Heme Activator Protein Research Articles

Nanopore sequencing improves construction of customized CRISPR-based gene activation libraries.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based screening has emerged as a powerful tool for identifying new gene targets for desired cellular phenotypes. The construction of guide RNA (gRNA) pools largely determines library quality and is usually performed using Golden Gate assembly or Gibson assembly. To date, library construction methods have not been systematically compared, and the quality check of each batch has been slow. In this study, an in-house nanopore sequencing workflow was established for assessing the current methods of gRNA pool construction. The bias of pool construction was reduced by employing the polymerase-mediated non-amplifying method. Then, a small gRNA pool was utilized to characterize stronger activation domains, specifically MED2 (a subunit of mediator complex) and HAP4 (a heme activator protein), as well as to identify better gRNA choices for dCas12a-based gene activation in Saccharomyces cerevisiae. Furthermore, based on the better CRISPRa tool identified in this study, a custom gRNA pool, which consisted of 99 gRNAs targeting central metabolic pathways, was designed and employed to screen for gene targets that could improve ethanol utilization in S. cerevisiae. The nanopore sequencing-based workflow demonstrated here should provide a cost-effective approach for assessing the quality of customized gRNA library, leading to faster and more efficient genetic and metabolic engineering in S. cerevisiae.

HapX-mediated H2B deub1 and SreA-mediated H2A.Z deposition coordinate in fungal iron resistance.

Plant pathogens are challenged by host-derived iron starvation or excess during infection, but the mechanism through which pathogens counteract iron stress is unclear. Here, we found that Fusarium graminearum encounters iron excess during the colonization of wheat heads. Deletion of heme activator protein X (FgHapX), siderophore transcription factor A (FgSreA) or both attenuated virulence. Further, we found that FgHapX activates iron storage under iron excess by promoting histone H2B deubiquitination (H2B deub1) at the promoter of the responsible gene. Meanwhile, FgSreA is shown to inhibit genes mediating iron acquisition during iron excess by facilitating the deposition of histone variant H2A.Z and histone 3 lysine 27 trimethylation (H3K27 me3) at the first nucleosome after the transcription start site. In addition, the monothiol glutaredoxin FgGrx4 is responsible for iron sensing and control of the transcriptional activity of FgHapX and FgSreA via modulation of their enrichment at target genes and recruitment of epigenetic regulators, respectively. Taken together, our findings elucidated the molecular mechanisms for adaptation to iron excess mediated by FgHapX and FgSreA during infection in F. graminearum and provide novel insights into regulation of iron homeostasis at the chromatin level in eukaryotes.

Role of the Heme Activator Protein Complex in the Sexual Development of Cryptococcus neoformans.

ABSTRACTThe CCAAT-binding heme activator protein (HAP) complex, comprising the DNA-binding heterotrimeric complex Hap2/3/5 and transcriptional activation subunit HapX, is a key regulator of iron homeostasis, mitochondrial functions, and pathogenicity in Cryptococcus neoformans, which causes fatal meningoencephalitis. However, its role in the development of human fungal pathogens remains unclear. To elucidate the role of the HAP complex in C. neoformans development, we constructed hap2Δ, hap3Δ, hap5Δ, and hapXΔ mutants and their complemented congenic MATα H99 and MATa YL99a strains. The HAP complex plays a conserved role in iron utilization and stress responses in cells of both mating types. Deletion of any of the HAP complex components markedly enhances filamentation during bisexual mating. However, the Hap2/3/5 complex, but not HapX, is crucial in repressing pheromone production and cell fusion and is thus a critical repressor of sexual differentiation of C. neoformans. Interestingly, deletion of the heterotrimeric complex transcriptionally regulated both positive and negative regulators in the pheromone-responsive Cpk1 mitogen-activated protein kinase (MAPK) pathway. Chromatin immunoprecipitation-quantitative PCR analysis revealed that the HAP complex physically bound to the CCAAT motif of the CRG1 and GPA2 promoter regions. Notably, the HAP complex was differentially localized depending on the mating type in basal conditions; it was enriched in the nuclei of MATα cells but diffused in the cytoplasm of MATa cells. Interestingly, however, a portion of the HAP complex in both mating types relocalized to the cell membrane during mating. In conclusion, the Hap2/3/5 heterotrimeric complex and HapX play major and minor roles, respectively, in repressing the sexual development of C. neoformans in association with the Cpk1 MAPK pathway.IMPORTANCE Cryptococcus neoformans isolates are of two mating types: MATα strains, which are predominant, and MATa strains, isolated from the sub-Saharan African region, where cryptococcosis is most abundant and severe. Here, we demonstrated the function of the CCAAT-binding HAP complex (Hap2/3/5/X) as a transcriptional repressor of Cpk1 pathway-related genes in cells of both mating types. Deletion of any HAP complex component markedly enhanced filamentation without affecting normal sporulation. In particular, deletion of the DNA-binding HAP complex components (Hap2/3/5), but not HapX, markedly enhanced pheromone production and cell fusion efficiency, validating its repressive role in the early stage of mating in C. neoformans. The HAP complex regulates the expression of both negative and positive mating regulators and is thus crucial for the regulation of the Cpk1 MAPK pathway during mating. This study provides insights into the complex signaling networks governing the sexual differentiation of C. neoformans.

