Shoko Kojima

Dual regulation of ETTIN (ARF3) gene expression by AS1-AS2, which maintains the DNA methylation level, is involved in stabilization of leaf adaxial-abaxial partitioning in Arabidopsis

Leaf primordia are generated at the periphery of the shoot apex, developing into flat symmetric organs with adaxial-abaxial polarity, in which the indeterminate state is repressed. Despite the crucial role of the ASYMMETRIC LEAVES1 (AS1)-AS2 nuclear-protein complex in leaf adaxial-abaxial polarity specification, information on mechanisms controlling their downstream genes has remained elusive. We systematically analyzed transcripts by microarray and chromatin immunoprecipitation assays and performed genetic rescue of as1 and as2 phenotypic abnormalities, which identified a new target gene, ETTIN (ETT)/AUXIN RESPONSE FACTOR3 (ARF3), which encodes an abaxial factor acting downstream of the AS1-AS2 complex. While the AS1-AS2 complex represses ETT by direct binding of AS1 to the ETT promoter, it also indirectly activates miR390- and RDR6-dependent post-transcriptional gene silencing to negatively regulate both ETT and ARF4 activities. Furthermore, AS1-AS2 maintains the status of DNA methylation in the ETT coding region. In agreement, filamentous leaves formed in as1 and as2 plants treated with a DNA methylation inhibitor were rescued by loss of ETT and ARF4 activities. We suggest that negative transcriptional, post-transcriptional and epigenetic regulation of the ARFs by AS1-AS2 is important for stabilizing early leaf partitioning into abaxial and adaxial domains.

Genetic networks regulated by ASYMMETRIC LEAVES1 (AS1) and AS2 in leaf development in Arabidopsis thaliana: KNOX genes control five morphological events

The asymmetric leaves 1 (as1) and as2 mutants of Arabidopsis thaliana exhibit pleiotropic phenotypes. Expression of a number of genes, including three class-1 KNOTTED-like homeobox (KNOX) genes (BP, KNAT2 and KNAT6) and ETTIN/ARF3, is enhanced in these mutants. In the present study, we attempted to identify the phenotypic features of as1 and as2 mutants that were generated by ectopic expression of KNOX genes, using multiple loss-of-function mutations of KNOX genes as well as as1 and as2. Our results revealed that the ectopic expression of class-1 KNOX genes resulted in reductions in the sizes of leaves, reductions in the size of sepals and petals, the formation of a less prominent midvein, the repression of adventitious root formation and late flowering. Our results also revealed that the reduction in leaf size and late flowering were caused by the repression, by KNOX genes, of a gibberellin (GA) pathway in as1 and as2 plants. The formation of a less prominent midvein and the repression of adventitious root formation were not, however, related to the GA pathway. The asymmetric formation of leaf lobes, the lower complexity of higher-ordered veins, and the elevated frequency of adventitious shoot formation on leaves of as1 and as2 plants were not rescued by multiple mutations in KNOX genes. These features must, therefore, be controlled by other genes in which expression is enhanced in the as1 and as2 mutants.

ASYMMETRIC LEAVES2 and Elongator, a Histone Acetyltransferase Complex, Mediate the Establishment of Polarity in Leaves of Arabidopsis thaliana

Leaf primordia are generated around the shoot apical meristem. Mutation of the ASYMMETRIC LEAVES2 (AS2) gene of Arabidopsis thaliana results in defects in repression of the meristematic and indeterminate state, establishment of adaxial-abaxial polarity and left-right symmetry in leaves. AS2 represses transcription of meristem-specific class 1 KNOX homeobox genes and of the abaxial-determinant genes ETTIN/ARF3, KANADI2 and YABBY5. To clarify the role of AS2 in the establishment of leaf polarity, we isolated mutations that enhanced the polarity defects associated with as2. We describe here the enhancer-of-asymmetric-leaves-two1 (east1) mutation, which caused the formation of filamentous leaves with abaxialized epidermis on the as2-1 background. Levels of transcripts of class 1 KNOX and abaxial-determinant genes were markedly higher in as2-1 east1-1 mutant plants than in the wild-type and corresponding single-mutant plants. EAST1 encodes the histone acetyltransferase ELONGATA3 (ELO3), a component of the Elongator complex. Genetic analysis, using mutations in genes involved in the biogenesis of a trans-acting small interfering RNA (ta-siRNA), revealed that ELO3 mediated establishment of leaf polarity independently of AS2 and the ta-siRNA-related pathway. Treatment with an inhibitor of histone deacetylases (HDACs) caused additive polarity defects in as2-1 east1-1 mutant plants, suggesting the operation of an ELO3 pathway, independent of the HDAC pathway, in the determination of polarity. We propose that multiple pathways play important roles in repression of the expression of class 1 KNOX and abaxial-determinant genes in the development of the adaxial domain of leaves and, thus, in the establishment of leaf polarity.

