Role of GARP family transcription factors in the regulatory network for nitrogen and phosphorus acquisition.

The GARP (Golden2, ARR-B, Psr1) family proteins with a conserved DNA-binding domain, called the B-motif, are plant-specific transcription factors involved in the regulation of various physiological processes. The GARP family proteins are divided into members that function as monomeric transcription factors, and members that function as transcription factors in the dimeric form, owing to the presence of a coiled-coil dimerization domain. Recent studies revealed that the dimer-forming GARP family members, which are further divided into the PHR1 and NIGT1 subfamilies, play critical roles in the regulation of phosphorus (P) and nitrogen (N) acquisition. In this review, we present a general overview of the GARP family proteins and discuss how several members of the PHR1 and NIGT1 subfamilies are involved in the coordinated acquisition of P and N in response to changes in environmental nutrient conditions, while mainly focusing on the recent findings that enhance our knowledge of the roles of PHR1 and NIGT1 in phosphate starvation signaling and nitrate signaling.

Differential requirement of MED14/17 recruitment for activation of heat inducible genes.

The mechanism of heat stress response in plants has been studied, focusing on the function of transcription factors (TFs). Generally, TFs recruit coactivators, such as Mediator, are needed to assemble the transcriptional machinery. However, despite the close relationship with TFs, how coactivators are involved in transcriptional regulation under heat stress conditions is largely unclear. We found a severe thermosensitive phenotype of Arabidopsis mutants of MED14 and MED17. Transcriptomic analysis revealed that a quarter of the heat stress (HS)-inducible genes were commonly downregulated in these mutants. Furthermore, chromatin immunoprecipitation assay showed that the recruitment of Mediator by HsfA1s, the master regulators of heat stress response, is an important step for the expression of HS-inducible genes. There was a differential requirement of Mediator among genes; TF genes have a high requirement whereas heat shock proteins (HSPs) have a low requirement. Furthermore, artificial activation of HsfA1d mimicking perturbation of protein homeostasis induced HSP gene expression without MED14 recruitment but not TF gene expression. Considering the essential role of MED14 in Mediator function, other coactivators may play major roles in HSP activation depending on the cellular conditions. Our findings highlight the importance of differential recruitment of Mediator for the precise control of HS responses in plants.

Cytosolic HSC70s repress heat stress tolerance and enhance seed germination under salt stress conditions.

Heat shock factor A1 (HsfA1) family proteins are the master regulators of the heat stress-responsive transcriptional cascade in Arabidopsis. Although 70 kDa heat shock proteins (HSP70s) are known to participate in repressing HsfA1 activity, the mechanisms by which they regulate HsfA1 activity have not been clarified. Here, we report the physiological functions of three cytosolic HSP70s, HSC70-1, HSC70-2 and HSC70-3, under normal and stress conditions. Expression of the HSC70 genes was observed in whole seedlings, and the HSC70 proteins were observed in the cytoplasm and nucleus under normal and stress conditions, as were the HsfA1s. hsc70-1/2 double and hsc70-1/2/3 triple mutants showed higher thermotolerance than the wild-type (WT) plants. Transcriptomic analysis revealed the upregulation of heat stress-responsive HsfA1-downstream genes in hsc70-1/2/3 mutants under normal growth conditions, demonstrating that these HSC70s redundantly function as repressors of HsfA1 activity. Furthermore, hsc70-1/2/3 plants showed a more severe growth delay during the germination stage than the WT plants under high-salt stress conditions, and many seed-specific cluster 2 genes that exhibited suppressed expression during germination were expressed in hsc70-1/2/3 plants, suggesting that these HSC70s also function in the developmental transition from seed to seedling under high-salt conditions by suppressing the expression of cluster 2 genes.

BPM-CUL3 E3 ligase modulates thermotolerance by facilitating negative regulatory domain-mediated degradation of DREB2A in Arabidopsis.

DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2A (DREB2A) acts as a key transcription factor in both drought and heat stress tolerance in Arabidopsis and induces the expression of many drought- and heat stress-inducible genes. Although DREB2A expression itself is induced by stress, the posttranslational regulation of DREB2A, including protein stabilization, is required for its transcriptional activity. The deletion of a 30-aa central region of DREB2A known as the negative regulatory domain (NRD) transforms DREB2A into a stable and constitutively active form referred to as DREB2A CA. However, the molecular basis of this stabilization and activation has remained unknown for a decade. Here we identified BTB/POZ AND MATH DOMAIN proteins (BPMs), substrate adaptors of the Cullin3 (CUL3)-based E3 ligase, as DREB2A-interacting proteins. We observed that DREB2A and BPMs interact in the nuclei, and that the NRD of DREB2A is sufficient for its interaction with BPMs. BPM-knockdown plants exhibited increased DREB2A accumulation and induction of DREB2A target genes under heat and drought stress conditions. Genetic analysis indicated that the depletion of BPM expression conferred enhanced thermotolerance via DREB2A stabilization. Thus, the BPM-CUL3 E3 ligase is likely the long-sought factor responsible for NRD-dependent DREB2A degradation. Through the negative regulation of DREB2A stability, BPMs modulate the heat stress response and prevent an adverse effect of excess DREB2A on plant growth. Furthermore, we found the BPM recognition motif in various transcription factors, implying a general contribution of BPM-mediated proteolysis to divergent cellular responses via an accelerated turnover of transcription factors.

Regulation of flowering time by SPL10/MED25 module in Arabidopsis.

Several SQUAMASA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors are involved in plant developmental transition from vegetative to reproductive growth. However, the function of SPL10 in regulating floral transition is largely unknown. It is also not known which Mediator subunit mediates SPL10 transcriptional activity. Here, we used overexpression lines and knockout mutants to examine the role of SPL10 in flowering-time regulation and we investigated possible interactions of SPL10 with several mediator subunits in vitro and in vivo. Plants overexpressing SPL10 showed precocious flowering, whereas the triple loss-of-function mutants of SPL10 and its two homologous genes, SPL2 and SPL11, flowered late compared with wild-type plants. We found that SPL10 interacts with MED25, a subunit of the Mediator complex, which bridges transcription factors and RNA polymerase II to facilitate transcription initiation. Genetic analysis showed that MED25 acts downstream of SPL10 to execute SPL10-regulated floral transition. Furthermore, SPL10 was required for MED25 association with the promoters of two target genes, FUL and LFY. We provide evidence that SPL10 recruits MED25 to the promoters of target genes to regulate flowering time. Our results on the SPL10/MED25 module are relevant to the molecular mechanism of other SPL family members.

The Transcriptional Cascade in the Heat Stress Response of Arabidopsis Is Strictly Regulated at the Level of Transcription Factor Expression.

Group A1 heat shock transcription factors (HsfA1s) are the master regulators of the heat stress response (HSR) in plants. Upon heat shock, HsfA1s trigger a transcriptional cascade that is composed of many transcription factors. Despite the importance of HsfA1s and their downstream transcriptional cascade in the acquisition of thermotolerance in plants, the molecular basis of their activation remains poorly understood. Here, domain analysis of HsfA1d, one of several HsfA1s in Arabidopsis thaliana, demonstrated that the central region of HsfA1d is a key regulatory domain that represses HsfA1d transactivation activity through interaction with HEAT SHOCK PROTEIN70 (HSP70) and HSP90. We designated this region as the temperature-dependent repression (TDR) domain. We found that HSP70 dissociates from HsfA1d in response to heat shock and that the dissociation is likely regulated by an as yet unknown activation mechanism, such as HsfA1d phosphorylation. Overexpression of constitutively active HsfA1d that lacked the TDR domain induced expression of heat shock proteins in the absence of heat stress, thereby conferring potent thermotolerance on the overexpressors. However, transcriptome analysis of the overexpressors demonstrated that the constitutively active HsfA1d could not trigger the complete transcriptional cascade under normal conditions, thereby indicating that other factors are necessary to fully induce the HSR. These complex regulatory mechanisms related to the transcriptional cascade may enable plants to respond resiliently to various heat stress conditions.

