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DOE OSTI2021

Modeling temporal and hormonal regulation of plant transcriptional response to wounding

Plants respond to wounding stress by changing gene expression patterns and inducing the production of hormones including jasmonic acid. This wounding transcriptional response activates specialized metabolism pathways such as the glucosinolate pathways in Arabidopsis thaliana. While the regulatory factors and sequences controlling a subset of wound-response genes are known, it remains unclear how wound response is regulated globally. Here, we how these responses are regulated by incorporating putative cis-regulatory elements, known transcription factor binding sites, in vitro DNA affinity purification sequencing, and DNase I hypersensitive sites to predict genes with different wound-response patterns using machine learning. We observed that regulatory sites and regions of open chromatin differed between genes upregulated at early and late wounding time-points as well as between genes induced by jasmonic acid and those not induced. Expanding on what we currently know, we identified cis-elements that improved model predictions of expression clusters over known binding sites. Using a combination of genome editing, in vitro DNA-binding assays, and transient expression assays using native and mutated cis-regulatory elements, we experimentally validated four of the predicted elements, three of which were not previously known to function in wound-response regulation. Our study provides a global model predictive of wound response and identifies new regulatory sequences important for wounding without requiring prior knowledge of the transcriptional regulators.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2000

Developmental regulation of collagenase-3 mRNA in normal, differentiating osteoblasts through the activator protein-1 and the runt domain binding sites

Collagenase-3 mRNA is initially detectable when osteoblasts cease proliferation, increasing during differentiation and mineralization. We showed that this developmental expression is due to an increase in collagenase-3 gene transcription. Mutation of either the activator protein-1 or the runt domain binding site decreased collagenase-3 promoter activity, demonstrating that these sites are responsible for collagenase-3 gene transcription. The activator protein-1 and runt domain binding sites bind members of the activator protein-1 and core-binding factor family of transcription factors, respectively. We identified core-binding factor a1 binding to the runt domain binding site and JunD in addition to a Fos-related antigen binding to the activator protein-1 site. Overexpression of both c-Fos and c-Jun in osteoblasts or core-binding factor a1 increased collagenase-3 promoter activity. Furthermore, overexpression of c-Fos, c-Jun, and core-binding factor a1 synergistically increased collagenase-3 promoter activity. Mutation of either the activator protein-1 or the runt domain binding site resulted in the inability of c-Fos and c-Jun or core-binding factor a1 to increase collagenase-3 promoter activity, suggesting that there is cooperative interaction between the sites and the proteins. Overexpression of Fra-2 and JunD repressed core-binding factor a1-induced collagenase-3 promoter activity. Our results suggest that members of the activator protein-1 and core-binding factor families, binding to the activator protein-1 and runt domain binding sites are responsible for the developmental regulation of collagenase-3 gene expression in osteoblasts.

NASA Discipline Musculoskeletal↗

DOE OSTI2020

Generation of ionic liquid tolerant Pseudomonas putida KT2440 strains via adaptive laboratory evolution

Although the use of ionic liquids (ILs) for the pretreatment of lignocellulosic biomass has been limited due to high costs, recent efforts to develop low-cost protic ILs show promise for achieving cost-effectiveness for biorefineries. However, an additional challenge remains in that ILs present in biomass hydrolysates are toxic to most microbial hosts, resulting in poor growth phenotypes. To address this issue, we applied an adaptive laboratory evolution (ALE) approach for tolerizing Pseudomonas putida KT2440, an industrially relevant bacterial host, to two low-cost ILs (triethanolammonium acetate [TEOH][OAc] and triethylammonium hydrogen sulfate [TEA][HS]). After continuous cultivations with gradually increased IL levels, we obtained evolved strains showing significant improvements in their growth performance under high concentrations of the ILs (maximum 4% [TEOH][OAc] and 8% [TEA][HS], in w/v) at which the wild-type strain cannot grow. Sequencing of evolved strains revealed multiple regions where mutations were associated with improved performance in minimal media conditions (relA, gacS, oprB/PP_1446, fleQ, tktA, and uvrY/PP_4100) and in IL-specific conditions (PP_5350, PP_4929/emrE, oprD, and PP_5324). We further validated the causality of the PP_5350 and emrE genes for improved IL tolerance via reverse engineering and transcriptomic analysis. Here, a common mutation in the PP_5350 gene, encoding a RpiR family transcriptional regulator, was shown to significantly upregulate the glyoxylate cycle for efficient acetate catabolism. In addition, it was suggested that the emrE gene encodes an efflux pump which can export [TEA][HS]. Finally, the cultivation of two of the best performing evolved strains with IL-treated biomass hydrolysates demonstrated their considerable potential to be used as platform strains. Taken as a whole, this work provides strains for utilization of IL-treated biomass and a mechanistic understanding that could be further leveraged to develop efficient microbial bioprocesses.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2021

