems and difficulties in applying epigenetic manipulation to rice and ensuring stable maintenance of the modified epigenetic states to secure given agricultural traits under natural conditions will then be discussed.

Posttranscriptional gene silencing via 21-nucleotide (nt) trans-acting small interfering RNAs (tasiRNAs) is a plant-specific RNA silencing mechanism. tasiRNAs are generated by RNA-DEPENDENT RNA POLYMERASE 6, SUPPRESSOR OF GENE SILENCING 3, and DICER-LIKE 4 using TAS transcripts as templates and degrade their target mRNA in a manner similar to micro-RNA. TAS gene-derived small RNAs also trigger cytosine methylation in cis together with known components of RNA-directed DNA methylation (RdDM). RdDM is known to be required for silencing of repetitive elements and transposons via a 24-nt small interfering RNA complementary to the target DNA. Plant-specific DNA-dependent RNA polymerase V (Pol V) is thought to be required for the recruitment of other RdDM components to the target region, together with the DDR complex, which contains DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1, DEFECTIVE IN MERISTEM SILENCING 3, and RNA-DIRECTED DNA METHYLATION 1. Although genome-wide studies indicate that Pol V and the DDR complex mostly share endogenous targets, the requirement for each component of RdDM at TAS genes remains obscure. Here, we investigated the involvement of components of the DDR complex in RdDM triggered by TAS gene-derived small RNAs. Our results show that methylation of the TAS3a gene was reduced in dms3 and drd1, whereas methylation of the TAS1c gene was decreased in dms3 but not in drd1. These findings indicate that Pol V and components of the DDR complex act separately in a locus-specific manner.

Genome editing in plants becomes popular since the advent of sequence-specific nucleases (SSNs) that are simple to set up and efficient in various plant species. Although transcription activator-like effector nucleases (TALENs) are one of the most prevalent SSNs and have a potential to provide higher target specificity by their dimeric property, TALENs are sensitive to methylated cytosines that are present not only in transposons but also in active genes in plants. In mammalian cells, the methylation sensitivity of TALENs could be overcome by using a base-recognition module (N*) that has a higher affinity to methylated cytosine. In contrast to mammals, plants carry DNA methylation at all cytosine contexts (CG, CHG, and CHH, where H represents A, C, or T) with various degrees and effectiveness of N* module in genome editing in plants has not been explored. In this study, we designed sets of TALENs with or without N* modules and examined their efficiency in genome editing of methylated regions in rice. Although improvement in genome editing efficiency was observed with N*-TALENs designed to a stably methylated target, another target carrying cytosines with various levels of methylation showed resistance to both normal and N*-TALENs. The results suggest that variability of cytosine methylation in target regions is an additional factor affecting the genome editing efficiency of TALENs.

Water deficit caused by global climate changes seriously endangers the survival of organisms and crop productivity, and increases environmental deterioration1,2. Plants' resistance to drought involves global reprogramming of transcription, cellular metabolism, hormone signalling and chromatin modification3-8. However, how these regulatory responses are coordinated via the various pathways, and the underlying mechanisms, are largely unknown. Herein, we report an essential drought-responsive network in which plants trigger a dynamic metabolic flux conversion from glycolysis into acetate synthesis to stimulate the jasmonate (JA) signalling pathway to confer drought tolerance. In Arabidopsis, the ON/OFF switching of this whole network is directly dependent on histone deacetylase HDA6. In addition, exogenous acetic acid promotes de novo JA synthesis and enrichment of histone H4 acetylation, which influences the priming of the JA signalling pathway for plant drought tolerance. This novel acetate function is evolutionarily conserved as a survival strategy against environmental changes in plants. Furthermore, the external application of acetic acid successfully enhanced the drought tolerance in Arabidopsis, rapeseed, maize, rice and wheat plants. Our findings highlight a radically new survival strategy that exploits an epigenetic switch of metabolic flux conversion and hormone signalling by which plants adapt to drought.