The complex of ASYMMETRIC LEAVES (AS) proteins plays a central role in antagonistic interactions of genes for leaf polarity specification in Arabidopsis

Leaf primordia are born around meristem‐containing stem cells at shoot apices, grow along three axes (proximal–distal, adaxial–abaxial, medial–lateral), and develop into flat symmetric leaves with adaxial–abaxial polarity. Axis development and polarity specification of Arabidopsis leaves require a network of genes for transcription factor‐like proteins and small RNAs. Here, we summarize present understandings of adaxial‐specific genes, ASYMMETRIC LEAVES1 (AS1) and AS2. Their complex (AS1–AS2) functions in the regulation of the proximal–distal leaf length by directly repressing class 1 KNOX homeobox genes (BP, KNAT2) that are expressed in the meristem periphery below leaf primordia. Adaxial–abaxial polarity specification involves antagonistic interaction of adaxial and abaxial genes including AS1 and AS2 for the development of two respective domains. AS1–AS2 directly represses the abaxial gene ETTIN/AUXIN RESPONSE FACTOR3 (ETT/ARF3) and indirectly represses ETT/ARF3 and ARF4 through tasiR‐ARF. Modifier mutations have been identified that abolish adaxialization and enhance the defect in the proximal–distal patterning in as1 and as2. AS1–AS2 and its modifiers synergistically repress both ARFs and class 1 KNOXs. Repression of ARFs is critical for establishing adaxial–abaxial polarity. On the other hand, abaxial factors KANADI1 (KAN1) and KAN2 directly repress AS2 expression. These data delineate a molecular framework for antagonistic gene interactions among adaxial factors, AS1, AS2, and their modifiers, and the abaxial factors ARFs as key regulators in the establishment of adaxial–abaxial polarity. Possible AS1–AS2 epigenetic repression and activities downstream of ARFs are discussed. WIREs Dev Biol 2015, 4:655–671. doi: 10.1002/wdev.196

Meta-Analyses of Microarrays of Arabidopsis asymmetric leaves1 (as1), as2 and Their Modifying Mutants Reveal a Critical Role for the ETT Pathway in Stabilization of Adaxial–Abaxial Patterning and Cell Division During Leaf Development

It is necessary to use algorithms to analyze gene expression data from DNA microarrays, such as in clustering and machine learning. Previously, we developed the knowledge-based fuzzy adaptive resonance theory (KB-FuzzyART), a clustering algorithm suitable for analyzing gene expression data, to find clues for identifying gene networks. Leaf primordia form around the shoot apical meristem (SAM), which consists of indeterminate stem cells. Upon initiation of leaf development, adaxial–abaxial patterning is crucial for lateral expansion, via cellular proliferation, and the formation of flat symmetric leaves. Many regulatory genes that specify such patterning have been identified. Analysis by the KB-FuzzyART and subsequent molecular and genetic analyses previously showed that ASYMMETRIC LEAVES1 (AS1) and AS2 repress the expression of some abaxial-determinant genes, such as AUXIN RESPONSE FACTOR3 (ARF3)/ETTIN (ETT) and ARF4, which are responsible for defects in leaf adaxial–abaxial polarity in as1 and as2. In the present study, genetic analysis revealed that ARF3/ETT and ARF4 were regulated by modifier genes, BOBBER1 (BOB1) and ELONGATA3 (ELO3), together with AS1–AS2. We analyzed expression arrays with as2 elo3 and as2 bob1, and extracted genes downstream of ARF3/ETT by using KB-FuzzyART and molecular analyses. The results showed that expression of Kip-related protein (KRP) (for inhibitors of cyclin-dependent protein kinases) and Isopentenyltransferase (IPT) (for biosynthesis of cytokinin) genes were controlled by AS1–AS2 through ARF3/ETT and ARF4 functions, which suggests that the AS1–AS2–ETT pathway plays a critical role in controlling the cell division cycle and the biosynthesis of cytokinin around SAM to stabilize leaf development in Arabidopsis thaliana.