The combined application of the anti- RAS1 and anti- RIM101 2'- O MethylRNA oligomers enhances Candida albicans filamentation control

Abstract Although antisense oligomers (ASOs) have been successfully utilized to control gene expression, they have been little exploited to control Candida albicans virulence's determinants. Filamentation is an important virulence factor of C. albicans, and RAS1 and RIM101 genes are involved in its regulation. Thus, the main goal of this work was to project ASOs, based on 2'-OMethyl chemical modification, to target RAS1 and RIM101 mRNA and to validate its application either alone or in combination, to reduce Candida filamentation in different human body fluids. It was verified that both, anti-RAS1 2'OMe and anti-RIM101 2'OMe oligomers, were able to reduce the levels of RAS1 and RIM101 genes’ expression and to significantly reduce C. albicans filamentation. Furthermore, the combined application of anti-RAS1 2'OMe oligomer and anti-RIM101 2'OMe oligomer enhances the control of C. albicans filamentation in artificial saliva and urine. Our work confirms that ASOs are useful tools for research and therapeutic development on the control of candidiasis.

Barbosa, Ana↗

NASA NTRS2010

High LET Radiation Can Enhance TGF(Beta) Induced EMT and Cross-Talk with ATM Pathways

The TGF(Beta) pathway has been shown to regulate or directly interact with the ATM pathway in the response to radiation in mammary epithelial cells. We investigated possible interactions between the TGF(Beta) and ATM pathways following simulated space radiation using hTERT immortalized human esophageal epithelial cells (EPC-hTERT), mink lung epithelial cells (Mv1lu), and several human fibroblast cell lines. TGF(Beta) is a key modulator of the Epithelial-Mesenchymal Transition (EMT), important in cancer progression and metastasis. The implication of EMT by radiation also has several lines of developing evidence, however is poorly understood. The identification of TGF(Beta) induced EMT can be shown in changes to morphology, related gene over expression or down regulation, which can be detected by RT-PCR, and immunostaining and western blotting. In this study, we have observed morphologic and molecular alternations consistent with EMT after Mv1lu cells were treated with TGF(Beta) High LET radiation enhanced TGF(Beta) mediated EMT with a dose as low as 0.1Gy. In order to consider the TGF(Beta) interaction with ATM we used a potent ATM inhibitor Ku55933 and investigated gene expression changes and Smad signaling kinetics. Ku559933 was observed to reverse TGF(Beta) induced EMT, while this was not observed in dual treated cells (radiation+TGF(Beta)). In EPC-hTERT cells, TGF(Beta) alone was not able to induce EMT after 3 days of application. A combined treatment with high LET, however, significantly caused the alteration of EMT markers. To study the function of p53 in the process of EMT, we knocked down P53 through RNA interference. Morphology changes associated with EMT were observed in epithelial cells with silenced p53. Our study indicates: high LET radiation can enhance TGF(Beta) induced EMT; while ATM is triggering the process of TGF(Beta)-induced EMT, p53 might be an essential repressor for EMT phenotypes.

