Akiko Kozaki

The maize INDETERMINATE1 flowering time regulator defines a highly conserved zinc finger protein family in higher plants.

BackgroundThe maize INDETERMINATE1 gene, ID1, is a key regulator of the transition to flowering and the founding member of a transcription factor gene family that encodes a protein with a distinct arrangement of zinc finger motifs. The zinc fingers and surrounding sequence make up the signature ID domain (IDD), which appears to be found in all higher plant genomes. The presence of zinc finger domains and previous biochemical studies showing that ID1 binds to DNA suggests that members of this gene family are involved in transcriptional regulation.ResultsComparison of IDD genes identified in Arabidopsis and rice genomes, and all IDD genes discovered in maize EST and genomic databases, suggest that ID1 is a unique member of this gene family. High levels of sequence similarity amongst all IDD genes from maize, rice and Arabidopsis suggest that they are derived from a common ancestor. Several unique features of ID1 suggest that it is a divergent member of the maize IDD family. Although no clear ID1 ortholog was identified in the Arabidopsis genome, highly similar genes that encode proteins with identity extending beyond the ID domain were isolated from rice and sorghum. Phylogenetic comparisons show that these putative orthologs, along with maize ID1, form a group separate from other IDD genes. In contrast to ID1 mRNA, which is detected exclusively in immature leaves, several maize IDD genes showed a broad range of expression in various tissues. Further, Western analysis with an antibody that cross-reacts with ID1 protein and potential orthologs from rice and sorghum shows that all three proteins are detected in immature leaves only.ConclusionComparative genomic analysis shows that the IDD zinc finger family is highly conserved among both monocots and dicots. The leaf-specific ID1 expression pattern distinguishes it from other maize IDD genes examined. A similar leaf-specific localization pattern was observed for the putative ID1 protein orthologs from rice and sorghum. These similarities between ID1 and closely related genes in other grasses point to possible similarities in function.

Activity of transcription factor JACKDAW is essential for SHR/SCR-dependent activation of SCARECROW and MAGPIE and is modulated by reciprocal interactions with MAGPIE, SCARECROW and SHORT ROOT

Two GRAS family transcription factors, SHORT-ROOT (SHR) and SCARECROW (SCR), are required for ground tissue and quiescent center formation in Arabidopsis roots. The action of SHR and SCR is regulated by two INDETERMINATE DOMAIN (IDD) family proteins, JACKDAW (JKD) and MAGPIE (MGP). Although the reciprocal interaction of these transcription factors is considered to be involved in the modulation of SHR and SCR action by JKD and MGP, the underlying mechanism remains unclear. In this study, we use a transient assay with Arabidopsis culture cells to show that the physical interaction of these transcription factors modulate their transcriptional activity. Transient expression of LUC reporter genes with the proximal sequences upstream from the ATG codon of SCR and MGP in protoplasts were activated by JKD. Moreover, promoter activities were enhanced further by the addition of SHR and SCR to JKD, but not by the combination of SHR and SCR in the absence of JKD. Yeast one-hybrid analysis showed that JKD binds to the SCR and MGP promoter sequences, indicating the existence of another binding sequences of JKD different from the previously determined IDD binding sequence. Our findings suggest that JKD directly regulates SCR and MGP expression in cooperation with SHR, SCR and MGP.

Evolutionary conservation of TORC1 components, TOR, Raptor, and LST8, between rice and yeast

Target of rapamycin (TOR) is a conserved eukaryotic serine/threonine kinase that functions as a central controller of cell growth. TOR protein is structurally defined by the presence several conserved domains such as the HEAT repeat, focal adhesion target (FAT), FKBP12/rapamycin binding (FRB), kinase, and FATC domains starting from the N-terminus. In most eukaryotes, TOR forms two distinct physical and functional complexes, which are termed as TOR complex 1 (TORC1) and TORC2. However, plants contain only TORC1 components, i.e., TOR, Raptor, and LST8. In this study, we analyzed the gene structure and functions of TORC components in rice to understand the properties of the TOR complex in plants. Comparison of the locations of introns in these genes among rice and other eukaryotes showed that they were well conserved among plants except for Chlamydomonas. Moreover, the intron positions in the coding sequence of human Raptor and LST8 were closer to those of plants than of fly or nematode. Complementation tests of rice TOR (OsTOR) components in yeast showed that although OsTOR did not complement yeast tor mutants, chimeric TOR, which consisted of the HEAT repeat and FAT domain from yeast and other regions from rice, rescued the tor mutants, indicating that the HEAT repeat and FAT domains are important for species-specific signaling. OsRaptor perfectly complemented a kog1 (yeast Raptor homolog) mutant, and OsLST8 partially complemented an lst8 mutant. Together, these data suggest the importance of the N-terminal region of the TOR, HEAT, and FAT domains for functional diversification of the TOR complex.

Design of a Seed-Specific Chimeric Promoter with a Modified Expression Profile to Improve Seed Oil Content

Increasing the yield of plant oil is an important objective to meet the demand for sustainable resources and energy. Some attempts to enhance the expression of genes involved in oil synthesis in seeds have succeeded in increasing oil content. In many cases, the promoters of seed-storage protein genes have been used as seed-specific promoters. However, conventional promoters are developmentally regulated and their expression periods are limited. We constructed a chimeric promoter that starts to express in the early stage of seed development, and high-level expression is retained until the later stage by connecting the promoters of the biotin carboxyl carrier protein 2 (BCCP2) gene encoding the BCCP2 subunit of acetyl-CoA carboxylase and the fatty acid elongase 1 (FAE1) gene from Arabidopsis. The constructed promoter was ligated upstream of the TAG1 gene encoding diacylglycerol acyltransferase 1 and introduced into Arabidopsis. Seeds from transgenic plants carrying AtTAG1 under the control of the chimeric promoter showed increased oil content (up by 18–73%) compared with wild-type seeds. The novel expression profile of the chimeric promoter showed that this could be a promising strategy to manipulate the content of seed-storage oils and other compounds.

Plant S6 kinases do not require hydrophobic motif phosphorylation for activity in yeast lacking Ypk3

The ribosomal protein S6 kinases (S6K) are among the major substrates and crucial effectors of the target of rapamycin (TOR) kinase, which is an evolutionarily conserved regulator of cell growth and proliferation. Recent research indicates that yeast Ypk3 is an ortholog of mammalian S6Ks. Here, we find that plant S6Ks restore ribosomal protein S6 phosphorylation in a rapamycin‐sensitive manner in yeast cells lacking Ypk3. However, phosphorylation of a hydrophobic motif, which is mediated through TOR signaling and essential for mammalian S6K activity, is not detected in plant S6Ks. Furthermore, deletion of the N‐terminal region of rice S6Ks shows phosphorylation of the hydrophobic motif and reduced rapamycin sensitivity. Our findings suggest a mechanism of plant S6K activation distinct from that of mammalian S6Ks.