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DOE OSTI2024

Chlamydomonas cells transition through distinct Fe nutrition stages within 48 h of transfer to Fe-free medium

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Chlamydomonas cells transition through distinct Fe nutrition stages within 48 h of transfer to Fe-free medium

Low iron (Fe) bioavailability can limit the biosynthesis of Fe-containing proteins, which are especially abundant in photosynthetic organisms, thus negatively affecting global primary productivity. Understanding cellular coping mechanisms under Fe limitation is therefore of great interest. We surveyed the temporal responses of Chlamydomonas (Chlamydomonas reinhardtii) cells transitioning from an Fe-rich to an Fe-free medium to document their short- and long-term adjustments. While slower growth, chlorosis and lower photosynthetic parameters are evident only after one or more days in Fe-free medium, the abundance of some transcripts, such as those for genes encoding transporters and enzymes involved in Fe assimilation, change within minutes, before changes in intracellular Fe content are noticeable, suggestive of a sensitive mechanism for sensing Fe. Promoter reporter constructs indicate a transcriptional component to this immediate primary response. With acetate provided as a source of reduced carbon, transcripts encoding respiratory components are maintained relative to transcripts encoding components of photosynthesis and tetrapyrrole biosynthesis, indicating metabolic prioritization of respiration over photosynthesis. In contrast to the loss of chlorophyll, carotenoid content is maintained under Fe limitation despite a decrease in the transcripts for carotenoid biosynthesis genes, indicating carotenoid stability. These changes occur more slowly, only after the intracellular Fe quota responds, indicating a phased response in Chlamydomonas, involving both primary and secondary responses during acclimation to poor Fe nutrition. Overall design: Sampling of Chlamydomonas CC-4532 cells cultivated photoheterotrophically (TAP) under Fe-starvation condition (0 uM Fe-EDTA). Samples were collected at multiple timepoints from biological duplicate cultures after washing in TAP medium lacking Fe. Two time courses were collected. A short time course with t=0 (pre-wash), 0 (post-wash), 5, 10, 15, 30, 60, 120, and 240 min. A long time course with t= 0, 0.5, 1, 2, 4, 8, 12, 24 and 48 hours. Please note that, for long time course, the GSE44611/PRJNA190650 samples were re-used/re-analyzed together with the short time course data: GSM1087792 C.reinhardtii_Fe_Long_0_hours SRX245324 SAMN01924672 GSM1087793 C.reinhardtii_Fe_Long_0.5_hours SRX245325 SAMN01924673 GSM1087794 C.reinhardtii_Fe_Long_1_hours SRX245326 SAMN01924674 GSM1087795 C.reinhardtii_Fe_Long_2_hours SRX245327 SAMN01924675 GSM1087796 C.reinhardtii_Fe_Long_4_hours SRX245328 SAMN01924676 GSM1087797 C.reinhardtii_Fe_Long_8_hours SRX245329 SAMN01924677 GSM1087798 C.reinhardtii_Fe_Long_12_hours SRX245330 SAMN01924678 GSM1087799 C.reinhardtii_Fe_Long_24_hours SRX245331 SAMN01924679 GSM1087800 C.reinhardtii_Fe_Long_48_hours SRX245332 SAMN01924680

Source record↗

DOE OSTI2024

Unveiling the Arsenal of Apple Bitter Rot Fungi: Comparative Genomics Identifies Candidate Effectors, CAZymes, and Biosynthetic Gene Clusters in Colletotrichum Species

The bitter rot of apple is caused by Colletotrichum spp. and is a serious pre-harvest disease that can manifest in postharvest losses on harvested fruit. In this study, we obtained genome sequences from four different species, C. chrysophilum, C. noveboracense, C. nupharicola, and C. fioriniae, that infect apple and cause diseases on other fruits, vegetables, and flowers. Our genomic data were obtained from isolates/species that have not yet been sequenced and represent geographic-specific regions. Genome sequencing allowed for the construction of phylogenetic trees, which corroborated the overall concordance observed in prior MLST studies. Bioinformatic pipelines were used to discover CAZyme, effector, and secondary metabolic (SM) gene clusters in all nine Colletotrichum isolates. We found redundancy and a high level of similarity across species regarding CAZyme classes and predicted cytoplastic and apoplastic effectors. SM gene clusters displayed the most diversity in type and the most common cluster was one that encodes genes involved in the production of alternapyrone. Our study provides a solid platform to identify targets for functional studies that underpin pathogenicity, virulence, and/or quiescence that can be targeted for the development of new control strategies. With these new genomics resources, exploration via omics-based technologies using these isolates will help ascertain the biological underpinnings of their widespread success and observed geographic dominance in specific areas throughout the country.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Wildfire impact on soil microbiome life history traits and roles in ecosystem carbon cycling