Discovery of novel rules for G-quadruplex-forming sequences in plants by using bioinformatics methods.

The G-quadruplex is one of the most frequently studied secondary DNA structures and consists of 4 guanine residues that interact through Watson-Crick and Hoogsteen pairing. The G-quadruplex formation is thought to be a molecular switch for gene expression. Genome-wide analyses of G-quadruplexes have been published for many species; however, only one genome-wide analysis of G-quadruplexes in plants has been reported. Here, we propose a new approach involving a two-step procedure for identifying G-quadruplex-forming sequences (potential G4 DNA motif regions: G4MRs) and classifying positional relationships between G4MRs and genes. By using this approach, we exhaustively searched for G4MRs in the whole genomes of 8 species: Arabidopsis thaliana, Oryza sativa subsp. japonica, Populus trichocarpa, Vitis vinifera, Homo sapiens, Danio rerio, Drosophila melanogaster, and Caenorhabditis elegans. We classified genes on the basis of their positional relationships to their proximal G4MRs. We identified novel rules for G4MRs in plants, such as G4MR-enrichment in the template strands at transcription start sites (TSSs). Next, we focused on the template strands of TSSs and conducted gene ontology (GO) analysis of genes proximal to G4MRs. We identified GO terms such as chloroplast and nucleosome (or histone) in O. sativa. Although these terms were strongly associated in O. sativa, weak associations were identified in other plants. These results will be helpful for elucidating the functional roles of G4 DNA.

ASYMMETRIC-LEAVES2 and an ortholog of eukaryotic NudC domain proteins repress expression of AUXIN-RESPONSE-FACTOR and class 1 KNOX homeobox genes for development of flat symmetric leaves in Arabidopsis

Summary Leaf primordia form around the shoot apical meristem, which consists of indeterminate stem cells. Upon initiation of leaf development, adaxial-abaxial patterning is crucial for appropriate lateral expansion, via cellular proliferation, and the formation of flat symmetric leaves. Many genes that specify such patterning have been identified, but regulation by upstream factors of the expression of relevant effector genes remains poorly understood. In Arabidopsis thaliana, ASYMMETRIC LEAVES2 (AS2) and AS1 play important roles in repressing transcription of class 1 KNOTTED1-like homeobox (KNOX) genes and leaf abaxial-determinant effector genes. We report here a mutation, designated enhancer of asymmetric leaves2 and asymmetric leaves1 (eal), that is associated with efficient generation of abaxialized filamentous leaves on the as2 or as1 background. Levels of transcripts of many abaxial-determinant genes, including ETTIN (ETT)/AUXIN RESPONSE FACTOR3 (ARF3), and all four class 1 KNOX genes were markedly elevated in as2 eal shoot apices. Rudimentary patterning in as2 eal leaves was suppressed by the ett mutation. EAL encodes BOBBER1 (BOB1), an Arabidopsis ortholog of eukaryotic NudC domain proteins. BOB1 was expressed in plant tissues with division potential and bob1 mutations resulted in lowered levels of transcripts of some cell-cycle genes and decreased rates of cell division in shoot and root apices. Coordinated cellular proliferation, supported by BOB1, and repression of all class 1 KNOX genes, ETT/ARF3 by AS2 (AS1) and BOB1 might be critical for repression of the indeterminate state and of aberrant abaxialization in the presumptive adaxial domain of leaf primordia, which might ensure the formation of flat symmetric leaves.

A genetic link between epigenetic repressor AS1-AS2 and a putative small subunit processome in leaf polarity establishment of Arabidopsis.