A new mechanistic insight into fate decisions during yeast cell aging process

Despite massive technological advances in mammalian models in recent years, studies in yeast still have the power to inform on the basic mechanisms of aging. Illustrating this, in Nan Hao’s recent article published in the journal Science, he and his lab use microfluidics and fluorescent imaging technology to analyze the dynamics and interactions of aging mechanisms within yeast cells. They focused in on the Sir2 gene and the heme activator protein and, through the manipulation of these two molecular aging pathways, were able to determine that yeast cells can undergo one of three modes of aging, with one of them having a significantly longer lifespan than the others. These findings provide unexpected insights into mechanisms of aging, apparently as regulated fate-decision process, and open up avenues for future research.

Cloning and functional analysis of LH2, a gene controlling late heading in rice

AbstractHeading date is an important agronomic trait in rice (Oryza sativa L.). We previously fine‐mapped two complementary genes, Late Heading Date 1 and Late Heading Date 2 (LH2), that control the late heading trait in rice. Here, we cloned and analyzed the function of LH2. LH2 was fine‐mapped to a 53‐kb genomic region. Sequencing analysis revealed that there were differences in the coding sequence of LOC_Os08g07740 between the varieties ‘Bo B’ and ‘Yuefeng B’. Therefore, LOC_Os08g07740 was identified as a candidate gene for LH2 and subsequently determined that LH2 is a Type 3 allele of Days To Heading 8. Transgenic plants of Yuefeng B carrying LH2 from Bo B significantly delayed the heading date under short‐day and long‐day conditions. A yeast two‐hybrid analysis revealed that LH2 physically interacted with Heme Activator Protein (HAP) 5A, ‐H, ‐I, ‐L, ‐K, and HAP5‐LIKE. The HAP5 domain of HAP5 family members has various functions. Further analysis revealed that LH2 represses photoperiodic flowering by controlling the expression of Early Heading Date 1, Heading date 3a, and Rice Flowering Locus T1 in rice.

Investigating the Antifungal Mechanism of Action of Polygodial by Phenotypic Screening in Saccharomyces cerevisiae.

Polygodial is a “hot” peppery-tasting sesquiterpenoid that was first described for its anti-feedant activity against African armyworms. Using the haploid deletion mutant library of Saccharomyces cerevisiae, a genome-wide mutant screen was performed to shed more light on polygodial’s antifungal mechanism of action. We identified 66 deletion strains that were hypersensitive and 47 that were highly resistant to polygodial treatment. Among the hypersensitive strains, an enrichment was found for genes required for vacuolar acidification, amino acid biosynthesis, nucleosome mobilization, the transcription mediator complex, autophagy and vesicular trafficking, while the resistant strains were enriched for genes encoding cytoskeleton-binding proteins, ribosomal proteins, mitochondrial matrix proteins, components of the heme activator protein (HAP) complex, and known regulators of the target of rapamycin complex 1 (TORC1) signaling. WE confirm that polygodial triggers a dose-dependent vacuolar alkalinization and that it increases Ca2+ influx and inhibits glucose-induced Ca2+ signaling. Moreover, we provide evidence suggesting that TORC1 signaling and its protective agent ubiquitin play a central role in polygodial resistance, suggesting that they can be targeted by polygodial either directly or via altered Ca2+ homeostasis.

Genome-wide identification and expression profile analysis of nuclear factor Y family genes in Sorghum bicolor L. (Moench).