Wang, Minli↗

DOE OSTI2021

Disrupted NOS2 metabolism drives myoblast response to wasting-associated cytokines

Highlights: • Amino acid metabolism is impacted in myoblasts treated with cancer cell conditioned media. • Inflammatory cytokines induce nitric oxide synthase 2 in myoblasts. • Elevated nitric oxide synthase 2 activity impairs myoblast proliferation and differentiation. Skeletal muscle wasting drives negative clinical outcomes and is associated with a spectrum of pathologies including cancer. Cancer cachexia is a multi-factorial syndrome that encompasses skeletal muscle wasting and remains understudied, despite being a frequent and serious co-morbidity. Deviation from the homeostatic balance between breakdown and regeneration leads to muscle wasting disorders, such as cancer cachexia. Muscle stem cells (MuSCs) are the cellular compartment responsible for muscle regeneration, which makes MuSCs an intriguing target in the context of wasting muscle. Molecular studies investigating MuSCs and skeletal muscle wasting largely focus on transcriptional changes, but our group and others propose that metabolic changes are another layer of cellular regulation underlying MuSC dysfunction in cancer cachexia. In the present study, we combined gene expression and non-targeted metabolomic profiling of myoblasts exposed to wasting conditions (cancer cell conditioned media, CC-CM) to derive a more complete picture of the myoblast response to wasting factors. After mapping these features to annotated pathways, we found that more than half of the mapped pathways were amino acid-related, linking global amino acid metabolic disruption to conditioned media-induced myoblast defects. Notably, arginine metabolism was a highly enriched pathway in combined metabolomic and transcriptomic data. Arginine catabolism generates nitric oxide (NO), an important signaling molecule known to have negative effects on mature muscle. We hypothesize that tumor-derived disruptions in Nitric Oxide Synthase (NOS)2-regulated arginine catabolism impair differentiation of MuSCs. The work presented here further investigates the effect of NOS2 overactivity on myoblast proliferation and differentiation. We show that NOS2 inhibition is sufficient to rescue wasting phenotypes associated with inflammatory cytokines. Ultimately, this work provides new insights into MuSC biology and opens up potential therapeutic avenues for addressing disrupted MuSC dynamics in cancer cachexia.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

Reactivation of a developmentally silenced embryonic globin gene

The α- and β-globin loci harbor developmentally expressed genes, which are silenced throughout post-natal life. Reactivation of these genes may offer therapeutic approaches for the hemoglobinopathies, the most common single gene disorders. Here, we address mechanisms regulating the embryonically expressed α-like globin, termed ζ-globin. We show that in embryonic erythroid cells, the ζ-gene lies within a ~65 kb sub-TAD (topologically associating domain) of open, acetylated chromatin and interacts with the α-globin super-enhancer. By contrast, in adult erythroid cells, the ζ-gene is packaged within a small (~10 kb) sub-domain of hypoacetylated, facultative heterochromatin within the acetylated sub-TAD and that it no longer interacts with its enhancers. The ζ-gene can be partially re-activated by acetylation and inhibition of histone de-acetylases. In addition to suggesting therapies for severe α-thalassemia, these findings illustrate the general principles by which reactivation of developmental genes may rescue abnormalities arising from mutations in their adult paralogues.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Engineering transcriptional regulation of pentose metabolism in Rhodosporidium toruloides for improved conversion of xylose to bioproducts

Abstract Efficient conversion of pentose sugars remains a significant barrier to the replacement of petroleum-derived chemicals with plant biomass-derived bioproducts. While the oleaginous yeast Rhodosporidium toruloides (also known as Rhodotorula toruloides ) has a relatively robust native metabolism of pentose sugars compared to other wild yeasts, faster assimilation of those sugars will be required for industrial utilization of pentoses. To increase the rate of pentose assimilation in R. toruloides, we leveraged previously reported high-throughput fitness data to identify potential regulators of pentose catabolism. Two genes were selected for further investigation, a putative transcription factor (RTO4_12978, Pnt1) and a homolog of a glucose transceptor involved in carbon catabolite repression (RTO4_11990). Overexpression of Pnt1 increased the specific growth rate approximately twofold early in cultures on xylose and increased the maximum specific growth by 18% while decreasing accumulation of arabitol and xylitol in fast-growing cultures. Improved growth dynamics on xylose translated to a 120% increase in the overall rate of xylose conversion to fatty alcohols in batch culture. Proteomic analysis confirmed that Pnt1 is a major regulator of pentose catabolism in R. toruloides. Deletion of RTO4_11990 increased the growth rate on xylose, but did not relieve carbon catabolite repression in the presence of glucose. Carbon catabolite repression signaling networks remain poorly characterized in R. toruloides and likely comprise a different set of proteins than those mainly characterized in ascomycete fungi.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1993