ABSTRACT Although the DEAD-box RNA helicase family is ubiquitous in eukaryotes, its developmental role remains unelucidated. Here, we report that cooperative action between the Arabidopsis nucleolar protein RH10, an ortholog of human DEAD-box RNA helicase DDX47, and the epigenetic repressor complex of ASYMMETRIC-LEAVES1 (AS1) and AS2 (AS1-AS2) is critical to repress abaxial (ventral) genes ETT/ARF3 and ARF4, which leads to adaxial (dorsal) development in leaf primordia at shoot apices. Double mutations of rh10-1 and as2 (or as1) synergistically up-regulated the abaxial genes, which generated abaxialized filamentous leaves with loss of the adaxial domain. DDX47 is part of the small subunit processome (SSUP) that mediates rRNA biogenesis. In rh10-1 we found various defects in SSUP-related events, such as: accumulation of 35S/33S rRNA precursors; reduction in the 18S/25S ratio; and nucleolar hypertrophy. Double mutants of as2 with mutations of genes that encode other candidate SSUP-related components such as nucleolin and putative rRNA methyltransferase exhibited similar synergistic defects caused by up-regulation of ETT/ARF3 and ARF4. These results suggest a tight link between putative SSUP and AS1-AS2 in repression of the abaxial-determining genes for cell fate decisions for adaxial development. Summary: This paper reports the importance of cooperative action between the nucleus-localized epigenetic repressor and the nucleolus-localized proteins involved in ribosomal RNA processing for polarity establishment of Arabidopsis leaves.

Berberine enhances defects in the establishment of leaf polarity in asymmetric leaves1 and asymmetric leaves2 of Arabidopsis thaliana

Leaves develop as flat lateral organs from the indeterminate shoot apical meristem. The establishment of polarity along three-dimensional axes, proximal–distal, medial–lateral, and adaxial–abaxial axes, is crucial for the growth of normal leaves. The mutations of ASYMMETRIC LEAVES1 (AS1) and AS2 of Arabidopsis thaliana cause defects in repression of the indeterminate state and the establishment of axis formation in leaves. Although many mutations have been identified that enhance the adaxial–abaxial polarity defects of as1 and as2 mutants, the roles of the causative genes in leaf development are still unknown. In this study, we found that wild-type plants treated with berberine produced pointed leaves, which are often observed in the single mutants that enhance phenotypes of as1 and as2 mutants. The berberine-treated as1 and as2 mutants formed abaxialized filamentous leaves. Berberine, an isoquinoline alkaloid compound naturally produced in various plant sources, has a growth inhibitory effect on plants that do not produce berberine. We further showed that transcript levels of meristem-specific class 1 KNOX homeobox genes and abaxial determinant genes were increased in berberine-treated as1 and as2. Berberine treated plants carrying double mutations of AS2 and the large subunit ribosomal protein gene RPL5B showed more severe defects in polarity than did the as2 single mutant plants. We suggest that berberine inhibits (a) factor(s) that might be required for leaf adaxial cell differentiation through a pathway independent of AS1 and AS2. Multiple pathways might play important roles in the formation of flat symmetric leaves.

Mechanisms of T-DNA transfer and integration into plant chromosomes: role of vir B , vir D4 and vir E2 and a short interspersed repetitive element (SINE) from tobacco

The present paper describes two topics on the early events during the formation of crown gall. (1) To measure an efficiency of T-DNA transfer from Agrobacterium cells to plant nuclei, we have developed a simple procedure which relies on Agrobacterium-mediated transient expression of the intron-GUS gene in plant cells. The results of experiments by this procedure indicate that products encoded by the virB locus and by the virD4 gene are necessary for transfer of T-DNA. VirE2 protein is also required for efficient transfer of T-DNA, although it is not absolutely essential. (2) We found a new family of short interspersed repetitive element (SINE) around the T-DNA integration target sites in the tobacco genome. SINEs are one of retroposons that is thought to originate from a tRNA or its gene. The SINE designated here as the TS family is the first example of a SINE family of plant bearing the significant homology to specific tRNAs including a vertebrate tRNALys which is thought to be a cognate molecule of most animal SINEs. The TS family occurs in approximately 5.0 104 copies per tobacco genome at least. Correlation between the presence of the TS family in tobacco chromosomes and integration of T-DNA will be discussed.