[Show abstract][Hide abstract] ABSTRACT: We recently demonstrated that cell elongation in plants is regulated by a triantagonistic bHLH system, in which three bHLH proteins, Activator of Cell Elongation 1 (ACE1), Arabidopsis ILI1 binding BHLH 1 (AtIBH1) and Paclobutrazol Resistance 1 (PRE1), competitively regulate the expression of genes for cell elongation. Here we show that ATBS1 Interacting Factor 2 (AIF2), AIF3 and AIF4 interact with PRE1 and ACE1, similar to AtIBH1, and also negatively regulate cell elongation in the triantagonistic bHLH system. The expression of each AIF is constitutive or induced by light, but AtIBH1 expression is dependent on BR signaling and developmental phase. These results indicate that AIFs and AtIBH1 may play different roles in cell elongation in different signaling pathways.
[Show abstract][Hide abstract] ABSTRACT: In plants, basic helix-loop-helix (bHLH) transcription factors play important roles in the control of cell elongation. Two bHLH proteins, PACLOBTRAZOL RESISTANCE1 (PRE1) and Arabidopsis ILI1 binding bHLH1 (IBH1), antagonistically regulate cell elongation in response to brassinosteroid and gibberellin signaling, but the detailed molecular mechanisms by which these factors regulate cell elongation remain unclear. Here, we identify the bHLH transcriptional activators for cell elongation (ACEs) and demonstrate that PRE1, IBH1, and the ACEs constitute a triantagonistic bHLH system that competitively regulates cell elongation. In this system, the ACE bHLH transcription factors directly activate the expression of enzyme genes for cell elongation by interacting with their promoter regions. IBH1 negatively regulates cell elongation by interacting with the ACEs and thus interfering with their DNA binding. PRE1 interacts with IBH1 and counteracts the ability of IBH1 to affect ACEs. Therefore, PRE1 restores the transcriptional activity of ACEs, resulting in induction of cell elongation. The balance of triantagonistic bHLH proteins, ACEs, IBH1, and PRE1, might be important for determination of the size of plant cells. The expression of IBH1 and PRE1 is regulated by brassinosteroid, gibberellins, and developmental phase dependent factors, indicating that two phytohormones and phase-dependent signals are integrated by this triantagonistic bHLH system.
[Show abstract][Hide abstract] ABSTRACT: Many eukaryotes have from one to three heat shock factors (Hsfs), but plants have more than 20 Hsfs, designated class A, B, and C. Class A Hsfs are activators of transcription, but details of the roles of individual Hsfs have not been fully characterized. We show here that Arabidopsis (Arabidopsis thaliana) HsfB1 and HsfB2b, members of class B, are transcriptional repressors and negatively regulate the expression of heat-inducible Hsfs (HsfA2, HsfA7a, HsfB1, and HsfB2b) and several heat shock protein genes. In hsfb1 hsfb2b double mutant plants, the expression of a large number of heat-inducible genes was enhanced in the non-heat condition (23°C) and the plants exhibited slightly higher heat tolerance at 42°C than the wild type, similar to Pro35S:HsfA2 plants. In addition, under extended heat stress conditions, expression of the heat-inducible Hsf genes remained consistently higher in hsfb1 hsfb2b than in the wild type. These data indicate that HsfB1 and HsfB2b suppress the general heat shock response under non-heat-stress conditions and in the attenuating period. On the other hand, HsfB1 and HsfB2b appear to be necessary for the expression of heat stress-inducible heat shock protein genes under heat stress conditions, which is necessary for acquired thermotolerance. We show that the heat stress response is finely regulated by activation and repression activities of Hsfs in Arabidopsis.