Members of the plant Heme Activator Protein (HAP) or NUCLEAR FACTOR Y (NF-Y) are trimeric transcription factor complexes composed of the NF-YA, NF-YB and NF-YC subfamilies. They bind to the CCAAT box in the promoter regions of the target genes and regulate gene expressions. Plant NF-Ys were reported to be involved in adaptation to several abiotic stresses as well as in development. In silico analysis of Sorghum bicolor genome resulted in the identification of a total of 42 NF-Y genes, among which 8 code for the SbNF-YA, 19 for SbNF-YB and 15 for the SbNF-YC subunits. Analysis was also performed to characterize gene structures, chromosomal distribution, duplication status, protein subcellular localizations, conserved motifs, ancestral protein sequences, miRNAs and phylogenetic tree construction. Phylogenetic relationships and ortholog predictions displayed that sorghum has additional NF-YB genes with unknown functions in comparison with Arabidopsis. Analysis of promoters revealed that they harbour many stress-related cis-elements like ABRE and HSE, but surprisingly, DRE and MYB elements were not detected in any of the subfamilies. SbNF-YA1, 2, and 6 were found upregulated under 200 mM salt and 200 mM mannitol stresses. While NF-YA7 appeared associated with high temperature (40°C) stress, NF-YA8 was triggered by both cold (4°C) and high temperature stresses. Among NF-YB genes, 7, 12, 15, and 16 were induced under multiple stress conditions such as salt, mannitol, ABA, cold and high temperatures. Likewise, NF-YC 6, 11, 12, 14, and 15 were enhanced significantly in a tissue specific manner under multiple abiotic stress conditions. Majority of the mannitol (drought)-inducible genes were also induced by salt, high temperature stresses and ABA. Few of the high temperature stress-induced genes are also induced by cold stress (NF-YA2, 4, 6, 8, NF-YB2, 7, 10, 11, 12, 14, 16, 17, NF-YC4, 6, 12, and 13) thus suggesting a cross talk among them. This work paves the way for investigating the roles of diverse sorghum NF-Y proteins during abiotic stress responses and provides an insight into the evolution of diverse NF-Y members.

Mitochondrial cysteinyl-tRNA synthetase is expressed via alternative transcriptional initiation regulated by energy metabolism in yeast cells

Eukaryotes typically utilize two distinct aminoacyl-tRNA synthetase isoforms, one for cytosolic and one for mitochondrial protein synthesis. However, the genome of budding yeast (Saccharomyces cerevisiae) contains only one cysteinyl-tRNA synthetase gene (YNL247W, also known as CRS1). In this study, we report that CRS1 encodes both cytosolic and mitochondrial isoforms. The 5' complementary DNA end method and GFP reporter gene analyses indicated that yeast CRS1 expression yields two classes of mRNAs through alternative transcription starts: a long mRNA containing a mitochondrial targeting sequence and a short mRNA lacking this targeting sequence. We found that the mitochondrial Crs1 is the product of translation from the first initiation AUG codon on the long mRNA, whereas the cytosolic Crs1 is produced from the second in-frame AUG codon on the short mRNA. Genetic analysis and a ChIP assay revealed that the transcription factor heme activator protein (Hap) complex, which is involved in mitochondrial biogenesis, determines the transcription start sites of the CRS1 gene. We also noted that Hap complex-dependent initiation is regulated according to the needs of mitochondrial energy production. The results of our study indicate energy-dependent initiation of alternative transcription of CRS1 that results in production of two Crs1 isoforms, a finding that suggests Crs1's potential involvement in mitochondrial energy metabolism in yeast.

Identification of a module of HAP transcription factors for seed development in rice

Heme activator protein (HAP) is a CCAAT box-binding factor family conserved in all higher eukaryotes. HAP transcription factors (TFs) consist of three subunits: HAP2, HAP3, and HAP5. Using recent rice genome information, we identified 11 HAP2 genes, 11 HAP3 genes, and 12 HAP5 genes. A HAP2 and five HAP5 genes were acknowledged more in this study than in the previous studies. Due to the functional association of HAP subunits, we carried out clustering analysis for expression patterns of all HAP members using anatomical meta-expression data of 983 rice Affymetrix arrays collected from the public database. Subsequently, we identified HAP members with endosperm and seed-preferred expression and focused on nine HAP family members associated with this expression pattern. We confirmed their expression patterns by RT-PCR analyses. The nine members were OsHAP2G, OsHAP2I, OsHAP3D, OsHAP3K, and five HAP5 genes (LOC_Os05g23910, LOC_Os10g11580, LOC_Os01g24460, LOC_Os01g01290, and LOC_Os01g39850). Promoter analyses using GUS activity again demonstrated that OsHAP3K is preferentially expressed in the aleurone layer of the seed and the LOC_Os05g23910 gene-encoding HAP5 showed endosperm-preferred expression. Network analysis using RiceNet suggests a functional gene network mediated by four rice HAP genes, further helping in the prediction of the biological function of OsHAP2G and OsHAP5 (LOC_Os10g11580). Our data provide new insight into HAP family-mediated regulatory pathways for endosperm or seed development in rice.