Sequence and Genetic Characterization of etrA, an fnr Analog that Regulates Anaerobic Respiration in Shewanella putrefaciens MR-1

An electron transport regulatory gene, etrA, has been isolated and characterized from the obligate respiratory bacterium Shewanella putrefaciens MR-l. The deduced amino acid sequence of etrA (EtrA) shows a high degree of identity to both the Fnr of Escherichia coli (73.6%) and the analogous protein (ANR) of Pseudomonas aeruginosa (50.8%). The four active cysteine residues of Fnr are conserved in EtrA, and the amino acid sequence of the DNA-binding domains of the two proteins are identical. Further, S.putrefaciens etrA is able to complement an fnr mutant of E.coli. In contrast to fnr, there is no recognizable Fnr box upstream of the etrA sequence. Gene replacement etr.A mutants of MR-1 were deficient in growth on nitrite, thiosulfate, sulfite, trimethylamine-N-oxide, dimethyl sulfoxide, Fe(III), and fumarate, suggesting that EtrA is involved in the regulation of the corresponding reductase genes. However, the mutants were all positive for reduction of and growth on nitrate and Mn(IV), indicating that EtrA is not involved in the regulation of these two systems. Southern blots of S.putrefaciens DNA with use of etrA as a probe revealed the expected etrA bands and a second set of hybridization signals whose genetic and functional properties remain to be determined.

Saffarini, Daad A.↗

NASA NTRS2016

Gene Expression and Structural Skeletal Responses to Long-Duration Simulated Microgravity in Rats

Spaceflight has deleterious effects on skeletal structure and function, specifically causingprofound loss in bone mass, density, and strength, as well as changes in expression levels of genes related to oxidative stress [Hyeon et al., Smith et al.]. It is known that bone resorption remains elevated after spaceflight and that bone density and strength fail to recover completely even years following spaceflight [Smith et al., Carpenter et al.]. However, our current understanding of the signaling pathways and molecular mechanisms that control bone loss and that link oxidative stress, bone resorption, and mechanical unloading of skeletal tissue is incomplete. Here, we aim to examine skeletal responses to simulated long-duration spaceflight on bone loss using the ground-based hindlimb unloading (HU) model in adult (9 months old) male rats. We hypothesized that simulated microgravity leads to the temporal regulation of oxidative-defense genes and pro-osteoclastogenic factors, showing progression and eventual plateau during long-term unloading, and that transient changes at early timepoints in these pathways precede skeletal adaptations to long-duration unloading. We will identify oxidativestress and bone resorption-related changes using global gene expression analysis (Affymetrix arrays) for both acute (within 14 days) and long-term timepoints (90 days). We will also use quantitative PCR to examine changes in expression of genes related to oxidative metabolism (e.g. Nrf2, SOD-1), bone turnover (resorption and formation markers, e.g. TRAP, osteocalcin respectively, SOST), and osteoclastogenesis (e.g. RANKL, OPG) at both early and late timepoints. We will then use detailed microarchitectural and structural analysis through microcomputed tomography to relate gene expression changes with structural changes in bone, expecting that plateaus in gene expression correlate with long-term changes in bone microarchitecture.