The natural bicolor floral traits of the horticultural petunia (Petunia hybrida) cultivars Picotee and Star are caused by the spatial repression of the chalcone synthase A (CHS-A) gene, which encodes an anthocyanin biosynthetic enzyme. Here we show that Picotee and Star petunias carry the same short interfering RNA (siRNA)-producing locus, consisting of two intact CHS-A copies, PhCHS-A1 and PhCHS-A2, in a tandem head-to-tail orientation. The precursor CHS mRNAs are transcribed from the two CHS-A copies throughout the bicolored petals, but the mature CHS mRNAs are not found in the white tissues. An analysis of small RNAs revealed the accumulation of siRNAs of 21 nucleotides that originated from the exon 2 region of both CHS-A copies. This accumulation is closely correlated with the disappearance of the CHS mRNAs, indicating that the bicolor floral phenotype is caused by the spatially regulated post-transcriptional silencing of both CHS-A genes. Linkage between the tandemly arranged CHS-A allele and the bicolor floral trait indicates that the CHS-A allele is a necessary factor to confer the trait. We suppose that the spatially regulated production of siRNAs in Picotee and Star flowers is triggered by another putative regulatory locus, and that the silencing mechanism in this case may be different from other known mechanisms of post-transcriptional gene silencing in plants. A sequence analysis of wild Petunia species indicated that these tandem CHS-A genes originated from Petunia integrifolia and/or Petunia inflata, the parental species of P. hybrida, as a result of a chromosomal rearrangement rather than a gene duplication event.

Small interfering RNAs (siRNAs) are widespread in various eukaryotes and are involved in maintenance of chromatin modifications, especially those for inert states represented by covalent modifications of cytosine and/or histones. In contrast to mammalian genomes, in which cytosine methylation is restricted mostly to CG dinucleotide sequences, inert chromatin in plants carries cytosine methylation in all sequence contexts, and siRNAs play a major role in directing cytosine methylation through the process of RNA-directed DNA methylation. Recent advances in this field have revealed that siRNA-mediated maintenance of inert chromatin has diverse roles in development as well as in plant responses to the environment. Various proteinaceous factors required for siRNA-mediated chromatin modification have been identified in Arabidopsis thaliana, and much effort has been invested in understanding their function and interaction, resulting in the assignment of many of these factors to specific biochemical activities and engagement with specific steps such as transcription of intergenic RNAs, RNA processing, and cytosine methylation. However, the precise functions of a number of factors remain undesignated, and interactions of distinct pathways for siRNA-mediated chromatin modification are largely unknown. In this review, we summarize the roles of siRNA-mediated chromatin modification in various biological processes of A. thaliana, and present some speculation on the functions and interactions of silencing factors that, while not yet assigned to defined biochemical activities, have been loosely assigned to specific events in siRNA-mediated chromatin modification pathways. Special Issue entitled: Epigenetic control of cellular and developmental processes in plants.

Morpheus' molecule1 (MOM1) is a plant-specific epigenetic regulator of transcriptional gene silencing. Mutants of MOM1 release silencing of subsets of endogenous repetitive elements and transgenes without affecting their cytosine methylation status. Although MOM1 is evolutionarily related to chromodomain helicase DNA binding protein3 (CHD3), a family of chromatin remodeling proteins involved in repression of gene expression, MOM1 does not carry the functional ATPase/helicase domain essential for chromatin remodeling activity, and therefore, its mode of action is unknown. We recently performed a genome-wide survey for endogenous targets silenced by MOM1 and identified loci that are concentrated around centromeres and rich in sequences homologous to the 24-nt small interfering RNAs (siRNAs) that accumulate in wild type plants. Further and independent analyses indicated that the degree of contribution of MOM1 to maintenance of the silent states varies in different loci and that other silencing machineries, including those in the RNA-directed DNA methylation (RdDM) pathway, interact genetically with MOM1. In this short article, I review what we know about MOM1 and discuss its possible functions in silencing through examination of other silencing factors that interact genetically with MOM1.

Epigenetic changes in chromatin can be induced upon hybridization, but their contribution to phenotypic changes in F1 hybrids is not known. In this study, we examined the effect of genome-wide alteration of epigenetic chromatin status on the growth of inbreds and their F1 hybrid by large-scale fine-time-lapse growth monitoring of rice (Oryza sativa L.) under a controlled environment. This allowed us to dissect seedling growth of inbreds and their hybrid into separate growth parameters, and to analyze the effect of disturbance of inert chromatin states on these parameters. We found that superior growth parameters are generally more sensitive to global inhibition of the activities of histone deacetylases (HDACs), but this higher sensitivity is not directly correlated to heterosis in F1. Unexpectedly, however, the rate of the initial exponential growth of shoots is sensitive to HDAC inhibition only in inbreds, but not in F1, irrespective of phenotypic superiority. Our phenomics approach has detected an inbred-specific dependence of basic growth on the inert state of chromatin that is lost in F1 hybrids.