Fine Mapping of Carbon Assimilation Rate 8, a Quantitative Trait Locus for Flag Leaf Nitrogen Content, Stomatal Conductance and Photosynthesis in Rice.

Increasing the rate of leaf photosynthesis is one important approach for increasing grain yield in rice (Oryza sativa). Exploiting the natural variation in CO2 assimilation rate (A) between rice cultivars using quantitative genetics is one promising means to identify genes contributing to higher photosynthesis. In this study, we determined precise location of Carbon Assimilation Rate 8 (CAR8) by crossing a high-yielding indica cultivar with a Japanese commercial cultivar. Fine mapping suggested that CAR8 encodes a putative Heme Activator Protein 3 (OsHAP3) subunit of a CCAAT-box-binding transcription factor called OsHAP3H. Sequencing analysis revealed that the indica allele of CAR8 has a 1-bp deletion at 322 bp from the start codon, resulting in a truncated protein of 125 amino acids. In addition, CAR8 is identical to DTH8/Ghd8/LHD1, which was reported to control rice flowering date. The increase of A is largely due to an increase of RuBP regeneration rate via increased leaf nitrogen content, and partially explained by reduced stomatal limitation via increased stomatal conductance relative to A. This allele also increases hydraulic conductivity, which would promote higher stomatal conductance. This indicates that CAR8 affects multiple physiological aspects relating to photosynthesis. The detailed analysis of molecular functions of CAR8 would help to understand the association between photosynthesis and flowering and demonstrate specific genetic mechanisms that can be exploited to improve photosynthesis in rice and potentially other crops.

The OsHAPL1-DTH8-Hd1 complex functions as the transcription regulator to repress heading date in rice.

Heading date is an important agronomic trait related to crop yield. Many genes related to heading date have already been identified in rice (Oryza sativa), and a complicated, preliminary regulatory genetic network has also already been established, but the protein regulatory network is poorly understood. We have identified a novel heading date regulator, Heme Activator Protein like 1 (OsHAPL1), which inhibits flowering under long-day conditions. OsHAPL1 is a nuclear-localized protein that is highly expressed in leaves in a rhythmic manner. OsHAPL1 can physically interact with Days To Heading on chromosome 8 (DTH8), which physically interacts with Heading date 1 (Hd1) both in vitro and in vivo. OsHAPL1 forms a complex with DTH8 and Hd1 in Escherichia coli. OsHAPL1, DTH8, and Hd1 physically interact with the HAP complex, and also with general transcription factors in yeast (Saccharomyces cerevisiae). Further studies showed that OsHAPL1 represses the expression of the florigen genes and FLOWERING LOCUS T 1 (RFT1) and Hd3a through Early heading date 1 (Ehd1). We propose that OsHAPL1 functions as a transcriptional regulator and, together with DTH8, Hd1, the HAP complex, and general transcription factors, regulates the expression of target genes and then affects heading date by influencing the expression of Hd3a and RFT1 through Ehd1.

Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors

Heme is an essential cofactor and signaling molecule. Heme acquisition by proteins and heme signaling are ultimately reliant on the ability to mobilize labile heme (LH). However, the properties of LH pools, including concentration, oxidation state, distribution, speciation, and dynamics, are poorly understood. Herein, we elucidate the nature and dynamics of LH using genetically encoded ratiometric fluorescent heme sensors in the unicellular eukaryote Saccharomyces cerevisiae We find that the subcellular distribution of LH is heterogeneous; the cytosol maintains LH at ∼20-40 nM, whereas the mitochondria and nucleus maintain it at concentrations below 2.5 nM. Further, we find that the signaling molecule nitric oxide can initiate the rapid mobilization of heme in the cytosol and nucleus from certain thiol-containing factors. We also find that the glycolytic enzyme glyceraldehyde phosphate dehydrogenase constitutes a major cellular heme buffer, and is responsible for maintaining the activity of the heme-dependent nuclear transcription factor heme activator protein (Hap1p). Altogether, we demonstrate that the heme sensors can be used to reveal fundamental aspects of heme trafficking and dynamics and can be used across multiple organisms, including Escherichia coli, yeast, and human cell lines.