Rael, Victoria E.↗

DOE OSTI2024

Cysteine Rich Intestinal Protein 2 is a copper-responsive regulator of skeletal muscle differentiation and metal homeostasis

Copper (Cu) is essential for respiration, neurotransmitter synthesis, oxidative stress response, and transcription regulation, with imbalances leading to neurological, cognitive, and muscular disorders. Here we show the role of a novel Cu-binding protein (Cu-BP) in mammalian transcriptional regulation, specifically on skeletal muscle differentiation using murine primary myoblasts. Utilizing synchrotron X-ray fluorescence-mass spectrometry, we identified murine cysteine-rich intestinal protein 2 (mCrip2) as a key Cu-BP abundant in both nuclear and cytosolic fractions. mCrip2 binds two to four Cu + ions with high affinity and presents limited redox potential. CRISPR/Cas9-mediated deletion of mCrip2 impaired myogenesis, likely due to Cu accumulation in cells. CUT&RUN and transcriptome analyses revealed its association with gene promoters, including MyoD1 and metallothioneins, suggesting a novel Cu-responsive regulatory role for mCrip2. Our work describes the significance of mCrip2 in skeletal muscle differentiation and metal homeostasis, expanding understanding of the Cu-network in myoblasts. Copper (Cu) is essential for various cellular processes, including respiration and stress response, but imbalances can cause serious health issues. This study reveals a new Cu-binding protein (Cu-BP) involved in muscle development in primary myoblasts. Using unbiased metalloproteomic techniques and high throughput sequencing, we identified mCrip2 as a key Cu-BP found in cell nuclei and cytoplasm. mCrip2 binds up to four Cu + ions and has a limited redox potential. Deleting mCrip2 using CRISPR/Cas9 disrupted muscle formation due to Cu accumulation. Further analyses showed that mCrip2 regulates the expression of genes like MyoD1, essential for muscle differentiation, and metallothioneins in response to copper supplementation. This research highlights the importance of mCrip2 in muscle development and metal homeostasis, providing new insights into the Cu-network in cells.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2009

Cell Cycle Progression of Human Cells Cultured in Rotating Bioreactor

Space flight has been shown to alter the astronauts immune systems. Because immune performance is complex and reflects the influence of multiple organ systems within the host, scientists sought to understand the potential impact of microgravity alone on the cellular mechanisms critical to immunity. Lymphocytes and their differentiated immature form, lymphoblasts, play an important and integral role in the body's defense system. T cells, one of the three major types of lymphocytes, play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptors. Reported studies have shown that spaceflight can affect the expression of cell surface markers. Cell surface markers play an important role in the ability of cells to interact and to pass signals between different cells of the same phenotype and cells of different phenotypes. Recent evidence suggests that cell-cycle regulators are essential for T-cell function. To trigger an effective immune response, lymphocytes must proliferate. The objective of this project is to investigate the changes in growth of human cells cultured in rotating bioreactors and to measure the growth rate and the cell cycle distribution for different human cell types. Human lymphocytes and lymphoblasts will be cultured in a bioreactor to simulate aspects of microgravity. The bioreactor is a cylindrical culture vessel that incorporates the aspects of clinostatic rotation of a solid fluid body around a horizontal axis at a constant speed, and compensates gravity by rotation and places cells within the fluid body into a sustained free-fall. Cell cycle progression and cell proliferation of the lymphocytes will be measured for a number of days. In addition, RNA from the cells will be isolated for expression of genes related in cell cycle regulations.

Parks, Kelsey↗

DOE OSTI2023

A Synthetic Transcription Factor and Core Promoter System in Picochlorum renovo Enables Tunable Gene Expression

Picochlorum renovo is a recently characterized microalga of industrial interest. Its rapid growth rate, and high temperature and salinity tolerances make P. renovo an attractive candidate for industrial scale cultivation and downstream production of sustainable fuels and chemicals. Currently, genetic tools for many non-model microalgae are limited and would greatly benefit from an orthogonal gene expression system to bypass host regulation. Additionally, the engineering of complex metabolic pathways in eukaryotic organisms to optimize growth or biosynthesize high value products often requires tunable expression of each gene in a pathway. Here we explore a tunable orthogonal gene expression system using a synthetic transcription factor (sTF) and core promoters (CPs) conferring expression of the fluorescent protein mCherry to quantify protein expression. The sTF paired with the relevant binding site (BS) led to an ~5X increase in reporter gene expression compared to the native RuBisCo promoter, however had limited tunability with increasing BS number. Quantification of mCherry expression under 34 different CPs paired with the sTF and BS showed an order of magnitude of expression tunability. Future work with this system will entail generation of an overexpression library via random integration of the relevant BS in an sTF expressing P. renovo strain. With this sTF and CP system we aim to greatly improve growth rates and product titers in photosynthetic organisms, while also providing a potentially universal gene expression system for microalgae.

