Hidekazu Iwakawa

Histone Deacetylases and ASYMMETRIC LEAVES2 Are Involved in the Establishment of Polarity in Leaves of Arabidopsis

We show that two Arabidopsis thaliana genes for histone deacetylases (HDACs), HDT1/HD2A and HDT2/HD2B, are required to establish leaf polarity in the presence of mutant ASYMMETRIC LEAVES2 (AS2) or AS1. Treatment of as1 or as2 plants with inhibitors of HDACs resulted in abaxialized filamentous leaves and aberrant distribution of microRNA165 and/or microRNA166 (miR165/166) in leaves. Knockdown mutations of these two HDACs by RNA interference resulted in phenotypes like those observed in the as2 background. Nuclear localization of overproduced AS2 resulted in decreased levels of mature miR165/166 in leaves. This abnormality was abolished by HDAC inhibitors, suggesting that HDACs are required for AS2 action. A loss-of-function mutation in HASTY, encoding a positive regulator of miRNA levels, and a gain-of-function mutation in PHABULOSA, encoding a determinant of adaxialization, suppressed the generation of abaxialized filamentous leaves by inhibition of HDACs in the as1 or as2 background. AS2 and AS1 were colocalized in subnuclear bodies adjacent to the nucleolus where HDT1/HD2A and HDT2/HD2B were also found. Our results suggest that these HDACs and both AS2 and AS1 act independently to control levels and/or patterns of miR165/166 distribution and the development of adaxial-abaxial leaf polarity and that there may be interactions between HDACs and AS2 (AS1) in the generation of those miRNAs.

Characterization of genes in the ASYMMETRIC LEAVES2/LATERAL ORGAN BOUNDARIES (AS2/LOB) family in Arabidopsis thaliana, and functional and molecular comparisons between AS2 and other family members.

The ASYMMETRIC LEAVES2 (AS2) gene is required for the generation of the flat and symmetrical shape of the leaf lamina in Arabidopsis. AS2 encodes a plant-specific protein with an AS2/LATERAL ORGAN BOUNDARIES (AS2/LOB) domain that includes a cysteine repeat, a conserved single glycine residue and a leucine-zipper-like sequence in its amino-terminal half. The Arabidopsis genome contains 42 genes, including AS2, that encode proteins with an AS2/LOB domain in their amino-terminal halves, and these genes constitute the AS2/LOB gene family. In the present study, we cloned and characterized cDNAs that covered the putative coding regions of all members of this family, and investigated patterns of transcription systematically in Arabidopsis plants. Comparisons among amino acid sequences that had been deduced from the cloned cDNAs revealed eight groups of genes, with two or three members each, and high degrees of identity among entire amino acid sequences, suggesting that some members of the AS2/LOB family might have redundant function(s). Moreover, no member of the family exhibited significant similarity, in terms of the deduced amino acid sequence of the carboxy-terminal half, to AS2. Results of domain swapping between AS2 and other members of the family showed that the AS2/LOB domain of AS2 cannot be functionally replaced by those of other members of the family, and that only a few dissimilarities among respective amino acid residues of the AS2/LOB domain of AS2 and those of other members are important for the specific functions of AS2.

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.

Transcriptional activation of tobacco E2F is repressed by co-transfection with the retinoblastoma-related protein: Cyclin D expression overcomes this repressor activity.

Evidence is emerging that the E2F family of transcription factors plays an important role in the regulation of gene expression at the G1/S transition in plants. Here, we show that in the tobacco proliferating cell nuclear antigen (PCNA), whose transcript is specifically expressed at G1/S phase, the two E2F binding sites are synergistically responsible for transcriptional activation at G1/S phase in synchronized tobacco BY-2 cells transformed with promoter constructs fused to a reporter gene. In addition, we have isolated the tobacco DP cDNA (NtDP) and showed that significant activation of the reporter gene was observed in transient expression assays by concomitantly transfecting with plasmids expressing NtE2F and NtDP. This transcriptional activation was repressed by co-transfection with a plasmid expressing NtRBR1; in vitro pull-down assays also revealed that NtRBR1 binds directly to NtE2F, thereby potentially blocking the transcriptional activation of NtE2F. Importantly, this repressor activity was cancelled when NtRBR1 was further co-transfected with a plasmid expressing cyclin D but not with cyclin A or cyclin B. These results are discussed with respect to the repression activity of NtRBR1 on the NtE2F/NtDP complex.

