Keiichiro Hiratsu

Repression Domains of Class II ERF Transcriptional Repressors Share an Essential Motif for Active Repression

We reported previously that three ERF transcription factors, tobacco ERF3 (NtERF3) and Arabidopsis AtERF3 and AtERF4, which are categorized as class II ERFs, are active repressors of transcription. To clarify the roles of these repressors in transcriptional regulation in plants, we attempted to identify the functional domains of the ERF repressor that mediates the repression of transcription. Analysis of the results of a series of deletions revealed that the C-terminal 35 amino acids of NtERF3 are sufficient to confer the capacity for repression of transcription on a heterologous DNA binding domain. This repression domain suppressed the intermolecular activities of other transcriptional activators. In addition, fusion of this repression domain to the VP16 activation domain completely inhibited the transactivation function of VP16. Comparison of amino acid sequences of class II ERF repressors revealed the conservation of the sequence motif L/FDLNL/F(x)P. This motif was essential for repression because mutations within the motif eliminated the capacity for repression. We designated this motif the ERF-associated amphiphilic repression (EAR) motif, and we identified this motif in a number of zinc-finger proteins from wheat, Arabidopsis, and petunia plants. These zinc finger proteins functioned as repressors, and their repression domains were identified as regions that contained an EAR motif.

AREB1 Is a Transcription Activator of Novel ABRE-Dependent ABA Signaling That Enhances Drought Stress Tolerance in Arabidopsis

ABSCISIC ACID–RESPONSIVE ELEMENT BINDING PROTEIN1 (AREB1) (i.e., ABF2) is a basic domain/leucine zipper transcription factor that binds to the abscisic acid (ABA)–responsive element (ABRE) motif in the promoter region of ABA-inducible genes. Here, we show that expression of the intact AREB1 gene on its own is insufficient to lead to expression of downstream genes under normal growth conditions. To overcome the masked transactivation activity of AREB1, we created an activated form of AREB1 (AREB1ΔQT). AREB1ΔQT-overexpressing plants showed ABA hypersensitivity and enhanced drought tolerance, and eight genes with two or more ABRE motifs in the promoter regions in two groups were greatly upregulated: late embryogenesis abundant class genes and ABA- and drought stress–inducible regulatory genes. By contrast, an areb1 null mutant and a dominant loss-of-function mutant of AREB1 (AREB1:RD) with a repression domain exhibited ABA insensitivity. Furthermore, AREB1:RD plants displayed reduced survival under dehydration, and three of the eight greatly upregulated genes were downregulated, including genes for linker histone H1 and AAA ATPase, which govern gene expression and multiple cellular activities through protein folding, respectively. Thus, these data suggest that AREB1 regulates novel ABRE-dependent ABA signaling that enhances drought tolerance in vegetative tissues.

The SUPERMAN protein is an active repressor whose carboxy-terminal repression domain is required for the development of normal flowers

SUPERMAN was identified as a putative regulator of transcription that acts in floral development, but its function remains to be clarified. We demonstrate here that SUPERMAN is an active repressor whose repression domain is located in the carboxy‐terminal region. Ectopic expression of SUPERMAN that lacked the repression domain resulted in a phenotype similar to that of superman mutants, demonstrating that the repression activity of SUPERMAN is essential for the development of normal flowers. Constitutive expression of SUPERMAN resulted in a severe dwarfism but did not affect cell size, indicating that SUPERMAN might regulate genes that are involved in cell division.

The Arabidopsis thaliana STYLISH1 Protein Acts as a Transcriptional Activator Regulating Auxin Biosynthesis

Biosynthesis of the plant hormone auxin must be tightly controlled. This work shows that the STYLISH1 protein of the plant species Arabidopsis thaliana plays an important role in this process by directly binding to and activating at least one of the auxin biosynthesis genes. The establishment and maintenance of auxin maxima in vascular plants is regulated by auxin biosynthesis and polar intercellular auxin flow. The disruption of normal auxin biosynthesis in mouse-ear cress (Arabidopsis thaliana) leads to severe abnormalities, suggesting that spatiotemporal regulation of auxin biosynthesis is fundamental for normal growth and development. We have shown previously that the induction of the SHORT-INTERNODES/STYLISH (SHI/STY) family member STY1 results in increased transcript levels of the YUCCA (YUC) family member YUC4 and also higher auxin levels and auxin biosynthesis rates in Arabidopsis seedlings. We have also shown previously that SHI/STY family members redundantly affect development of flowers and leaves. Here, we further examine the function of STY1 by analyzing its DNA and protein binding properties. Our results suggest that STY1, and most likely other SHI/STY members, are DNA binding transcriptional activators that target genes encoding proteins mediating auxin biosynthesis. This suggests that the SHI/STY family members are essential regulators of auxin-mediated leaf and flower development. Furthermore, the lack of a shoot apical meristem in seedlings carrying a fusion construct between STY1 and a repressor domain, SRDX, suggests that STY1, and other SHI/STY members, has a role in the formation and/or maintenance of the shoot apical meristem, possibly by regulating auxin levels in the embryo.