DECREASE IN DNA METHYLATION1 (DDM1) is a chromatin remodeling protein required for maintenance of cytosine and histone H3K9 methylation in heterochromatin regions in the genome of Arabidopsis thaliana (Gendrel et al., 2002). A ddm1 mutant also increases the rate of meiotic recombination in euchromatic but not heterochromatic regions of A. thaliana (Melamed-Bessudo and Levy, 2012), and changes the state of cytosine methylation of a gene flanked by a LINE element located in a region of euchromatin (Saze and Kakutani, 2007).

The low frequency of meiotic recombination in chromosomal regions other than hotspots is a general obstacle to efficient breeding. A number of active genes are present in recombination-repressed centromeric regions in higher eukaryotes, suggesting that suppression of meiotic recombination prevents shuffling of genes within a centromeric region. In this study, by using an inter-subspecific cross of Oryza sativa L., we show that modification of inactive chromatin states by either genetic or chemical inhibition of chromatin modifying proteins induced changes in both the position of meiotic recombination and, unexpectedly, the pattern of segregation distortion of parental alleles. Antisense knockdown of rice homologues of DECREASE IN DNA METHYLATION1, which is required for the maintenance of heterochromatin in Arabidopsis thaliana, induced a recombination hotspot in a centromeric region accompanied by a steep increase in the proportion of heterozygotes. Our results describe a previously undocumented phenomenon in which artificial chromatin modification could be used to change the pattern of segregation distortion in rice and open up novel possibilities for efficient crop breeding.

In vegetatively propagated plants, branches of periclinal chimera give rise to bud mutants, or “sports”, which have been used to breed new cultivars. Here, we have examined DNA methylation profiles in the cells of the L1 and L2+3 layers from single plants of vegetatively propagated carnations. The band patterns of methylation-sensitive amplified fragment polymorphism (M-AFLP) of the genomic DNAs in the L1 and L2+3 layers of the carnation cultivar, “White Sim”, vegetatively propagated over the past 50 years, were completely different. The bud mutant, “White Mind”, obtained from “White Sim” about 25 years ago, also had very different M-AFLP patterns to the parental “White Sim” line. The cultivar, “Red”, which was separated from “Satisfaction” as a bud mutant two years ago, showed similar patterns to “Satisfaction” in each corresponding layer. However, we were able to detect minor, but significantly different M-AFLP patterns between the cultivars, “Red” and “Satisfaction”. The number of bands that differed between these cultivars was larger in the L2+3 layers than in the L1 layers. The results indicate that the DNA methylation profiles differ between each cell layer derived from distinct cell lineages of vegetatively propagated plants, and that changes in these profiles occur frequently and accumulate in the cells of the L2+3 layers, rather than in the L1 layer.

RNA-directed modification of histones is essential for maintenance of heterochromatin in higher eukaryotes. In plants, cytosine methylation, especially in non-CG sequence contexts, is tightly related to inactive chromatin, but the mechanisms regulating the coexistence of cytosine methylation and repressive histone modification remain obscure. We recently revealed that MORPHEUS' MOLECULE1 (MOM1) of Arabidopsis thaliana silences endogenous loci related to transposons and homologous to the 24-nt siRNAs accumulated in wild type plants, and suggested that MOM1 transduces RNA-directed DNA methylation (RdDM) signals to repressive histone modification. In this addendum, we focus on the involvement of MOM1 in multiple transcriptional gene silencing (TGS) pathways.

A synthetic gene, mwti1b, coding for a winged bean trypsin inhibitor WTI-1B, has been introduced and expressed in rice plants, Oryza sativa. Protein extracts from transgenic rice plants expressing the trypsin inhibitor inhibited the gut proteases of larvae of the serious insect pest, the rice stem borer, Chilo suppressalis (Lepidoptera: Pyralidae) in vitro. The growth of larvae reared on transgenic rice plants expressing WTI-1B at more than 1 ng/10 μg total protein was significantly retarded compared to that on non-transgenic control plants.