[Show abstract][Hide abstract] ABSTRACT: Heat shock transcription factor A2 (HsfA2) acts as a key component of the Hsf signaling network involved in cellular responses to various types of environmental stress. However, the mechanism governing the regulation of HsfA2 expression is still largely unknown. We demonstrated here that a heat shock element (HSE) cluster in the 5'-flanking region of the HsfA2 gene is involved in high light (HL)-inducible HsfA2 expression. Accordingly, to identify the Hsf regulating the expression of HsfA2, we analyzed the effect of loss-of-function mutations of class A Hsfs on the expression of HsfA2 in response to HL stress. Overexpression of an HsfA1d or HsfA1e chimeric repressor and double knockout of HsfA1d and HsfA1e Arabidopsis mutants (KO-HsfA1d/A1e) significantly suppressed the induction of HsfA2 expression in response to HL and heat shock (HS) stress. Transient reporter assays showed that HsfA1d and HsfA1e activate HsfA2 transcription through the HSEs in the 5'-flanking region of HsfA2. In the KO-HsfA1d/A1e mutants, 560 genes, including a number of stress-related genes and several Hsf genes, HsfA7a, HsfA7b, HsfB1 and HsfB2a, were down-regulated compared with those in the wild-type plants under HL stress. The PSII activity of KO-HsfA1d/A1e mutants decreased under HL stress, while the activity of wild-type plants remained high. Furthermore, double knockout of HsfA1d and HsfA1e impaired tolerance to HS stress. These findings indicated that HsfA1d and HsfA1e not only regulate HsfA2 expression but also function as key regulators of the Hsf signaling network in response to environmental stress.
Plant and Cell Physiology 04/2011; 52(5):933-45. · 4.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chimeric REpressor gene Silencing Technology (CRES-T) is a useful tool for functional analysis of plant transcription factors. In this system, a chimeric repressor that is produced by fusion of a transcription factor to the plant-specific EAR-motif repression domain (SRDX) suppresses target genes of a transcription factor dominantly over the activity of endogenous and functionally redundant transcription factors. As a result, the transgenic plants that express a chimeric repressor exhibit phenotypes similar to loss-of-function of the alleles of the gene encoding the transcription factor. This system is simple and effective and can be used as a powerful tool not only for functional analysis of redundant transcription factors but also for the manipulation of plant traits by active suppression of the gene expression. Strategies for construction of the chimeric repressors and their expression in transgenic plants are described. Transient effector-reporter assays for functional analysis of transcription factors and detection of protein-protein interactions using the trans-repressive activity of SRDX repression domain are also described.
[Show abstract][Hide abstract] ABSTRACT: Yeast one-hybrid screening is widely used for the identification of transcription factors (TFs) that interact with specific DNA sequences. However, screening a whole cDNA library is not efficient for the identification of TFs because TF genes represent only a small percentage of clones in a cDNA library. Here, we present the development of an efficient yeast one-hybrid screening system using a prey library composed only of approximately 1,500 TF cDNAs of Arabidopsis thaliana. This library enabled us to isolate a TF that binds to a specific promoter sequence with high efficiency, even when the promoter region of the gene of interest was directly employed as bait. Furthermore, this library was also successfully applied as a yeast two-hybrid library to find TFs that interact with specific proteins. This efficient system will contribute to the elucidation of gene regulatory networks in plants.
Plant and Cell Physiology 10/2010; 51(12):2145-51. · 4.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Silene latifolia is a model dioecious plant with morphologically distinguishable XY sex chromosomes. The end of the Xq arm is quite different from that of the Yp arm, although both are located at opposite ends of their respective chromosomes relative to a pseudo-autosomal region. The Xq arm does not seem to originate from the same autosome as the Yp arm. Bacterial artificial chromosome clone #15B12 has an insert containing a 130-kb stretch in which a 313-bp satellite DNA is repeated 420 times. PCR with a single primer revealed that this 130-kb stretch consists of three reversals of the orientation of the satellite DNA. A non-long terminal repeat retroelement and two sequences that share homology with an Oryza sativa RING zinc finger and a putative Arabidopsis thaliana protein, respectively, were found in the sequences that flank the satellite DNA. Fluorescence in situ hybridization carried out using this low-copy region of #15B12 as a probe confirmed that these sequences originated from the X chromosome and that homologous sequences exist at the end of chromosome 7. The region distal to DD44X on the Xq arm is postulated to have recombined with a region containing satellite DNA on chromosome 7 during the process of sex chromosome evolution.