Duplication ofOsHAPfamily genes and their association with heading date in rice

Heterotrimeric Heme Activator Protein (HAP) family genes are involved in the regulation of flowering in plants. It is not clear how many HAP genes regulate heading date in rice. In this study, we identified 35 HAP genes, including seven newly identified genes, and performed gene duplication and candidate gene-based association analyses. Analyses showed that segmental duplication and tandem duplication are the main mechanisms of HAP gene duplication. Expression profiling and functional identification indicated that duplication probably diversifies the functions of HAP genes. A nucleotide diversity analysis revealed that 13 HAP genes underwent selection. A candidate gene-based association analysis detected four HAP genes related to heading date. An investigation of transgenic plants or mutants of 23 HAP genes confirmed that overexpression of at least four genes delayed heading date under long-day conditions, including the previously cloned Ghd8/OsHAP3H. Our results indicate that the large number of HAP genes in rice was mainly produced by gene duplication, and a few HAP genes function to regulate heading date. Selection of HAP genes is probably caused by their diverse functions rather than regulation of heading.

Overexpression of a rice heme activator protein gene (OsHAP2E) confers resistance to pathogens, salinity and drought, and increases photosynthesis and tiller number.

Heme activator protein (HAP), also known as nuclear factor Y or CCAAT binding factor (HAP/NF-Y/CBF), has important functions in regulating plant growth, development and stress responses. The expression of rice HAP gene (OsHAP2E) was induced by probenazole (PBZ), a chemical inducer of disease resistance. To characterize the gene, the chimeric gene (OsHAP2E::GUS) engineered to carry the structural gene encoding β-glucuronidase (GUS) driven by the promoter from OsHAP2E was introduced into rice. The transgenic lines of OsHAP2Ein::GUS with the intron showed high GUS activity in the wounds and surrounding tissues. When treated by salicylic acid (SA), isonicotinic acid (INA), abscisic acid (ABA) and hydrogen peroxide (H2 O2 ), the lines showed GUS activity exclusively in vascular tissues and mesophyll cells. This activity was enhanced after inoculation with Magnaporthe oryzae or Xanthomonas oryzae pv. oryzae. The OsHAP2E expression level was also induced after inoculation of rice with M. oryzae and X. oryzae pv. oryzae and after treatment with SA, INA, ABA and H2 O2, respectively. We further produced transgenic rice overexpressing OsHAP2E. These lines conferred resistance to M. oryzae or X. oryzae pv. oryzae and to salinity and drought. Furthermore, they showed a higher photosynthetic rate and an increased number of tillers. Microarray analysis showed up-regulation of defence-related genes. These results suggest that this gene could contribute to conferring biotic and abiotic resistances and increasing photosynthesis and tiller numbers.

Deletion of the Cardiolipin-specific Phospholipase Cld1 Rescues Growth and Life Span Defects in the Tafazzin Mutant: IMPLICATIONS FOR BARTH SYNDROME

Cardiolipin (CL) that is synthesized de novo is deacylated to monolysocardiolipin (MLCL), which is reacylated by tafazzin. Remodeled CL contains mostly unsaturated fatty acids. In eukaryotes, loss of tafazzin leads to growth and respiration defects, and in humans, this results in the life-threatening disorder Barth syndrome. Tafazzin deficiency causes a decrease in the CL/MLCL ratio and decreased unsaturated CL species. Which of these biochemical outcomes contributes to the physiological defects is not known. Yeast cells have a single CL-specific phospholipase, Cld1, that can be exploited to distinguish between these outcomes. The cld1Δ mutant has decreased unsaturated CL, but the CL/MLCL ratio is similar to that of wild type cells. We show that cld1Δ rescues growth, life span, and respiratory defects of the taz1Δ mutant. This suggests that defective growth and respiration in tafazzin-deficient cells are caused by the decreased CL/MLCL ratio and not by a deficiency in unsaturated CL. CLD1 expression is increased during respiratory growth and regulated by the heme activator protein transcriptional activation complex. Overexpression of CLD1 leads to decreased mitochondrial respiration and growth and instability of mitochondrial DNA. However, ATP concentrations are maintained by increasing glycolysis. We conclude that transcriptional regulation of Cld1-mediated deacylation of CL influences energy metabolism by modulating the relative contribution of glycolysis and respiration.