The CHS genes encoding chalcone synthase for flavonoid biosynthesis in the common and Japanese morning glories comprise a multigene family. Among these Ipomoea CHS genes, the CHS-D gene is the most abundantly expressed in the pigmented young flower buds and is primarily responsible for flower pigmentation. Majority of the remaining CHS transcripts in the flower buds are produced from the CHS-E gene. We characterized the genomic DNA segments of these CHS-D and CHS-E genes. Both genes have two exons with identical intron positions and carry several copies of two mobile element-like sequences with short terminal inverted repeats, MELS3 and MELS6 of around 200-300 bp. Small tandem repeats were also found in these CHS gene regions. The CHS-D and CHS-E genes are expressed predominantly in flower limbs and tubes, respectively. These structural and functional features and their evolutionary implications are discussed.

Summary The organ-specific and temporally regulated expression of the genes for Kunitz chymotrypsin inhibitor-3 in winged bean is known to be governed by its 5′ flanking region. Two nuclear factors that bind to AT-rich sequences in this region were detected in gel retardation assays. The binding activity of one of these factors, band 1-forming factor, was inhibited by phosphorylation, as is the AT-binding protein ATBP-1 of tobacco, which functions as an activator of transcription. In addition, the binding of band 1-forming factor was reduced in the presence of a low concentration of the minor groove-binding dye, Hoechst 33258, suggesting that band 1-forming factor recognizes the minor groove of the probe, as does ATBP-1. The binding of another factor, band 3-forming factor, was also reduced by Hoechst 33258, but the binding activity of this factor was not affected by phosphorylation. During partial purification by gel-filtration and affinity chromatography, the binding activity of band 3-forming factor was cofractionated with a 75-ku (kDa) DNA-binding protein. These results indicate that band 3-forming factor is a new DNA-binding factor that recognizes the minor groove of AT-rich DNA.

New cDNA sequences for chalcone synthase (CHS), a key enzyme in the flavonoid biosynthesis, were obtained from the Japanese and common morning glories; they are more closely related to other CHS sequences than the six previously described CHS genes from the same plants. The newly isolated CHS-D gene is abundantly expressed in the pigmented flower buds, while its expression is drastically reduced in the white flower buds. Thus CHS-D appears to produce major CHS transcripts for flower pigmentation.

A winged bean Kunitz-type chymotrypsin inhibitor (WCI) is expressed in seeds and tuberous roots. In seeds, the expression of WCI is restricted to the period between the mid- and late-maturation stage. To understand the mechanisms that regulate the expression of WCI genes, we analyzed the promoter activity of the upstream region of the WCI-3b gene, which encodes a major WCI protein, in transgenic tobacco plants. By using a series of constructs with 5′ deletions in the upstream sequences, the region between -882 and -623, relative to the transcription start site, was shown to contain multiple sequences which are responsible for high level expression in mid-maturation stage seeds. However, when this region was fused to the cauliflower mosaic virus 35S core promoter in both orientations, the chimeric promoters showed only a weak transcription activity in transgenic tobacco plants. Further analyses using internal deletion constructs revealed that the region between -882 and -174 is required for the transcription activation. Disruption of the RY sequence at -517, which is conserved in many seed protein genes, resulted in a drastic reduction of the transcription activity in seeds. These results suggest that sequences necessary for high level induction of the WCI-3b gene transcription in developing seeds are dispersed in the region between -882 and -174, and that the RY sequence is one of these sequences.

Plasticity of root growth in response to environmental cues and stresses is a fundamental characteristic of land plants. However, the molecular basis underlying the regulation of root growth under stressful conditions is poorly understood. Here, we report that a rice nuclear factor, RICE SALT SENSITIVE3 (RSS3), regulates root cell elongation during adaptation to salinity. Loss of function of RSS3 only moderately inhibits cell elongation under normal conditions, but it provokes spontaneous root cell swelling, accompanied by severe root growth inhibition, under saline conditions. RSS3 is preferentially expressed in the root tip and forms a ternary complex with class-C basic helix-loop-helix (bHLH) transcription factors and JASMONATE ZIM-DOMAIN proteins, the latter of which are the key regulators of jasmonate (JA) signaling. The mutated protein arising from the rss3 allele fails to interact with bHLH factors, and the expression of a significant portion of JA-responsive genes is upregulated in rss3. These results, together with the known roles of JAs in root growth regulation, suggest that RSS3 modulates the expression of JA-responsive genes and plays a crucial role in a mechanism that sustains root cell elongation at appropriate rates under stressful conditions.