Abstract Wildfires, which are increasing in frequency and severity with climate change, reduce soil microbial biomass and alter microbial community composition and function. The soil microbiome plays a vital role in carbon (C) and nitrogen (N) cycling, but its complexity makes it challenging to predict post-wildfire soil microbial dynamics and resulting impacts on ecosystem biogeochemistry. The application of biogeochemically relevant conceptual trait-based frameworks to the soil microbiome can distill this complexity, enabling enhanced predictability of soil microbiome recovery following wildfire and subsequent impacts to biogeochemical cycles. Conceptual frameworks that have direct links to soil C and N cycling have been developed for the soil microbiome; the Y-A-S framework overviews soil microbiome life history strategies that have tradeoffs with one another and others have proposed frameworks specific to wildfire. Here, we aimed to delineate post-wildfire changes of bacterial traits in western US coniferous forests to inform how severe wildfire influences soil microbiome recovery and resultant biogeochemical cycling. We utilized a comprehensive metagenome-assembled genome catalog from post-wildfire soils representing 1 to 11 years following low- and high-severity burning to identify traits that enable the persistence of microbial taxa in burned soils and influence ecosystem C and N cycling. We found that high-severity wildfire initially selects for fast growers and, up to a decade post-fire, taxa that invest in genes for acquiring diverse resources from the external environment, which in combination could increase soil C losses. This work begins to disentangle how climate change–induced shifts in wildfire behavior might alter microbially mediated soil biogeochemical cycling.

Nelson, Amelia R.↗

DOE OSTI2024

Biosensors for the detection of chorismate and cis,cis -muconic acid in Corynebacterium glutamicum

Abstract Corynebacterium glutamicum ATCC 13032 is a promising microbial chassis for industrial production of valuable compounds, including aromatic amino acids derived from the shikimate pathway. In this work, we developed two whole-cell, transcription factor based fluorescent biosensors to track cis,cis-muconic acid (ccMA) and chorismate in C. glutamicum. Chorismate is a key intermediate in the shikimate pathway from which value-added chemicals can be produced, and a shunt from the shikimate pathway can divert carbon to ccMA, a high value chemical. We transferred a ccMA-inducible transcription factor, CatM, from Acinetobacter baylyi ADP1 into C. glutamicum and screened a promoter library to isolate variants with high sensitivity and dynamic range to ccMA by providing benzoate, which is converted to ccMA intracellularly. The biosensor also detected exogenously supplied ccMA, suggesting the presence of a putative ccMA transporter in C. glutamicum, though the external ccMA concentration threshold to elicit a response was 100-fold higher than the concentration of benzoate required to do so through intracellular ccMA production. We then developed a chorismate biosensor, in which a chorismate inducible promoter regulated by natively expressed QsuR was optimized to exhibit a dose-dependent response to exogenously supplemented quinate (a chorismate precursor). A chorismate–pyruvate lyase encoding gene, ubiC, was introduced into C. glutamicum to lower the intracellular chorismate pool, which resulted in loss of dose dependence to quinate. Further, a knockout strain that blocked the conversion of quinate to chorismate also resulted in absence of dose dependence to quinate, validating that the chorismate biosensor is specific to intracellular chorismate pool. The ccMA and chorismate biosensors were dually inserted into C. glutamicum to simultaneously detect intracellularly produced chorismate and ccMA. Biosensors, such as those developed in this study, can be applied in C. glutamicum for multiplex sensing to expedite pathway design and optimization through metabolic engineering in this promising chassis organism. One-Sentence Summary High-throughput screening of promoter libraries in Corynebacterium glutamicum to establish transcription factor based biosensors for key metabolic intermediates in shikimate and β-ketoadipate pathways.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Two related families of metal transferases, ZNG1 and ZNG2, are involved in acclimation to poor Zn nutrition in Arabidopsis