The roles of two transcription factors, ABI4 and CBFA, in ABA and plastid signalling and stress responses

Genetic and physiological studies have revealed evidences for multiple signaling pathways by which the plastid exerts retrograde control over photosynthesis-associated-nuclear-genes. In this study we have examined the mechanisms of control of transcription by plastid signals, focusing on transcription factors. We have also further addressed the physical nature of plastid signals and the physiological role, in stress acclimation of this regulatory pathway. ABI4, a master Apetala 2 (AP2)-type transcription factor (TF), is targeted by multiple signalling pathways in plant cells, such as abscisic acid (ABA) signals, sugar signals and plastid signals derived from reactive oxygen species (ROS) and chlorophyll intermediates. ABI4 binds the promoter of target genes to prevent their transcription by competing with other competitive TFs. However, we found that once ABI4 bound the element (CCACGT), it may not be bound by other TFs, therefore making the signalling long-lasting. Downstream of ABI4, CBFA (CCAAT binding factor A) is a subunit of the HAP2/HAP3/HAP5 (Heme activator protein) trimeric transcription complex. CBFA however is a redundant HAP3 subunit. When emergency occurs (such as herbicide treatments or environmental stresses followed by ABA and ROS accumulation), the master transcription factor ABI4 down-regulates some TFs, like CBFA, and then some other TF subunits enter the transcription complex and transcriptional efficiency of stress-responsive genes (including the transcription co-factor CBP) is improved instantaneously. abi4, cbfA and cbp mutants showed weaker drought-tolerance after a herbicide norflurazon treatment, which indicated the physiological role of these key transcription factors.

Quantitative RT-PCR analysis of 20 transcription factor genes of MADS, ARF, HAP2, MBF and HB families in moisture stressed shoot and root tissues of sorghum

Transcription factors (TFs) are an important target in understanding the regulation of plant responses to environmental stress including moisture stress. Members of the same TF family may differ in their response to moisture stress. The expression pattern could vary between shoot and root tissues depending on level of moisture stress. A set of five rarely studied TF families viz., MADS-box (MCM1, AGAMOUS, DEFICIENS and SRF), Auxin Responsive Factor (ARF), Heme Activator Protein 2 (HAP2), Multiprotein Bridging Factor (MBF) and Homeobox (HB) together having 20 members in sorghum, were expression analyzed through quantitative real-time PCR (qRT-PCR) in well watered and moisture stressed shoot and root tissues of sorghum using SYBR Green® to quantify dsDNA synthesis. Fluorescence values were used to calculate PCR efficiency by using LinRegPCR. The PTSb00029.1 and PTSb00033.1 of ARF family and PTSb00174.1 and PTSb00175.1 of HB family recorded 2 to 5, PTSb00221.1 and PTSb00208.1 of MADS family and PTSb00128.1 of HAP2 family recorded 5 to 10 fold up-regulation under moisture stress regimes. The PTSb00128.1, a HAP2 family member, recorded 15 fold up-regulation in mild moisture stressed root tissues. TF genes such as PTSb00218.1, PTSb00220.1, PTSb00031.1, PTSb00032.1, PTSb00034.1 and PTSb00223.1 were found down regulating in both tissues types under moisture stress condition. However, the PTSb00128.1, PTSb00221.1, PTSb00029.1, PTSb00033.1 and PTSb00174.1 TFs were found up-regulating to varied levels in mild and severe moisture stressed root tissues only. Verification of qRT-PCR results was done by in situ hybridization (ISH) of randomly selected two TF genes in shoot and root tissues of sorghum. Taken together, moisture stress triggered up-regulation of more genes in root tissue compared to shoot tissue in sorghum.