[Show abstract][Hide abstract] ABSTRACT: Most transcription factors act either as activators or repressors, and no such factors with dual function have been unequivocally identified and characterized in plants. We demonstrate here that the Arabidopsis thaliana protein WUSCHEL (WUS), which regulates the maintenance of stem cell populations in shoot meristems, is a bifunctional transcription factor that acts mainly as a repressor but becomes an activator when involved in the regulation of the AGAMOUS (AG) gene. We show that the WUS box, which is conserved among WOX genes, is the domain that is essential for all the activities of WUS, namely, for regulation of stem cell identity and size of floral meristem. All the known activities of WUS were eliminated by mutation of the WUS box, including the ability of WUS to induce the expression of AG. The mutation of the WUS box was complemented by fusion of an exogenous repression domain, with resultant induction of somatic embryogenesis in roots and expansion of floral meristems as observed upon ectopic expression of WUS. By contrast, fusion of an exogenous activation domain did not result in expanded floral meristems but induced flowers similar to those induced by the ectopic expression of AG. Our results demonstrate that WUS acts mainly as a repressor and that its function changes from that of a repressor to that of an activator in the case of regulation of the expression of AG.
The Plant Cell 11/2009; 21(11):3493-505. · 9.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We showed previously that the ERF-associated amphiphilic repression (EAR) motif is a plant-specific repression domain that contains the conserved amino acid sequence LXLXL. In this report, we describe the identification of a novel repression domain, L/VR/KLFGVXM/V/L, which is different from known EAR motifs, in B3 DNA-binding domain transcription factors in Arabidopsis. Database analysis revealed that 29 Arabidopsis transcription factors, which included members of the RAV, ARF, Hsf and MYB families, contain the R/KLFGV conserved motif found in the novel repression domain. We demonstrated that factors that contain the R/KLFGV motif, namely, RAV1, RAV2, HsfB1 and HsfB2b, exhibited the repressive activity.
Plant and Cell Physiology 04/2009; 50(5):970-5. · 4.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In seed plants, embryogenesis is an important process to produce a new generation. It comprises three steps: establishment of organization as an embryo, accumulation of storage substances in the embryo, and acquisition of desiccation tolerance and seed dormancy. These steps are accurately regulated by many factors, including phytohormones, proteins, transcription factors, and other substances related to embryogenesis. The embryogenesis mechanism has been analyzed through biochemical, biological, and molecular approaches using embryo-defective mutants or somatic embryogenesis whose traits are similar to zygotic embryogenesis, both morphologically and physiologically. Appropriate auxin transport plays an important role in the formation of cotyledon and meristem during early embryo-genesis. Some transcription factors (LEC1, ABI3, LEC2, and FUS3) that have been isolated from embryo-defective mutants are charac-terized as embryo-related genes. Among them, some transcription factors are related to phytohormone signaling. ABI3 and ABA regulate the expression of the LEA gene, whose proteins are accumulated during late embryogenesis. Also, LEC2 and FUS3 negatively regulate bioactive GA synthesis. On the other hand, some regulatory factors have been isolated and identified from culture medium during somatic embryogenesis. The factors are low molecular substances such as the phenolic compounds (4HBA, VBE and 4PMP) that inhibit somatic embryogenesis, or the peptidyl growth factor, PSK, which stimulates somatic embryogenesis. Here, we review recent findings of various factors regulating plant embryogenesis.
[Show abstract][Hide abstract] ABSTRACT: Somatic embryogenesis has been used as amodel system to understand the mechanisms regulating
plant embryogenesis. The morphological and physiological characteristics of somatic embryos are similar
to those of zygotic embryos. However, what are the patterns of gene expression during somatic embryogenesis?