trans -acting small interfering RNAs (tasiRNAs) are plant-specific endogenous siRNAs produced via a unique pathway whose first step is the microRNA (miRNA)-programmed RNA-induced silencing complex (RISC)–mediated cleavage of tasiRNA gene ( TAS ) transcripts. One of the products is subsequently transformed into tasiRNAs by a pathway that requires several factors including SUPPRESSOR OF GENE SILENCING3 (SGS3) and RNA-DEPENDENT RNA POLYMERASE6. Here, using in vitro assembled ARGONAUTE (AGO)1–RISCs, we show that SGS3 is recruited onto RISCs only when they bind target RNA. Following cleavage by miRNA173 (miR173)-programmed RISC, SGS3 was found in complexes containing cleaved TAS2 RNA and RISC. The 3′ cleavage fragment (the source of tasiRNAs) was protected from degradation in this complex. Depletion of SGS3 did not affect TAS2 RNA cleavage by miR173-programmed RISC, but did affect the stability of the 3′ cleavage fragment. When the 3′ nucleotide of 22-nt miR173 was deleted or the corresponding nucleotide in TAS2 RNA was mutated, the complex was not observed and the 3′ cleavage fragment was degraded. Importantly, these changes in miR173 or TAS2 RNA are known to lead to a loss of tasiRNA production in vivo. These results suggest that ( i ) SGS3 associates with AGO1–RISC via the double-stranded RNA formed by the 3′-terminal nucleotides of 22-nt miR173 and corresponding target RNA, which probably protrudes from the AGO1–RISC molecular surface, ( ii ) SGS3 protects the 3′ cleavage fragment of TAS2 RNA from degradation, and ( iii ) the observed SGS3-dependent stabilization of the 3′ fragment of TAS2 RNA is key to tasiRNA production.

Winged bean Kunitz chymotrypsin inhibitor (WCI) accumulates abundantly in seeds and tuberous roots, and small amounts of the WCI protein and mRNA can also be detected in stems. In this study, we analyzed the localization of the WCI protein in stems of winged bean. The results demonstrated that the WCI protein was localized in sieve tubes. Furthermore, we showed that the 5' region of the WCI-3b gene, which exhibited strong transcriptional activity in developing seeds, also promoted transcription of a reporter gene in the phloem of stems of transgenic tobacco.

Regulation of cytosine methylation in the plant genome is of pivotal in determining the epigenetic states of chromosome regions. Relative tolerance of plant to deficiency in cytosine methylation provides unparalleled opportunities to study the mechanism for regulation of cytosine methylation. The Decrease in DNA Methylation 1 (DDM1) of Arabidopsis thaliana is one of the best characterized plant epigenetic regulators that are necessary for maintenance of cytosine methylation in genomic DNA. Although cytosine methylation could affect various aspects of plant growth and development including those related to agricultural importance, orthologs of DDM1 in plants other than Arabidopsis has not been studied in detail. In this study, we identified two rice genes with similarity to Arabidopsis DDM1 and designated them OsDDM1a and OsDDM1b. Both of the rice DDM1 homologs are transcribed during development and their amino acid sequences are 93 % identical to each other. Transgenic rice lines expressing the OsDDM1a cDNA in the antisense orientation exhibited genomic DNA hypomethylation. In those lines, repeated sequences were more severely affected than a single copy sequence as is the case in Arabidopsis ddm1 mutants. Transcripts derived from endogenous transposon-related loci were up-regulated in the antisense OsDDM1 lines, opening a possibility to identify and utilize potentially active transposons for rice functional genomics.

The natural bicolor floral traits of the horticultural petunia (Petunia hybrida) cultivars Picotee and Star are caused by the spatial repression of the chalcone synthase A (CHS-A) gene, which encodes an anthocyanin biosynthetic enzyme. Here we show that Picotee and Star petunias carry the same short interfering RNA (siRNA)-producing locus, consisting of two intact CHS-A copies, PhCHS-A1 and PhCHS-A2, in a tandem head-to-tail orientation. The precursor CHS mRNAs are transcribed from the two CHS-A copies throughout the bicolored petals, but the mature CHS mRNAs are not found in the white tissues. An analysis of small RNAs revealed the accumulation of siRNAs of 21 nucleotides that originated from the exon 2 region of both CHS-A copies. This accumulation is closely correlated with the disappearance of the CHS mRNAs, indicating that the bicolor floral phenotype is caused by the spatially regulated post-transcriptional silencing of both CHS-A genes. Linkage between the tandemly arranged CHS-A allele and the bicolor floral trait indicates that the CHS-A allele is a necessary factor to confer the trait. We suppose that the spatially regulated production of siRNAs in Picotee and Star flowers is triggered by another putative regulatory locus, and that the silencing mechanism in this case may be different from other known mechanisms of post-transcriptional gene silencing in plants. A sequence analysis of wild Petunia species indicated that these tandem CHS-A genes originated from Petunia integrifolia and/or Petunia inflata, the parental species of P. hybrida, as a result of a chromosomal rearrangement rather than a gene duplication event.

