ACRE, a class of AP2/ERF transcription factors, activates the expression of sweet potato ß-amylase and sporamin genes through the sugar-responsible element CMSRE-1.

Sugars, synthesized by photosynthesis in source organs, are loaded and utilized as an energy source and carbon skeleton in sink organs, and also known to be important signal molecules regulating gene expression in higher plants. The expression of genes coding for sporamin and ß-amylase, the two most abundant proteins in storage roots of sweet potato, is coordinately induced by sugars. We previously reported on the identification of the carbohydrate metabolic signal-responsible element-1 (CMSRE-1) essential for the sugar-responsible expression of two genes. However, transcription factors that bind to this sequence have not been identified. In this study, we performed yeast one-hybrid screening using the sugar-responsible minimal promoter region of the ß-amylase gene as bait and a library composed only transcription factor cDNAs of Arabidopsis. Two clones, named Activator protein binding to CMSRE-1 (ACRE), encoding AP2/ERF transcription factors were isolated. ACRE showed transactivation activity of the sugar-responsible minimal promoter in a CMSRE-1-dependent manner in Arabidopsis protoplasts. Electric mobility shift assay (EMSA) using recombinant proteins and transient co-expression assay in Arabidopsis protoplasts revealed that ACRE could actually act to the CMSRE-1. Among the DEHYDRATION -RESPONSIVE ELEMENT BINDING FACTOR (DREB) subfamily, almost all homologs including ACRE, could act on the DRE, while only three ACREs could act to the CMSRE-1. Moreover, ACRE-homologs of Japanese morning glory also have the same property of DNA-binding preference and transactivation activity through the CMSRE-1. These findings suggested that ACRE plays an important role in the mechanism regulating the sugar-responsible gene expression through the CMSRE-1 conserved across plant species.

A hierarchical transcriptional network activates specific CDK inhibitors that regulate G2 to control cell size and number in Arabidopsis.

How cell size and number are determined during organ development remains a fundamental question in cell biology. Here, we identified a GRAS family transcription factor, called SCARECROW-LIKE28 (SCL28), with a critical role in determining cell size in Arabidopsis. SCL28 is part of a transcriptional regulatory network downstream of the central MYB3Rs that regulate G2 to M phase cell cycle transition. We show that SCL28 forms a dimer with the AP2-type transcription factor, AtSMOS1, which defines the specificity for promoter binding and directly activates transcription of a specific set of SIAMESE-RELATED (SMR) family genes, encoding plant-specific inhibitors of cyclin-dependent kinases and thus inhibiting cell cycle progression at G2 and promoting the onset of endoreplication. Through this dose-dependent regulation of SMR transcription, SCL28 quantitatively sets the balance between cell size and number without dramatically changing final organ size. We propose that this hierarchical transcriptional network constitutes a cell cycle regulatory mechanism that allows to adjust cell size and number to attain robust organ growth.

Gateway binary vectors with organelle-targeted fluorescent proteins for highly sensitive reporter assay in gene expression analysis of plants.

Fluorescent proteins are valuable tools in the bioscience field especially in subcellular localization analysis of proteins and expression analysis of genes. Fusion with organelle-targeting signal accumulates fluorescent proteins in specific organelles, increases local brightness, and highlights the signal of fluorescent proteins even in tissues emitting a high background of autofluorescence. For these advantages, organelle-targeted fluorescent proteins are preferably used for promoter:reporter assay to define organ-, tissue-, or cell-specific expression pattern of genes in detail. In this study, we have developed a new series of Gateway cloning technology-compatible binary vectors, pGWBs (attR1-attR2 acceptor sites) and R4L1pGWB (attR4-attL1 acceptor sites), carrying organelle-targeted synthetic green fluorescent protein with S65T mutation (sGFP) (ER-, nucleus-, peroxisome-, and mitochondria-targeted sGFP) and organelle-targeted tag red fluorescent protein (TagRFP) (nucleus-, peroxisome-, and mitochondria-targeted TagRFP). These are available for preparation of promoter:reporter constructs by an LR reaction with a promoter entry clone attL1-promoter-attL2 (for pGWBs) or attL4-promoter-attR1 (for R4L1pGWBs), respectively. A transient expression experiment with particle bombardment using cauliflower mosaic virus 35S promoter-driven constructs has confirmed the correct localization of newly developed organelle-targeted TagRFPs by a co-localization analysis with the previously established organelle-targeted sGFPs. More intense and apparent fluorescence signals were detected by the nucleus- and peroxisome-targeted sGFPs than by the normal sGFPs in the promoter assay using transgenic Arabidopsis thaliana. The new pGWBs and R4L1pGWBs developed here are highly efficient and may serve as useful platforms for more accurate observation of GFP and RFP signals in gene expression analyses of plants.

The PLETHORA Gene Regulatory Network Guides Growth and Cell Differentiation in Arabidopsis Roots.

Organ formation in animals and plants relies on precise control of cell state transitions to turn stem cell daughters into fully differentiated cells. In plants, cells cannot rearrange due to shared cell walls. Thus, differentiation progression and the accompanying cell expansion must be tightly coordinated across tissues. PLETHORA (PLT) transcription factor gradients are unique in their ability to guide the progression of cell differentiation at different positions in the growing Arabidopsis thaliana root, which contrasts with well-described transcription factor gradients in animals specifying distinct cell fates within an essentially static context. To understand the output of the PLT gradient, we studied the gene set transcriptionally controlled by PLTs. Our work reveals how the PLT gradient can regulate cell state by region-specific induction of cell proliferation genes and repression of differentiation. Moreover, PLT targets include major patterning genes and autoregulatory feedback components, enforcing their role as master regulators of organ development.

