Characterization of autonomous Dart1 transposons belonging to the hAT superfamily in rice

An endogenous 0.6-kb rice DNA transposon, nDart1-0, was found as an active nonautonomous element in a mutable virescent line, pyl-v, displaying leaf variegations. Here, we demonstrated that the active autonomous element aDart in pyl-v corresponds to Dart1-27 on chromosome 6 in Nipponbare, which carries no active aDart elements, and that aDart and Dart1-27 are identical in their sequences and chromosomal locations, indicating that Dart1-27 is epigenetically silenced in Nipponbare. The identification of aDart in pyl-v was first performed by map-based cloning and by detection of the accumulated transposase transcripts. Subsequently, various transposition activities of the cloned Dart1-27 element from Nipponbare were demonstrated in Arabidopsis. Dart1-27 in Arabidopsis was able to excise nDart1-0 and Dart1-27 from cloned sites, generating footprints, and to integrate into new sites, generating 8-bp target site duplications. In addition to Dart1-27, Nipponbare contains 37 putative autonomous Dart1 elements because their putative transposase genes carry no apparent nonsense or frameshift mutations. Of these, at least four elements were shown to become active aDart elements in transgenic Arabidopsis plants, even though considerable sequence divergence arose among their transposases. Thus, these four Dart1 elements and Dart1-27 in Nipponbare must be potential autonomous elements silenced epigenetically. The regulatory and evolutionary implications of the autonomous Dart1 elements and the development of an efficient transposon-tagging system in rice are discussed

Characterization of autonomous Dart1 transposons belonging to the hAT superfamily in rice

　　As a major provision for more than half of the world’s population, rice (Oryza sativa L.) is one of the most important plant species and has great economic importance. Also, rice has been focused on as an excellent model plant for cereal genomics studies due to its following features; 1) the smallest genome size (389 Mb) among the cereal grasses, 2) syntenic relation with other agronomically important Poaceae species such as maize, barley and wheat, 3) available varied resources including a large number of genetic markers, genomic libraries, many mutant lines and retrotransposon tagged lines, 4) there exists well developed technologies for rice genome manipulation such as Agrobacterium tumefaciens-mediated gene transformation and gene targeting with homologous recombination. Consequently, fine quality map-based genome sequencing of rice was completed in 2005.　　One of the most challenging goals for the plant-research community going forward is to identify the function and regulation of rice genes. Thus, both forward and reverse genetic approaches have been developed to elucidate these functions. However, because the tissue culture process is a necessary step in most of the currently available procedures used in rice genome research, somaclonal variations, which refer to genetic and epigenetic mutations induced by tissue culture, can hamper these approaches. Therefore, a lot of attention has been given to recently identified endogenous DNA transposons that are active under natural growth conditions, a character that is quite useful to development of more efficient rice transposon tagging as a functional genomics tool free from somaclonal variation.　　One such DNA transposon, nDart1-0 (non-autonomous DNA-based active rice transposon one-zero) in the hAT superfamily, had been identified as a causative element of spontaneous leaf variegation shown in the mutant line pyl-v (pale-yellow-leaf variegated). This mutable phenotype is caused by the disruption and restoration of the nuclear-coded essential chloroplast protease gene, OsClpP5, due to the insertion and subsequent excision of nDart1-0. As a typical non-autonomous transposon in the hATsuperfamily, nDart1-0 can transpose only when the trans-acting transposase is supplied from an autonomous element, aDart (active autonomous Dart). On the other hand, an indicator line, pyl-stb (pyl-stable) shows uniform pale-yellow leaves with no nDart1-0 excision due to a lack of an aDart. The result of test crosses between pyl-v and pyl-stb lines indicated that the pyl-v line carries an aDart element in its genome.　　In the published genomic sequence of the cultivar Nipponbare, there are 38 candidate autonomous Dart elements that have putative transposase genes with no apparent nonsense or frameshift mutations. However, from the result of test crosses with the pyl -stb line, it was shown that Nipponbare carries no aDart elements in its genome. Meanwhile, the excision of some endogenous nDart1 elements in Nipponbare and pyl- stb was induced by treatment with a DNA methylation inhibitor, 5-azacytidine. Hence, these lines were predicted to carry epigenetically silenced autonomous elements, iDarts (inactive autonomous Darts).　　The first aims of this study were identifying the aDart element in the pyl-v line and demonstrating its molecular criteria as an autonomous element. To this end, I performed map-based cloning and revealed that the aDart element in the pyl-v line coincides with one of the 38 candidate autonomous elements, iDart1-27, residing on chromosome 6 in Nipponbare. Also, I have found that all of the examined transcripts of the Dart transposase gene were derived from Dart1-27in the pyl-v line. These results strongly suggested that Dart1-27in pyl-v acts on nDart1-0 as an active aDart element. Then, I demonstrated that iDart1-27 cloned from the Nipponbare genome can be converted to an active aDart element in Arabidopsis thaliana plants when its methylation status was eliminated during the cloning process; Dart1-27 excised nDart1-0 as well as itself from the introduced vectors and integrated into various sites of the A. thalianagenome. These results clearly indicated that Dart1-27 is a functional autonomous element, and it is active as an aDart element in the pyl-v line whereas epigenetically silenced as iDart1- 27 in Nipponbare. Furthermore, I showed other Dart elements, Dart1-1, Dart1-20, Dart1-28 and Dart1-52 are also functional autonomous elements, but they are epigenetically silenced as iDarts in Nipponbare. 　Next, in order to study if there are any regulatory mechanisms that control the activity of the Dart/nDart system in the pyl-stb line, I introduced Dart1-27 derivatives into the pyl-stb line and evaluated their activity. As a prerequisite for this transgenic approach, I carefully confirmed that during each step of the A. tumefaciens-mediated transformation process the endogenous iDart elements in the pyl-stb genome are almost never activated (0.1%). Based on this confirmation, I introduced Dart1-27 derivatives into pyl-stb and demonstrated that they can mobilize nDart1-0 elements from the OsClpP5 gene as well as from an introduced GUSPlus gene at a high frequency in transgenic pyl-stb plants. This result reconfirmed that Dart1-27 is a functional autonomous element able to act on nDart elements when its methylation status is eliminated, as shown in A. thalianaplants. From the results of phenotypic analysis of transgenic pyl-stb plants, it was suggested that there is a development-dependent regulation of Dartactivity in regenerated pyl-stb plants; most of the transgenic pyl-stb plants introduced with Dart1-27 derivatives were the pyl-stb phenotype at their 4-6 leaves stage, but almost all of them became the pyl-v phenotype at their 7-10 leaves stage.　　In this manuscript, I have unambiguously demonstrated that the active autonomous element in the pyl-v line is Dart1-27 on chromosome 6 and that the rice genome contains multiple potential autonomous Dart elements silenced epigenetically. From analysis of transgenic pyl-stb plants, I have also indicated a development-dependent regulation that could be a key to further elucidating Dart/nDart regulation mechanisms in the rice genome. I believe these results will facilitate an effective gene tagging system using the Dart/nDart elements in rice