Histone modification is the key event in epigenetic regulation of gene expression in most eukaryotes. Plants, fungi, and vertebrates have machineries for DNA methylation as an additional layer of epigenetic modification, and the presence of CHH and CHG (H = A, T, or C) methylation sites are characteristics of plants. In Arabidopsis thaliana, both CHH and CHG methylation sites are enriched with sequences related to transposons in heterochromatin regions, and the RNA-directed DNA methylation pathway plays a major role in asymmetric cytosine (CHH) methylation. Heterochromatin regions are also rich in dimethylated histone H3 lysine 9 (H3K9me2) – a histone mark of inert chromatin. Although enzymes transferring methyl groups to DNA or H3K9 have been identified, mechanisms ­linking these two methylation steps specifically to inert chromatin are obscure. In this chapter, we review characteristics of the machineries governing DNA methylation and histone modification in A. thaliana and discuss the possible interplay between these two epigenetic marks in relation to suppression of non coding RNA production.

Winged bean Kunitz chymotrypsin inhibitor (WCI) is encoded by a multigene family and accumulation of its mRNA is restricted in mid-maturation stage seeds and tuberous roots. In this paper, we analyzed the accumulation of mRNA derived from each WCI gene using a novel method: sequence-specific termination analysis. The results demonstrated that the accumulation of each WCI mRNA was differentially regulated in winged bean plants.

We analyzed the structure and the expression of Kunitz chymotrypsin inhibitor (WCI) genes in winged bean. WCI was encoded by a multigene family which comprised at least seven members. From their primary structures, four genes (WCI-2, WCI-3a, WCI-3b, and WCI-x) were expected to be functional ones and the other three (WCI-P1, WCI-P2, and WCI-P3) to be pseudogenes. The nucleotide sequences of the WCI-3a and WCI-3b genes were nearly identical, and they encoded the WCI-3 protein, the major chymotrypsin inhibitor in seeds. The WCI-2 gene also encoded the chymotrypsin inhibitor found in seeds and the WCI-x gene was expected to encode an unidentified chymotrypsin inhibitor. WCI messenger RNA and protein accumulated mainly in developing seeds and tuberous roots, small amounts of WCI mRNA being present in stems and pericarps. In seeds, transient accumulation of WCI mRNA was observed during the seed maturation period. These results suggest that the expression of WCI genes is regulated organ-specifically and developmentally in winged bean.

We analyzed the inheritance of DNA methylation in the first filial generation(F1) hybrid of Oryza sativa L. ("Nipponbare" x "Kasalath") by restriction landmark genome scanning (RLGS). Most parental RLGS spots were found in the F1, but eight spots (4%) showed abnormal inheritance: seven of the eight spots were missing in the F1, and one was newly detected in the F1. Here we show demethylation at restriction enzyme sites in the F1. We also found a candidate site of stable heterozygous methylation in the genome. These results show the applicability of the RLGS method for analysis of the inheritance and alteration of methylation in F1 hybrid plants.

International audience; Constitutive heterochromatin is a compact, transcriptionally inert structure formed in gene-poor and repeat- and transposon-rich regions. In Arabidopsis, constitutive heterochromatin is characterized by hypermethylated DNA and histone H3 dimethylated at lysine (K) 9 (H3K9me2) together with depletion of histone H3 dimethylated at lysine 4 (H3K4me2). Here, we describe loci with intermediate properties of heterochromatin in which transcription downregulation is inherited in a manner similar to constitutive heterochromatin, although the loci are associated with opposing histone marks--H3K4me2 and H3K9me2. In the ddm1 (decrease in DNA methylation 1) mutants, their transcriptional activation is accompanied by the expected shift in the H3 modifications--depletion of H3K9me2 and enrichment in H3K4me2. In mom1 (Morpheus' molecule 1) mutants, however, a marked increase in transcription is not accompanied by detectable changes in the levels of H3K4me2 and H3K9me2. Therefore, transcriptional regulation in the intermediate heterochromatin involves two distinct epigenetic mechanisms. Interestingly, silent transgenic inserts seem to acquire properties characteristic of the intermediate heterochromatin.