DELLA protein functions as a transcriptional activator through the DNA binding of the indeterminate domain family proteins.

DELLA protein is a key negative regulator of gibberellin (GA) signaling. Although how DELLA regulates downstream gene expression remains unclear, DELLA has been proposed to function as a transcriptional activator. However, because DELLA lacks a DNA-binding domain, intermediate protein(s) mediating the DELLA/DNA interaction are believed to be necessary for activating DELLA target genes. Here, using yeast hybrid screenings, we identified five members of indeterminate domain (IDD) protein family which bind physically to both DELLA and the promoter sequence of the GA-positive regulator SCARECROW-LIKE 3 (SCL3), which previously was characterized as a DELLA direct target gene. Transient assays using Arabidopsis protoplasts demonstrated that a luciferase reporter controlled by the SCL3 promoter was additively transactivated by REPRESSOR of ga1-3 (RGA) and IDDs. Phenotypic analysis of transgenic plants expressing AtIDD3 (one of the 16 IDDs in the Arabidopsis genome) fused with the plant-specific repression domain (SRDX) supported the possibility that AtIDD3 is positively involved in GA signaling. In addition, we found that SCL3 protein also interacts with IDDs, resulting in the suppression of its target gene expression. In this context, DELLA and SCL3 interact competitively with IDD proteins to regulate downstream gene expression. These results suggest that the coregulators DELLA and SCL3, using IDDs as transcriptional scaffolds for DNA binding, antagonistically regulate the expression of their downstream targets to control the GA signaling pathway.

Mutations in MYB3R1 and MYB3R4 cause pleiotropic developmental defects and preferential down-regulation of multiple G2/M-specific genes in Arabidopsis.

R1R2R3-Myb proteins represent an evolutionarily conserved class of Myb family proteins important for cell cycle regulation and differentiation in eukaryotic cells. In plants, this class of Myb proteins are believed to regulate the transcription of G2/M phase-specific genes by binding to common cis-elements, called mitosis-specific activator (MSA) elements. In Arabidopsis (Arabidopsis thaliana), MYB3R1 and MYB3R4 act as transcriptional activators and positively regulate cytokinesis by activating the transcription of KNOLLE, which encodes a cytokinesis-specific syntaxin. Here, we show that the double mutation myb3r1 myb3r4 causes pleiotropic developmental defects, some of which are due to deficiency of KNOLLE whereas other are not, suggesting that multiple target genes are involved. Consistently, microarray analysis of the double mutant revealed altered expression of many genes, among which G2/M-specific genes showed significant overrepresentation of the MSA motif and a strong tendency to be down-regulated by the double mutation. Our results demonstrate, on a genome-wide level, the importance of the MYB3R-MSA pathway for regulating G2/M-specific transcription. In addition, MYB3R1 and MYB3R4 may have diverse roles during plant development by regulating G2/M-specific genes with various functions as well as genes possibly unrelated to the cell cycle.

An AP2-type transcription factor, WRINKLED1, of Arabidopsis thaliana binds to the AW-box sequence conserved among proximal upstream regions of genes involved in fatty acid synthesis.

Although an APETALA2 (AP2)-type transcription factor, WRINKLED1 (WRI1), has been shown to be required for accumulation of triacylglycerols (TAGs) in Arabidopsis seeds, its direct target genes have not been established. Overexpression of WRI1 up-regulated a set of genes involved in fatty acid (FA) synthesis in plastids, including genes for a subunit of pyruvate kinase (Pl-PKbeta1), acetyl-CoA carboxylase (BCCP2), acyl carrier protein (ACP1), and ketoacyl-acyl carrier protein synthase (KAS1), while expression of these genes is reduced in mutants with reduced WRI1 expression. Transient expression of LUC reporter genes with the proximal sequences upstream from the ATG codon of Pl-PKbeta1, BCCP2, and KAS1 in protoplasts was activated by co-expression of WRI1, and recombinant WRI1 bound to these upstream sequences in vitro. The seven WRI1 binding sites shared a sequence [CnTnG](n)(7)[CG], where n is any nucleotide designated as the AW-box, and mutations in AW-boxes near the transcription start site and in the 5'-untranslated region of Pl-PKbeta1 abolished activation by WRI1 in protoplasts and expression during seed maturation. Although expression of genes for the synthesis of TAGs and packaging into oil bodies in the endoplasmic reticulum in developing seeds required WRI1, their expression was not up-regulated by WRI1 overexpression. Thus, WRI1 promotes the flow of carbon to oil during seed maturation by directly activating genes involved in FA synthesis and controlling genes for assembly and storage of TAG.

Improved Gateway binary vectors: high-performance vectors for creation of fusion constructs in transgenic analysis of plants.

We made a series of improved Gateway binary vectors (pGWBs) for plant transformation. Fifteen different reporters and tags, sGFP, GUS, LUC, EYFP, ECFP, G3GFP, mRFP, 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7, and TAP, were employed. Some vectors carry the 2x35S-Omega promoter for higher-level expression. The kanamycin- and hygromycin-resistant markers are independently available for each of the 43 types of vectors, thus an additional transformation of once-transformed plants can be carried out easily. Their small size and high-copy number in Escherichia coli make possible easier handling at plasmid preparation and sequencing. Improved pGWBs should be a powerful tool for transgenic research in plants.