Metal homeostasis has evolved to tightly modulate the availability of metals within the cell, avoiding cytotoxic interactions due to excess and protein inactivity due to deficiency. Even in the presence of homeostatic processes, however, low bioavailability of these essential metal nutrients in soils can negatively impact crop health and yield. While research has largely focused on how plants assimilate metals, acclimation to metal-limited environments requires a suite of strategies that are not necessarily involved in metal transport across membranes. The identification of these mechanisms provides a new opportunity to improve metal-use efficiency and develop plant foodstuffs with increased concentrations of bioavailable metal nutrients. Here, we investigate the function of two distinct subfamilies of the nucleotide-dependent metallochaperones (NMCs), named ZNG1 and ZNG2, that are found in plants, using Arabidopsis thaliana as a reference organism. AtZNG1 (AT1G26520) is an ortholog of human and fungal ZNG1, and like its previously characterized eukaryotic relatives, localizes to the cytosol and physically interacts with methionine aminopeptidase type I (AtMAP1A). Analysis of At ZNG1 , At MAP1A , At MAP2A , and At MAP2B transgenic mutants are consistent with the role of Arabidopsis ZNG1 as a Zn transferase for AtMAP1A, as previously described in yeast and zebrafish. Structural modeling reveals a flexible cysteine-rich loop that we hypothesize enables direct transfer of Zn from AtZNG1 to AtMAP1A during GTP hydrolysis. Based on proteomics and transcriptomics, loss of this ancient and conserved mechanism has pleiotropic consequences impacting the expression of hundreds of genes, including those involved in photosynthesis and vesicle transport. Members of the plant-specific family of NMCs, ZNG2A1 (AT1G80480) and ZNG2A2 (AT1G15730), are also required during Zn deficiency, but their target protein(s) remain to be discovered. In conclusion, RNA-seq analyses reveal wide-ranging impacts across the cell when the genes encoding these plastid-localized NMCs are disrupted.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

T-Toxin Virulence Genes: Unconnected Dots in a Sea of Repeats

In 1970 a corn disease epidemic ravaged fields in the United States to great economic loss. The outbreak was caused by a never-before seen, supervirulent strain of the fungal pathogen Cochliobolus heterostrophus. This was a plant disease epidemic, however, the current COVID-19 pandemic of humans is a stark reminder that novel, highly virulent, pathogens evolve with devastating consequences, no matter what the host-animal, plant, or other organism.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Deletion of the A137R Gene from the Pandemic Strain of African Swine Fever Virus Attenuates the Strain and Offers Protection against the Virulent Pandemic Virus

African swine fever virus (ASFV) is causing a devastating pandemic in domestic and wild swine within an extended geographical area from Central Europe to East Asia, resulting in economic losses for the regional swine industry. There are no commercial vaccines; therefore, disease control relies on identification and culling of infected animals. We report here that the deletion of the ASFV gene A137R from the highly virulent ASFV-Georgia2010 (ASFV-G) isolate induces a significant attenuation of virus virulence in swine. A recombinant virus lacking the A137R gene, ASFV-G-DA137R, was developed to assess the role of this gene in ASFV virulence in domestic swine. Animals inoculated intramuscularly with 102 50% hemadsorption doses (HAD50) of ASFV-G-DA137R remained clinically healthy during the 28-day observational period. All animals inoculated with ASFV-G-DA137R had medium to high viremia titers and developed a strong virus-specific antibody response. Importantly, all ASFV-G-DA137Rinoculated animals were protected when challenged with the virulent parental strain ASFV-G. No evidence of replication of challenge virus was observed in the ASFV-GDA137R-inoculated animals. Therefore, ASFV-G-DA137R is a novel potential live attenuated vaccine candidate and one of the few experimental vaccine strains reported to induce protection against the highly virulent ASFV Georgia virus that is the cause of the current Eurasian pandemic.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Deletion of a Peptidylprolyl Isomerase Gene Results in the Inability of Caldicellulosiruptor bescii To Grow on Crystalline Cellulose without Affecting Protein Glycosylation or Growth on Soluble Substrates