algae↗

DOE OSTI2021

Protective effect of SIRT6 on cholesterol crystal-induced endothelial dysfunction via regulating ACE2 expression

Highlights: • ACE2 was involved in CC-induced endothelial dysfunction. • SIRT1 and SIRT6 contributed to regulating the protein expression of ACE2 in CC-stimulated endothelial cells. • SIRT6 regulated ACE2 with intranuclear p-ATF2, but not affecting p-ATF2 expression in the cytoplasm. Sirtuins are a family of highly conserved nicotinamide adenine dinucleotide (NAD{sup +})-dependent enzymes. Among the sirtuins, SIRT1 and SIRT6 participate in the regulation of endothelial functions and play significant roles in the physiological and pathological processes of cardiovascular diseases (CVD). Recently, our study found that minute cholesterol crystals (CC) can be endocytosed by endothelial cells and further impair endothelial functions. Since previous studies have reported that angiotensin-converting enzyme (ACE2) involves Angiotensin (Ang) II-induced inflammation in endothelial cells, this study was designed to investigate the role of SIRT1 and SIRT6 in CC-induced variation of ACE2 expression and the related mechanism between SIRT6 and ACE2. We found that ACE2 is involved in CC-induced endothelial dysfunction, which inhibits decreases in nitric oxide (NO) level and endothelial nitric oxide synthase (eNOS) activity and increases in inflammatory factors and adhesion molecules. Besides, SIRT1 and SIRT6 regulated the protein expression of ACE2 in CC-stimulated human umbilical vein endothelial cells (HUVECs). Moreover, bioinformatics analysis from the Enrichr database indicated that activating transcription factor 2 (ATF2), is highly correlated with genes that significantly upregulated after infection with the SIRT6 adenovirus vector. In CC-induced HUVECs, ACE2 expression was up-regulated in cells transfected with ATF2 siRNA. However, further mechanism studies revealed that overexpression of SIRT6 decreases the accumulation of p-ATF2 in the nucleus, but did not affect p-ATF2 expression in the cytoplasm. Taken together, these data indicated that SIRT6 regulates ACE2 might via inhibiting the accumulation of nucleus p-ATF2 in CC-induced endothelial dysfunction.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

REC protein family expansion by the emergence of a new signaling pathway

This report presents multi-genome evidence that REC protein family expansion occurs when the emergence of new pathways gives rise to functional discordance. Specificity between residues in REC domain containing response regulators with paired histidine kinases is under negative purifying selection, constrained by the presence of other bacterial two-component systems signaling cascades that share sequence and structural identity. Presuming that the two-component systems can evolve by neutral amino acid changes (neutral drift) when purifying evolutionary constraints are relaxed, how might the REC protein family expand by amino acid changes when these constraints remain intact? Using an unsupervised machine learning approach to observe the sequence landscape of REC domains across long phylogenetic distances, we find that within-gene recombination, a subcategory of gene conversion, switched the effector domain and, consequently, the regulatory context of a duplicated response regulator from transcriptional regulation by σ54 to that by σ70. We determined that the recombined response regulator diverged from its parent by episodic diversifying selection and neutral drift. Functional experiments of the parent of recombined response regulators in a model Pseudomonas putida KT2440 model system revealed that the parent and recombined response regulators sense and respond to different carboxylic acids. Finally, a residue-switching experiment using structural predictions and functional characterization suggests that the new residues in the recombined regulator could form a new interaction interface and mediate condition-specific phosphotransfer. Overall, our study finds that genetic perturbations can create conditions of functional discordance, whereby the REC protein family can evolve by episodic diversifying selection.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Chlamydomonas CHT7 is involved in repressing DNA replication and mitotic genes during synchronous growth