Loss of function of the HSFA9 seed longevity program

Gain of function approaches that have been published by our laboratory determined that HSFA9 (Heat Shock Factor A9) activates a genetic program contributing to seed longevity and to desiccation tolerance in plant embryos. We now evaluate the role(s) of HSFA9 by loss of function using different modified forms of HaHSFA9 (sunflower HSFA9), which were specifically overexpressed in seeds of transgenic tobacco. We used two inactive forms (M1, M2) with deletion or mutation of the transcription activation domain of HaHSFA9, and a third form (M3) with HaHSFA9 converted to a potent active repressor by fusion of the SRDX motif. The three forms showed similar protein accumulation in transgenic seeds; however, only HaHSFA9-SRDX showed a highly significant reduction of seed longevity, as determined by controlled deterioration tests, a rapid seed ageing procedure. HaHSFA9-SRDX impaired the genetic program controlled by the tobacco HSFA9, with a drastic reduction in the accumulation of seed heat shock proteins (HSPs) including seed-specific small HSP (sHSP) belonging to cytosolic (CI, CII) classes. Despite such effects, the HaHSFA9-SRDX seeds could survive developmental desiccation during embryogenesis and their subsequent germination was not reduced. We infer that the HSFA9 genetic program contributes only partially to seed-desiccation tolerance and longevity.

Aberrant vegetative and reproductive development by overexpression and lethality by silencing of OsHAP3E in rice

We generated transgenic rice plants overexpressing OsHAP3E which encodes a subunit of a CCAAT-motif binding HAP complex. The OsHAP3E-overexpressing plants showed various abnormal morphologies both in their vegetative and reproductive phases. The OsHAP3E-overexpressing plants were dwarf with erected leaves and similar to brassinosteroid mutants in the vegetative phase. In the reproductive phase, dense panicle was developed, and occasionally successive generation of lateral rachises and formation of double flowers were observed. These phenotypes indicate association of OsHAP3E with determination of floral meristem identity. On the other hand, repression of OsHAP3E by RNAi or by overexpressing chimeric repressor fusion constructs brought about lethality to transformed cells, and almost no transformant was obtained. This suggests that the OsHAP3E function is essential for rice cells. Altogether, our loss-of-function and gain-of-function analyses suggest that OsHAP3E plays important pleiotropic roles in vegetative and reproductive development or basic cellular processes in rice.

Loss of function of the HSFA9 seed longevity program: HSFA9 loss of function

Gain of function approaches that have been published by our laboratory determined that HSFA9 (Heat Shock Factor A9) activates a genetic program contributing to seed longevity and to desiccation tolerance in plant embryos. We now evaluate the role(s) of HSFA9 by loss of function using different modified forms of HaHSFA9 (sunflower HSFA9), which were specifically overexpressed in seeds of transgenic tobacco. We used two inactive forms (M1, M2) with deletion or mutation of the transcription activation domain of HaHSFA9, and a third form (M3) with HaHSFA9 converted to a potent active repressor by fusion of the SRDX motif. The three forms showed similar protein accumulation in transgenic seeds; however, only HaHSFA9-SRDX showed a highly significant reduction of seed longevity, as determined by controlled deterioration tests, a rapid seed ageing procedure. HaHSFA9-SRDX impaired the genetic program controlled by the tobacco HSFA9, with a drastic reduction in the accumulation of seed heat shock proteins (HSPs) including seed-specific small HSP (sHSP) belonging to cytosolic (CI, CII) classes. Despite such effects, the HaHSFA9-SRDX seeds could survive developmental desiccation during embryogenesis and their subsequent germination was not reduced. We infer that the HSFA9 genetic program contributes only partially to seed-desiccation tolerance and longevity.