Here, we review molecular events involved in embryogenesis. Four important transcription factors
were isolated from adefective-embryo mutant (LEC1, LEC2, FUS3
and ABI3), and three factors were isolated using deferential screening
(SERK, AGL15 and BBM);
all are expressed during both somatic and zygotic embryo development. These genes may be important
in regulating phytohormone synthesis and phytohormone response during somatic and zygotic embryogenesis.
Regulation of embryo-specific LEA gene expression is similar in
both somatic and zygotic embryos. Recent research involves examination of new mutants that form embryonic
[Show abstract][Hide abstract] ABSTRACT: When seed coats (pericarps) were picked from 14-day-old carrot (Daucus carota) seedlings and cultured on agar plates, embryogenic cell clusters were produced very rapidly at a high frequency on the open side edge. Embryo induction progressed without auxin treatment; indeed treatment caused the formation of non-embryogenic callus. The embryogenic tissues (primary embryos) developed normally until the torpedo stage; however, after this a number of secondary somatic embryos were produced in the hypocotyl and root regions. "Tertiary" embryos were formed on some of the secondary embryos, but many developed into normal plantlets. The primary embryos contained significantly higher levels of abscisic acid (ABA) than the hypocotyl-derived normal and seed-coat-derived secondary embryos. Fluridone inhibited the induction of secondary embryogenesis, while exogenously supplied ABA induced not only "tertiary" embryogenesis on the seed-coat-derived secondary embryos, but also secondary embryos on the hypocotyl-derived normal somatic embryos. These results indicate that ABA is one of the important endogenous factors for the induction of secondary embryogenesis on carrot somatic embryos. Higher levels of indole-3-acetic acid (IAA) in primary embryos also suggest the presence of some concerted effect of ABA and IAA on the induction of secondary embryogenesis in primary embryos.
[Show abstract][Hide abstract] ABSTRACT: We examined the applicability of small and branching varieties of sunflower (Helianthus annuus L. cv. Sonja, Valentine, and Pacino) for plant regeneration and gene introduction. Some small and branching sunflower varieties have advantages as transformation materials. The frequency of shoot regeneration in MS� 0.1 mg l� 1 BA medium was high in Sonja and Valentine and many shoots formed on each explant. Roots formed easily from regenerated shoots in MS� 0.1 mg l� 1 NAA medium and the regenerated plants grew normally and formed flowers and seeds in Pacino. In addition, the introduction of genes into branching sunflowers was shown to be straightforward. About 20-50% of regenerated shoots displayed GUS expression over wide areas of tissue in all three varieties. In Pacino, some uniformly transformed shoots were observed after simple infection with Agrobacterium. All of the steps required to obtain transformants of branching sunflowers are simple and straightforward. These small and easily transformed sunflower varieties are therefore useful subjects for molecular genetic experiments.
[Show abstract][Hide abstract] ABSTRACT: Manipulation of horticultural plants' traits using genetic engineering has been a challenge because of gene redundancy and limited information concerning genome or other factors necessary for successful engineering. Recently we have developed a powerful tool with potential to overcome these difficulties, a novel gene silencing technology targeting transcription factor, which is designated Chimeric REpressor gene-Silencing Technology (CRES-T). Using this system, we are now analyzing biological functions of transcription factors in Arabidopsis and trying to manipulate morphological traits of various floricultural plants. To provide these information for genetic engineering of horticultural plants, we have developed the 'FioreDB' database in a web-based interface (http://www.cres-t.org/fiore/public_db/), which stores phenotypic information induced by various chimeric repressors in Arabidopsis and six floricultural plants, namely torenia, chrysanthemum, gentian, cyclamen, eustoma, morning glory. Users can find gene constructs that induce their preferred phenotype in Arabidopsis using simple searches, and can browse induced phenotypes in floricultural plants. Most phenotypic information has photo data. FioreDB is continually updated by addition of new data derived from the CRES-T analyses. FioreDB will help to improve traits of horticultural plants using the CRES-T system.