Caldicellulosiruptor bescii secretes a large number of complementary multifunctional enzymes with unique activities for biomass deconstruction. The most abundant enzymes in the C. bescii secretome are found in a unique gene cluster containing a glycosyl transferase (GT39) and a putative peptidyl prolyl cis-trans isomerase. Deletion of the glycosyl transferase in this cluster resulted in loss of detectable protein glycosylation in C. bescii, and its activity has been shown to be responsible for the glycosylation of the proline-threonine rich linkers found in many of the multifunctional cellulases. The presence of a putative peptidyl prolyl cis-trans isomerase within this gene cluster suggested that it might also play a role in cellulase modification. Here, we identify this gene as a putative prsA prolyl cis-trans isomerase. Deletion of prsA2 leads to the inability of C. bescii to grow on insoluble substrates such as Avicel, the model cellulose substrate, while exhibiting no differences in phenotype with the wild-type strain on soluble substrates. Finally, we provide evidence that the prsA2 gene is likely needed to increase solubility of multifunctional cellulases and that this unique gene cluster was likely acquired by members of the Caldicellulosiruptor genus with a group of genes to optimize the production and activity of multifunctional cellulases.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Second-Phase Hepatitis C Plasma Viral Kinetics Directly Reflects Reduced Intrahepatic Burden of Hepatitis C Virus

Mathematical models explain how antivirals control viral infections. Hepatitis C virus (HCV) treatment results in at least 2 phases of decline in viremia. Here, the first phase reflects clearance of rapidly produced virions. The second phase is hypothesized to derive from loss of infected cells but has been challenging to prove. Using single-cell methods, we quantified the number of hepatitis C virus (HCV)-infected hepatocytes in liver biopsies taken before and within 7 days of initiating direct-acting antivirals (DAAs) in a double-blinded randomized controlled trial testing 2 (sofosbuvir-velpatasvir) versus 3 (sofosbuvir-velpatasvir-voxilaprevir) DAAs. We employed thousands of intrahepatic measurements in 10 persons with chronic genotype 1a HCV infection: median proportion of infected hepatocytes declined from 11.3% (range, 1.3%–59%) to 0.6% (range, <0.3%–5.8%), a loss of 75%–95% infected hepatocytes. Plasma viremia correlated with numbers of HCV-infected hepatocytes (r = 0.77; P < .0001). Second-phase plasma dynamics and changes in infected hepatocytes were indistinct (P = .16), demonstrating that second-phase viral dynamics derive from loss of infected cells. DAAs led to a decline in intracellular HCV RNA and interferon-stimulated gene expression (P < .05 for both). We proved that second-phase viral dynamics reflect decay of intrahepatic burden of HCV, partly due to clearance of HCV RNA from hepatocytes.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

A DE1 BINDING FACTOR 1–GLABRA2 module regulates rhamnogalacturonan I biosynthesis in Arabidopsis seed coat mucilage

Abstract The mucilage surrounding hydrated Arabidopsis thaliana seeds is a specialized extracellular matrix composed mainly of the pectic polysaccharide rhamnogalacturonan I (RG-I). Although, several genes responsible for RG-I biosynthesis have been identified, the transcriptional regulatory mechanisms controlling RG-I production remain largely unknown. Here we report that the trihelix transcription factor DE1 BINDING FACTOR 1 (DF1) is a key regulator of mucilage RG-I biosynthesis. RG-I biosynthesis is significantly reduced in loss-of-function mutants of DF1. DF1 physically interacts with GLABRA2 (GL2) and both proteins transcriptionally regulate the expression of the RG-I biosynthesis genes MUCILAGE MODIFIED 4 (MUM4) and GALACTURONOSYLTRANSFERASE-LIKE5 (GATL5). Through chromatin immunoprecipitation-quantitative PCR and transcriptional activation assays, we uncover a cooperative mechanism of the DF1–GL2 module in activating MUM4 and GATL5 expression, in which DF1 binds to the promoters of MUM4 and GATL5 through interacting with GL2 and facilitates the transcriptional activity of GL2. The expression of DF1 and GL2 is directly regulated by TRANSPARENT TESTA GLABRA2 (TTG2) and, in turn, DF1 directly represses the expression of TTG2. Taken together, our data reveal that the transcriptional regulation of mucilage RG-I biosynthesis involves a regulatory module, comprising DF1, GL2, and TTG2.