Heme regulates protein homeostasis at transcription, protein translation, and degradation levels

Heme, as a prosthetic group of proteins, is an iron-protoporphyrin involved in a wide range of cellular functions. Cellular heme levels vary due to the accurate balance of its synthesis and degradation. The “heme sensor protein” is currently a focus of investigation because heme has been found as a cellular signaling messenger involved in various biologic processes, including gene expression, protein localization, protein stability and microRNA processing. Several eukaryotic transcriptional factors can be regulated by heme, including heme activator protein (Hap1), Bach1, REV-erbα, and neuronal PAS domain protein 2 (NPAS2). Especially, the two circadian transcriptional factors serving as the heme sensor, REV-erbα and NPAS2, coordinate the circadian clock with metabolic pathways. It is well established that heme regulates the activity of heme-regulated eukaryotic initiation factor 2α (eIF2α) kinase (HRI), which serves as a feedback inhibitor of protein translation in both erythroid and non-erythroid cells. Additionally, heme is involved in protein degradation by inducing the degradation of several proteins such as the iron response regulator (Irr), iron regulatory protein 2 (IRP2), Bach1, and circadian factor period 2 (Per2). The N-end rule ubiquitin-dependent protein degradation pathway has also been identified as a sensor of heme, which blocks the function of arginyl-tRNA protein transferase (ATE1) and E3 ubiquitin ligase. In this review, we summarize the regulatory roles of heme at the levels of transcription, protein translation, and protein degradation, highlighting the role of heme in maintaining cellular homeostasis.

Analysis of a post-translational steroid induction system for GIGANTEA in Arabidopsis

BackgroundTo investigate the link between the flowering time gene GIGANTEA (GI) and downstream genes, an inducible GI system was developed in Arabidopsis thaliana L. Heynh. Transgenic Arabidopsis plant lines were generated with a steroid-inducible post-translational control system for GI. The gene expression construct consisted of the coding region of the GI protein fused to that of the ligand binding domain of the rat glucocorticoid receptor (GR). This fusion gene was expressed from the constitutive cauliflower mosaic virus 35S promoter and was introduced into plants carrying the gi-2 mutation. Application of the steroid dexamethasone (DEX) was expected to result in activation of the GI-GR protein and its relocation from the cytoplasm to the nucleus.ResultsApplication of DEX to the transgenic plant lines rescued the late flowering phenotype conferred by the gi-2 mutation. However, despite their delayed flowering in the absence of steroid, the transgenic lines expressed predicted GI downstream genes such as CONSTANS (CO) to relatively high levels. Nevertheless, increased CO and FLOWERING LOCUS T (FT) transcript accumulation was observed in transgenic plants within 8 h of DEX treatment compared to controls which was consistent with promotion of flowering by DEX. Unlike CO and FT, there was no change in the abundance of transcript of two other putative GI downstream genes HEME ACTIVATOR PROTEIN 3A (HAP3A) or TIMING OF CHLOROPHYLL A/B BINDING PROTEIN 1 (TOC1) after DEX application.ConclusionThe post-translational activation of GI and promotion of flowering by steroid application supports a nuclear role for GI in the floral transition. Known downstream flowering time genes CO and FT were elevated by DEX treatment, but not other proposed targets HAP3A and TOC1, indicating that the expression of these genes may be less directly regulated by GI.

A Unique Mechanism of Chaperone Action: Heme Regulation of Hap1 Activity Involves Separate Control of Repression and Activation

The Hsp90 and Hsp70 molecular chaperones play important roles in the folding and proper functioning of diverse cellular proteins, including transcriptional regulators and protein kinases. In yeast, several transcriptional regulators and protein kinases are known to be substrates for Hsp90 and Hsp70 molecular chaperones. The yeast heme activator protein Hap1 promotes transcription of many genes in response to heme. It requires Hsp90 and Hsp70 molecular chaperones for its activity to be precisely regulated by heme concentration. The mechanism by which molecular chaperones promote heme regulation of Hap1 activity is distinct from the mechanism by which molecular chaperones promote steroid signaling. Hsp70 and Hsp90 molecular chaperones act separately to promote Hap1 repression in heme-deficient cells and heme activation of Hap1 in heme-sufficient cells. Likewise, distinct Hap1 elements or domains act to mediate Hap1 repression and heme activation separately. In this review, we summarize the current knowledge about the molecular mechanism governing heme regulation of Hap1 activity, and we compare this mechanism to the molecular mechanism by which Hsp90 and Hsp70 molecular chaperones promote the regulation of glucocorticoid receptor, the most extensively studied substrate of Hsp90 and Hsp70 molecular chaperones.