Functional analysis of the Hikeshi-like protein and its interaction with HSP70 in Arabidopsis.

Heat shock proteins (HSPs) refold damaged proteins and are an essential component of the heat shock response. Previously, the 70 kDa heat shock protein (HSP70) has been reported to translocate into the nucleus in a heat-dependent manner in many organisms. In humans, the heat-induced translocation of HSP70 requires the nuclear carrier protein Hikeshi. In the Arabidopsis genome, only one gene encodes a protein with high homology to Hikeshi, and we named this homolog Hikeshi-like (HKL) protein. In this study, we show that two Arabidopsis HSP70 isoforms accumulate in the nucleus in response to heat shock and that HKL interacts with these HSP70s. Our histochemical analysis revealed that HKL is predominantly expressed in meristematic tissues, suggesting the potential importance of HKL during cell division in Arabidopsis. In addition, we show that HKL regulates HSP70 localization, and HKL overexpression conferred thermotolerance to transgenic Arabidopsis plants. Our results suggest that HKL plays a positive role in the thermotolerance of Arabidopsis plants and cooperatively interacts with HSP70.

Tissue-specific transcriptomic analysis uncovers potential roles of natural antisense transcripts in Arabidopsis heat stress response.

Natural antisense transcripts (NATs) are an important class of non-coding ribonucleic acids (RNAs) that have been shown to regulate gene expression. Using strand-specific RNA sequencing, 36,317 NAT pairs were identified, and 5,536 were specifically expressed under heat stress. We found distinct expression patterns between vegetative and reproductive tissues for both coding genes and genes encoding NATs. Genes for heat-responsive NATs are associated with relatively high levels of H3K4me3 and low levels of H3K27me2/3. On the other hand, small RNAs are significantly enriched in sequence overlapping regions of NAT pairs, and a large number of heat-responsive NATs pairs serve as potential precursors of nat-siRNAs. Collectively, our results suggest epigenetic modifications and small RNAs play important roles in the regulation of NAT expression, and highlight the potential significance of heat-inducible NATs.

Epigenetic regulation of ecotype-specific expression of the heat-activated transposon ONSEN.

Transposable elements are present in a wide variety of organisms; however, our understanding of the diversity of mechanisms involved in their activation is incomplete. In this study, we analyzed the transcriptional activation of the ONSEN retrotransposon, which is activated by high-temperature stress in Arabidopsis thaliana. We found that its transcription is significantly higher in the Japanese ecotype Kyoto. Considering that transposons are epigenetically regulated, DNA methylation levels were analyzed, revealing that CHH methylation was reduced in Kyoto compared to the standard ecotype, Col-0. A mutation was also detected in the Kyoto CMT2 gene, encoding a CHH methyltransferase, suggesting that it may be responsible for increased expression of ONSEN. CHH methylation is controlled by histone modifications through a self-reinforcing loop between DNA methyltransferase and histone methyltransferase. Analysis of these modifications revealed that the level of H3K9me2, a repressive histone marker for gene expression, was lower in Kyoto than in Col-0. The level of another repressive histone marker, H3K27me1, was decreased in Kyoto; however, it was not impacted in a Col-0 cmt2 mutant. Therefore, in addition to the CMT2 mutation, other factors may reduce repressive histone modifications in Kyoto.

Arabidopsis HsfA1 transcription factors function as the main positive regulators in heat shock-responsive gene expression.

Arabidopsis DREB2A is a key transcription factor of heat- and drought-responsive gene expression, and DREB2A expression is induced by these stresses. We analyzed the DREB2A promoter and found a heat shock element that functions as a cis-acting element in the heat shock (HS)-responsive expression of DREB2A. Among the 21 Arabidopsis heat shock factors, we chose 4 HsfA1-type proteins as candidate transcriptional activators (HsfA1a, HsfA1b, HsfA1d, and HsfA1e) based on transactivation activity and expression patterns. We generated multiple mutants and found that the HS-responsive expression of DREB2A disappeared in hsfa1a/b/d triple and hsfa1a/b/d/e quadruple mutants. Moreover, HS-responsive gene expression, including that of molecular chaperones and transcription factors, was globally and drastically impaired in the hsfa1a/b/d triple mutant, which exhibited greatly reduced tolerance to HS stress. HsfA1 protein accumulation in the nucleus was negatively regulated by their interactions with HSP90, and other factors potentially strongly activate the HsfA1 proteins under HS stress. The hsfa1a/b/d/e quadruple mutant showed severe growth retardation, and many genes were downregulated in this mutant even under non-stress conditions. Our study indicates that HsfA1a, HsfA1b, and HsfA1d function as main positive regulators in HS-responsive gene expression and four HsfA1-type proteins are important in gene expression for normal plant growth.