Knowledge-based Fuzzy Adaptive Resonance Theory and Its Application to the Analysis of Gene Expression in Plants

Gene expression data obtained from DNA microarrays are very useful in revealing the mechanisms that drive life. It is necessary to analyze these data through the use of algorithms, as in clustering and machine-learning. In a previous study, we developed fuzzy adaptive resonance theory (FuzzyART) and applied it to gene expression data, to identify genetic networks. FuzzyART was used as a clustering algorithm that is very suitable for the analysis of biological data; however, although FuzzyART is very useful in the analysis of dozens of gene expression profiles, it is difficult to apply this method to thousands of gene expression profiles, owing to inherent category proliferation and long calculation time. In the present study, we developed a knowledge-based FuzzyART (KB-FuzzyART) to mitigate these problems. We first constructed a gene list-1 from the gene database of Arabidopsis thaliana as knowledge for KB-FuzzyART, because KB-FuzzyART requires any knowledge as input. This method was applied to gene expression data obtained via the microarray analysis of A. thaliana, to identify the downstream genes of ASYMMETRIC LEAVES1 (AS1) and ASYMMETRIC LEAVES2 (AS2), both of which are involved in leaf development. The results of the analysis using KB-FuzzyART showed that the KNAT6 and YABBY5 (YAB5) genes are candidates for downstream factors, after a short calculation time for analysis. These results suggest that our gene list-1 is a very useful database for analyzing the expression profiles of genes that are related to the development of A. thaliana; they also suggest that the KB-FuzzyART has the high potential to function as a new method by which one can select candidate genes from thousands of genes, using gene expression data on mutant strains.

High-frequency genetic transformation of Phalaenopsis amabilis orchid using tomato extract-enriched medium for the pre-culture of protocorms

Summary The development of an efficient methodology for the genetic transformation of orchids is needed in order to support thegenetic engineering of orchids. It is therefore important to identify those factors affecting the transformation process.Previously, we reported a convenient method for the transformation of Phalaenopsis amabilis using Agrobacterium tumefaciens, in which intact protocorms were used. We also found that embryos cultured on a medium containing tomato extract grew more rapidly than those cultured on a medium with coconut water. When we used protocorms grown on a medium containing tomato extract, we obtained regenerated shoots that had been transformed with a kanamycin resistance gene at relatively high frequencies (7 – 17%). These results suggest that the rate of growth of pre-cultured protocorms may be important for the successful regeneration of transformed shoots. We also obtained regenerated shoots that had been transformed with the green fluorescent protein (GFP) gene at a high frequency (10 – 14%). Both the presence and expression of these transgenes were confirmed in transformed plants by molecular analyses and by the detection of green fluorescence following excitation with blue light.

Formation of a Symmetric Flat Leaf Lamina in Arabidopsis

The ASYMMETRIC LEAVESI (AS1) and ASYMMETRIC LEAVES2 (AS2) genes of Arabidopsis thatiana are involved in the establishment of the leaf venation system, which includes the prominent midvein, as well as in the development of a symmetric lamina. The gene product also represses the expression of class 1 knox homeobox genes in leaves. We have characterized the AS2 gene, which appears to encode a novel protein with cysteine repeats (designated the C-motif), conserved glycine, and a leucine-zipper-like sequence in the amino-terminal half of the primary sequence. The Arabidopsis genome contains 42 putative genes that potentially encode proteins with conserved amino acid sequences in the amino-terminal half. Thus, the AS2 protein belongs to a novel family of proteins that we have designated the AS2 family. Members of this family except AS2 also have been designated ASLs (AS2-like proteins). Overexpression of AS2 cDNA in transgenic Arabidopsis plants resulted in upwardly curled leaves, which differed markedly from the downwardly curled leaves generated by loss-of-function mutation of AS2. Our results suggest that AS2 functions in the transcription of a certain gene(s) in plant nuclei and thereby controls the formation of a symmetric flat leaf lamina and the establishment of a prominent midvein and other patterns of venation.

The Asymmetric Leaves2 (AS2) Gene of Arabidopsis Thaliana Regulates Lamina Formation and is Required for Patterning of Leaf Venation

ABSTRACT To understand the molecular mechanisms behind symmetrical development of leaf, we have analyzed the asymmetric leaves2 (as2) mutant of A. thaliana, which generated leaf lobes and leaflet-like structures from the petioles of leaves in a bilaterally asymmetric manner. The delayed formation of the primary vein and the asymmetric formation of secondary veins were apparent in leaf primordia of as2 plants. A distinct midvein, which is the thickest vein and is located in the longitudinal center of the leaf lamina of wild-type plants, was often rudimentary even in mature as2 leaves. However, several parallel veins of very similar thickness were evident in such leaves. The malformed veins were visible prior to the development of asymmetry of the leaf lamina, and were maintained in the mature as2 leaves. Culture in vitro on phytohormone-free medium of leaf sections from the as2 mutants and from the asymmetric leavesl (as1) mutant, which has a phenotype similar to that of as2, revealed an elevated potential in both cases for regeneration of shoots from leaf cells. Analysis by the reverse transcription-polymerase chain reaction showed that AS2 and AS1 negatively regulates the homeobox genes KNAT1, KNAT2 and KNAT6 in leaves. Taken together, our results suggest that AS2 and AS1 are involved in establishment of leaf venation and the formation of symmetric leaf lamina, which might be related to repression of expression of the homeobox genes in leaves.