Search

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.

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.

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.

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.

The genomic nucleotide sequences of japonica rice (Sasanishiki and Nipponbare) contained about 2.7-kb unique region at the point of 0.4-kb upstream of the OsPsbS1 gene. In this study, we found that japonica rice with a few exceptions possessing such DNA sequences [denoted to OsMULE-japonica specific sequence (JSS)] is distinct by the presence of Mutator-like-element (MULE). Such sequence was absent in most of indica cultivars and Oryza glaberrima. In OsMULE-JSS1, we noted the presence of possible target site duplication (TSD; CTTTTCCAG) and about 80-bp terminal inverted repeat (TIR) near TSD. We also found the enhancement ofOsPsbS1 mRNA accumulation by intensified light, which was not associated with the DNA methylation status in OsMULE/JSS. In addition, O. rufipogon, possible ancestor of modern rice cultivars was found to compose PsbS gene of either japonica (minor) or indica (major) type. Transient gene expression assay showed that the japonica type promoter elevated a reporter gene activity than indica type.

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.

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.

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.

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.

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.

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.

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.

A winged bean Kunitz-type chymotrypsin inhibitor (WCI) accumulates abundantly in developing embryos and tuberous roots, and also in a small amount in stems. Promoter activity of the 1.0 kbp upstream region of the WCI-3b gene, which encodes a major WCI protein in seeds, was analyzed in transgenic tobacco plants. The 1.0 kbp upstream region directed strong expression of a reporter gene (s-glucuronidase; GUS) in developing seeds of the transgenic tobacco. The GUS activity was also detected in stems, although no significant GUS activity was observed in leaves. Histochemical analysis revealed that the GUS gene was mainly expressed in embryos and in phloem of stems. These results suggested that the transcription regulation plays an important role in the expression of the WCI-3b gene in embryos and in phloem of stems. The cis-elements promoting transcription of the WCI-3b gene in these cells are included within the 1.0 kbp upstream region.