Transcriptional Regulatory Network of Plant Heat Stress Response.

Heat stress (HS) is becoming an increasingly significant problem for food security as global warming progresses. Recent studies have elucidated the complex transcriptional regulatory networks involved in HS. Here, we provide an overview of current knowledge regarding the transcriptional regulatory network and post-translational regulation of the transcription factors involved in the HS response. Increasing evidence suggests that epigenetic regulation and small RNAs are important in heat-induced transcriptional responses and stress memory. It remains to be elucidated how plants sense and respond to HS. Several recent reports have discussed the heat sensing and signaling that activate transcriptional cascades; thus, we also highlight future directions of promoting crop tolerance to HS using these factors or other strategies for agricultural applications.

A small RNA mediated regulation of a stress-activated retrotransposon and the tissue specific transposition during the reproductive period in Arabidopsis.

Transposable elements (TEs) are key elements that facilitate genome evolution of the host organism. A number of studies have assessed the functions of TEs, which change gene expression in the host genome. Activation of TEs is controlled by epigenetic modifications such as DNA methylation and histone modifications. Several recent studies have reported that TEs can also be activated by biotic or abiotic stress in some plants. We focused on a Ty1/copia retrotransposon, ONSEN, that is activated by heat stress (HS) in Arabidopsis. We found that transcriptional activation of ONSEN was regulated by a small interfering RNA (siRNA)-related pathway, and the activation could also be induced by oxidative stress. Mutants deficient in siRNA biogenesis that were exposed to HS at the initial stages of vegetative growth showed transgenerational transposition. The transposition was also detected in the progeny, which originated from tissue that had differentiated after exposure to the HS. The results indicated that in some undifferentiated cells, transpositional activity could be maintained quite long after exposure to the HS.

HsfA1d, a protein identified via FOX hunting using Thellungiella salsuginea cDNAs improves heat tolerance by regulating heat-stress-responsive gene expression.

Thellungiella salsuginea (formerly T. halophila), a species closely related to Arabidopsis (Arabidopsis thaliana), is tolerant not only to high salt levels, but also to chilling, freezing, and ozone. Here, we report that T. salsuginea also shows greater heat tolerance than Arabidopsis. We identified T. salsuginea HsfA1d (TsHsfA1d) as a gene that can confer marked heat tolerance on Arabidopsis. TsHsfA1d was identified via Full-length cDNA Over-eXpressing gene (FOX) hunting from among a collection of heat-stress-related T. salsuginea cDNAs. Transgenic Arabidopsis overexpressing TsHsfA1d showed constitutive up-regulation of many genes in the Arabidopsis AtHsfA1 regulon under normal growth temperature. In Arabidopsis mesophyll protoplasts, TsHsfA1d was localized in both the nucleus and the cytoplasm. TsHsfA1d also interacted with AtHSP90, which negatively regulates AtHsfA1s by forming HsfA1-HSP90 complexes in the cytoplasm. It is likely that the partial nuclear localization of TsHsfA1d induced the expression of the AtHsfA1d regulon in the transgenic plants at normal temperature. We also discovered that transgenic Arabidopsis plants overexpressing AtHsfA1d were more heat-tolerant than wild-type plants and up-regulated the expression of the HsfA1d regulon, as was observed in TsHsfA1d-overexpressing plants. We propose that the products of both TsHsfA1d and AtHsfA1d function as positive regulators of Arabidopsis heat-stress response and would be useful for the improvement of heat-stress tolerance in other plants.