In the green alga Chlamydomonas reinhardtii, regulation of the cell cycle in response to external cues is critical for survival in a changing environment. The loss of the nuclear COMPROMISED HYDROLYSIS OF TRIACYLGLYCEROLS 7 (CHT7) protein affects the expression of many genes especially in response to nitrogen availability. Cells lacking CHT7 exhibit abnormal cell morphology following nitrogen deprivation and fail to resume normal cell division after N resupply. To investigate the function of CHT7 in the regulation of cell cycle-related pathways, cells were synchronized, and RNA-seq analysis was performed during various stages of the cell cycle. In the cht7 mutant following nitrogen deprivation, the cells were not dividing, but a subset of cell cycle genes involved in DNA replication and mitosis were found to be derepressed, suggesting that the CHT7 protein plays a role in cell cycle regulation that is opposite to that of the mitotic cyclin-dependent kinases. Furthermore, genes for cell wall synthesis and remodeling were found to be abnormally induced in nondividing cht7 cells; this misregulation may deplete cellular resources and thus contribute to cell death following nitrogen deprivation. Lastly, 43 minimally characterized kinases were found to be highly misregulated in cht7. Further analysis suggested that some of these CHT7-regulated kinases may be related to the MAP3K and Aurora-like kinases, while others are unique. Together, these results suggest a role of CHT7 in transcriptional regulation of the cell cycle and reveal several pathways and genes whose expression appears to be subject to a CHT7-mediated regulatory network.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Data for Characterization of the Ghd8 Flowering Time Gene in a Mini-Core Collection of Miscanthus sinensis

The optimal flowering time for bioenergy crop miscanthus is essential for environmental adaptability and biomass accumulation. However, little is known about how genes controlling flowering in other grasses contribute to flowering regulation in miscanthus. Here, we report on the sequence characterization and gene expression of Miscanthus sinensisGhd8 , a transcription factor encoding a HAP3/NF-YB DNA-binding domain, which has been identified as a major quantitative trait locus in rice, with pleiotropic effects on grain yield, heading date and plant height. In M. sinensis , we identified two homoeologous loci, MsiGhd8A located on chromosome 13 and MsiGhd8B on chromosome 7, with one on each of this paleo-allotetraploid species’ subgenomes. A total of 46 alleles and 28 predicted protein sequence types were identified in 12 wild-collected accessions. Several variants of MsiGhd8 showed a geographic and latitudinal distribution. Quantitative real-time PCR revealed that MsiGhd8 expressed under both long days and short days, and MsiGhd8B showed a significantly higher expression than MsiGhd8A . The comparison between flowering time and gene expression indicated that MsiGhd8B affected flowering time in response to day length for some accessions. This study provides insight into the conserved function of Ghd8 in the Poaceae, and is an important initial step in elucidating the flowering regulatory network of Miscanthus.

Feedstock Production↗

DOE OSTI2021

Characterization of the Ghd8 Flowering Time Gene in a Mini-Core Collection of Miscanthus sinensis

The optimal flowering time for bioenergy crop Miscanthus is essential for environmental adaptability and biomass accumulation. However, little is known about how genes controlling flowering in other grasses contribute to flowering regulation in Miscanthus. Here, we report on the sequence characterization and gene expression of Miscanthus sinensisGhd8, a transcription factor encoding a HAP3/NF-YB DNA-binding domain, which has been identified as a major quantitative trait locus in rice, with pleiotropic effects on grain yield, heading date and plant height. In M. sinensis, we identified two homoeologous loci, MsiGhd8A located on chromosome 13 and MsiGhd8B on chromosome 7, with one on each of this paleo-allotetraploid species’ subgenomes. A total of 46 alleles and 28 predicted protein sequence types were identified in 12 wild-collected accessions. Several variants of MsiGhd8 showed a geographic and latitudinal distribution. Quantitative real-time PCR revealed that MsiGhd8 expressed under both long days and short days, and MsiGhd8B showed a significantly higher expression than MsiGhd8A. The comparison between flowering time and gene expression indicated that MsiGhd8B affected flowering time in response to day length for some accessions. This study provides insight into the conserved function of Ghd8 in the Poaceae, and is an important initial step in elucidating the flowering regulatory network of Miscanthus.

geographic distribution↗