Herbicide tolerance-assisted multiplex targeted nucleotide substitution in rice

Acetolactate synthase (ALS) catalyzes the initial step in the biosynthesis of branched-chain amino acids, and is highly conserved from bacteria to higher plants. ALS is encoded by a single copy gene in rice genome and is a target enzyme of several classes of herbicides. Although ALS mutations conferring herbicide-resistance property to plants are well documented, effect of Imazamox (IMZ) on rice and the mutations in ALS correlated with IMZ tolerance were unclear. In this article, the effect of IMZ on rice calli and seedlings in tissue culture conditions were evaluated. Also, the ALS A96V mutation was confirmed to improve IMZ tolerance of rice calli. Based on these results, ALS-assisted multiplex targeted base editing in rice was demonstrated in combination with Target-AID, a CRISPR/Cas9-cytidine deaminase fusion system [1], [2]

Ehd1, a B-type response regulator in rice, confers short-day promotion of flowering and controls FT-like gene expression independently of Hd1

Two evolutionarily distant plant species, rice (Oryza sativa L.), a short-day (SD) plant, and Arabidopsis thaliana, a long-day plant, share a conserved genetic network controlling photoperiodic flowering. The orthologous floral regulators—rice Heading date 1 (Hd1) and Arabidopsis CONSTANS (CO)—integrate circadian clock and external light signals into mRNA expression of the FLOWERING LOCUS T (FT) group floral inducer. Here, we report that the rice Early heading date 1 (Ehd1) gene, which confers SD promotion of flowering in the absence of a functional allele of Hd1, encodes a B-type response regulator that might not have an ortholog in the Arabidopsis genome. Ehd1 mRNA was induced by 1-wk SD treatment, and Ehd1 may promote flowering by inducing FT-like gene expression only under SD conditions. Microarray analysis further revealed a few MADS box genes downstream of Ehd1. Our results indicate that a novel two-component signaling cascade is integrated into the conserved pathway in the photoperiodic control of flowering in rice

BRASSINOSTEROID UPREGULATED1, Encoding a Helix-Loop-Helix Protein, Is a Novel Gene Involved in Brassinosteroid Signaling and Controls Bending of the Lamina Joint in Rice1[W][OA]

Brassinosteroids (BRs) are involved in many developmental processes and regulate many subsets of downstream genes throughout the plant kingdom. However, little is known about the BR signal transduction and response network in monocots. To identify novel BR-related genes in rice (Oryza sativa), we monitored the transcriptomic response of the brassinosteroid deficient1 (brd1) mutant, with a defective BR biosynthetic gene, to brassinolide treatment. Here, we describe a novel BR-induced rice gene BRASSINOSTEROID UPREGULATED1 (BU1), encoding a helix-loop-helix protein. Rice plants overexpressing BU1 (BU1:OX) showed enhanced bending of the lamina joint, increased grain size, and resistance to brassinazole, an inhibitor of BR biosynthesis. In contrast to BU1:OX, RNAi plants designed to repress both BU1 and its homologs displayed erect leaves. In addition, compared to the wild type, the induction of BU1 by exogenous brassinolide did not require de novo protein synthesis and it was weaker in a BR receptor mutant OsbriI (Oryza sativa brassinosteroid insensitive1, d61) and a rice G protein alpha subunit (RGA1) mutant d1. These results indicate that BU1 protein is a positive regulator of BR response: it controls bending of the lamina joint in rice and it is a novel primary response gene that participates in two BR signaling pathways through OsBRI1 and RGA1. Furthermore, expression analyses showed that BU1 is expressed in several organs including lamina joint, phloem, and epithelial cells in embryos. These results indicate that BU1 may participate in some other unknown processes modulated by BR in rice