In mammals and plants, formation of heterochromatin is associated with hypermethylation of DNA at CpG sites and histone H3 methylation at lysine 9. Previous studies have revealed that maintenance of DNA methylation in Neurospora and Arabidopsis requires histone H3 methylation. A feedback loop from DNA methylation to histone methylation, however, is less understood. Its recent examination in Arabidopsis with a partial loss of function in DNA methyltransferase 1 (responsible for maintenance of CpG methylation) yielded conflicting results. Here we report that complete removal of CpG methylation in an Arabidopsis mutant null for DNA maintenance methyltransferase results in a clear loss of histone H3 methylation at lysine 9 in heterochromatin and also at heterochromatic loci that remain transcriptionally silent. Surprisingly, these dramatic alterations are not reflected in heterochromatin relaxation.

Sequential deletions of the promoter region of the WCI-3b gene, which encodes the major chymotrypsin inhibitor of winged bean, were constructed and their expression was analyzed in transgenic tobacco plants and in bombarded winged bean seeds. In transgenic tobacco plants, a critical promoter region which is important for high levels of expression in seeds was identified, but deletion of this region had essentially no effect when bombarded into winged bean seeds.

Mitotic and meiotic inheritance of epigenetic information is coupled to the reproduction of chromatin conformation and DNA methylation patterns. This implies that the S phase of the cell cycle provides a window of opportunity for changes in epigenetic determination. Recent studies, however, have suggested that chromatin structure is also rather dynamic in quiescent cells of multicellular eukaryotes and that silent heterochromatic regions can become accessible to transcription. Such epigenetic flexibility in differentiated tissues could be of physiological importance. The mechanisms and molecular components involved are of great interest but as yet unknown. We examined MOM1 (Morpheus' Molecule 1), a regulator of transcriptional gene silencing (TGS) that acts independently of DNA methylation, for its role in the maintenance of TGS in non-dividing, differentiated cells. The results provide evidence that TGS maintenance mediated by MOM1 is a dynamic process that can be modified in non-dividing cells of mature plant organs by depletion of MOM1.

Genetic approaches to understanding the role of epigenetic regulation of gene expression in plants and its mechanisms have revealed several new components. Rapidly accumulating information from other eukaryotes provides complementary knowledge with important implications for plant research. Comparison of epigenetic events across species is proving critical for defining the mechanisms and functions of epigenetic modification, including those specific to plants.

The amplified restriction fragment length polymorphism (AFLP)-based mRNA fingerprinting (AMF) method makes it possible systematically and conveniently to identify differentially expressed cDNAs with high reproducibility. We have applied the AMF method to the cloning of the Q gene of common wheat, which is located on the long arm of chromosome 5A and pleiotropically controls the spike morphology and the threshing character of seeds. Using the AMF method, we compared the fingerprints of mRNA samples extracted from the young spikes of Triticum aestivum cv. Chinese Spring (CS) carrying the Q gene to those of a chromosome deletion line of CS, namely, q5, which lacks 15% of 5AL including the Q gene. Approximately 12,200 fragments were produced after PCR with 256 primer combinations. Of these, 92 fragments were differentially expressed between CS and q5. Northern and Southern analyses showed that 16 fragments gave specific or relatively stronger transcript signals in CS, and these clones were present in single copy or in low copy numbers in the wheat genome. Four clones were genetically mapped to the region deleted in q5. Subsequently, one clone, pTaQ22, was mapped at the same locus as the Q gene, indicating that pTaQ22 corresponds to the Q gene or is tightly linked to it. DNA sequence data showed that pTaQ22 had no homology to any known genes, thus suggesting a novel function for this gene in flower morphogenesis. This AMF method might provide a straightforward method for isolating genes in the hexaploid background of common wheat.

Epigenetic modifications change transcription patterns in multicellular organisms to achieve tissue-specific gene expression and inactivate alien DNA such as transposons or transgenes. In plants and animals, DNA methylation is involved in heritability and flexibility of epigenetic states, although its function is far from clear. We have isolated an Arabidopsis gene, MOM, whose product is required for the maintenance of transcriptional gene silencing. Mutation of this gene or depletion of its transcript by expression of antisense RNA reactivates transcription from several previously silent, heavily methylated loci. Despite this, the dense methylation at these reactivated loci is maintained even after nine generations, indicating that transcriptional activity and methylation pattern are inherited independently. The predicted MOM gene product is a nuclear protein of 2,001 amino acids containing a region similar to part of the ATPase region of the SWI2/SNF2 family, members of which are involved in chromatin remodelling. MOM is the first known molecular component that is essential for transcriptional gene silencing and does not affect methylation pattern. Thus, it may act downstream of methylation in epigenetic regulation, or be part of a new pathway that does not require methylation marks.

The Japanese morning glory has an extensive history of genetic studies and over 200 different spontaneous mutant lines have been described. Of these, we identified that two mutable alleles, flecked and speckled, for flower variegations are caused by integration of transposable elements, belonging to the En/Spm family, into the DFR-B and CHI genes for flower pigmentation, respectively. The mutable flaked allele of the common morning glory bearing variegated flowers is caused by insertion of a new transposable element, Tip 100, into one of the CHS genes for pigmentation and that Tip 100 belongs to the Ac/Ds family. These results are discussed with regard to spontaneous transposon mutagenesis and generation of floricultural traits of morning glories.

The mutable flaked (or af) lines of the common morning glory bear white flowers with colored flakes and sectors. The af allele shows incomplete dominance. Plants in the heterozygous state A/af bear lightly colored flowers with intensely colored flakes and occasionally with white sectors. We showed that the mutable af allele is caused by insertion of a new transposable element, Tip100, into the CHS-D gene intron. Tip100 is 3.9 kb long and belongs to the Ac/Ds family. Although the timing and frequency of the flower variegation vary in different lines, they carry an identical mutable allele. We also noticed that a flaked subline, bearing variegated flowers, carries a Tip100 derivative, Tip100-1. The structure of Tip100-1 contains an additional 48 bp terminal sequence as tandem repeats and its integration site is identical to that of Tip100. Another line, with stable white flowers, is a double mutant carrying two copies of Tip100 in the CHS-D gene. These results are discussed with regard to the variegated phenotypes of flowers in various mutable lines.

Using amplified restriction fragment length polymorphism (AFLP) technology, we have developed a new protocol for the fingerprinting of mRNA that allows systematic comparison of the differential expression of genes between mRNA samples. The major advantage of our protocol is the use of only a single restriction enzyme that recognizes a 4-bp sequence but allows screening of large numbers of different cDNAs. Using this new protocol, we compared mRNA samples obtained from the flower buds of two lines of the common morning glory (Ipomoea purpurea) with red and white flowers, respectively. Approximately 50 bands were observed in each lane of a denaturing polyacrylamide gel and the results were highly reproducible, as indicated by the results of analysis of two sets of independent mRNA samples. Two cDNA fragments, which were differentially amplified in the samples from the two lines, were shown to have been derived from a single gene that was actively expressed in the buds of red flowers but not in those of white flowers. A full-length cDNA of this gene was cloned from a bud cDNA library. Sequence analysis showed that this cDNA carries a sequence highly homologous to the chalcone synthase (CHS) genes, the key enzyme in the flavonoid biosynthetic pathway.

Winged bean Kunitz chymotrypsin inhibitor (WCI) accumulates in an organ-specific and temporally regulated manner. The protein is encoded by a multigene family that includes at least four putative inhibitor-coding genes and three pseudogenes. The structure of the WCI genes indicates that an insertion at a 5′ proximal site occurred after duplication of the ancestral WCI gene and that several gene conversion events subsequently contributed to the evolution of this gene family. Analysis of the promoter activity of the 5′ regions of the WCI genes in transgenic tobacco showed that only the 5′ regions of the WCI-3a and WCI-3b genes, which encode the major WCI protein in winged bean, promoted the organ-specific and temporally regulated expression of a reporter gene. The 5′ region of a pseudogene, the WCI-P1 gene which contains frameshift mutations, exhibited constitutive promoter activity in tobacco, an indication that the 5′ region of the WCI-P1 gene might spontaneously have acquired new regulatory sequences during evolution. Since gene conversion is a relatively frequent event and since the homology between the WCI-P1 and WCI-3a/b genes is disrupted at a 5′ proximal site by remnants of an inserted sequence, the WCI-P1 gene appears to be a possible intermediate that could be converted into a new functional gene with a distinct pattern of expression by a single gene-conversion event.