Biochemistry & Molecular Biology↗

DOE OSTI2023

Sugar accumulation enhancement in sorghum stem is associated with reduced reproductive sink strength and increased phloem unloading activity

Sweet sorghum has emerged as a promising source of bioenergy mainly due to its high biomass and high soluble sugar yield in stems. Studies have shown that loss-of-function Dry locus alleles have been selected during sweet sorghum domestication, and decapitation can further boost sugar accumulation in sweet sorghum, indicating that the potential for improving sugar yields is yet to be fully realized. To maximize sugar accumulation, it is essential to gain a better understanding of the mechanism underlying the massive accumulation of soluble sugars in sweet sorghum stems in addition to the Dry locus. We performed a transcriptomic analysis upon decapitation of near-isogenic lines for mutant (d, juicy stems, and green leaf midrib) and functional (D, dry stems and white leaf midrib) alleles at the Dry locus. Our analysis revealed that decapitation suppressed photosynthesis in leaves, but accelerated starch metabolic processes in stems. SbbHLH093 negatively correlates with sugar levels supported by genotypes (DD vs. dd), treatments (control vs. decapitation), and developmental stages post anthesis (3d vs.10d). D locus gene SbNAC074A and other programmed cell death-related genes were downregulated by decapitation, while sugar transporter-encoding gene SbSWEET1A was induced. Both SbSWEET1A and Invertase 5 were detected in phloem companion cells by RNA in situ assay. Loss of the SbbHLH093 homolog, AtbHLH093, in Arabidopsis led to a sugar accumulation increase. This study provides new insights into sugar accumulation enhancement in bioenergy crops, which can be potentially achieved by reducing reproductive sink strength and enhancing phloem unloading.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

HPRT promotes proliferation and metastasis in head and neck squamous cell carcinoma through direct interaction with STAT3

Highlights: • The higher expression of HPRT in HNSCC tissue was related to poor prognosis of patients. • Overexpression of HPRT increased the gene expression of epithelial mesenchymal transition markers. • HPRT directly interacted with STAT3. • Knocking down HPRT significantly decreased tumour growth and enhanced the anticancer effect against HNSCC xenografts. Increasing effort has been put into finding novel molecular pathways to improve the efficiency of EGFR inhibitors against head and neck squamous cell cancer (HNSCC). In this study, we performed data mining and bioinformatically analysed RNA-Seq data downloaded from TCGA and confirmed that higher expression of HPRT in HNSCC tissue was related to poor prognosis of patients. Then, we conducted in vitro and in vivo loss- and gain-of-function experiments to demonstrate the role of HPRT in HNSCC cell lines. Overexpression of HPRT increased the gene expression of epithelial mesenchymal transition markers via direct interaction with STAT3. Knocking down HPRT significantly decreased tumour growth and enhanced the anticancer effect of EGFR inhibitors against HNSCC xenografts. In conclusion, HPRT is a binding partner of STAT3 that promotes EMT and proliferation. Our findings support HPRT as a promising prognostic indicator and potential therapeutic target for HNSCC.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2022

Low-abundance populations distinguish microbiome performance in plant cell wall deconstruction

Abstract Background Plant cell walls are interwoven structures recalcitrant to degradation. Native and adapted microbiomes can be particularly effective at plant cell wall deconstruction. Although most understanding of biological cell wall deconstruction has been obtained from isolates, cultivated microbiomes that break down cell walls have emerged as new sources for biotechnologically relevant microbes and enzymes. These microbiomes provide a unique resource to identify key interacting functional microbial groups and to guide the design of specialized synthetic microbial communities. Results To establish a system assessing comparative microbiome performance, parallel microbiomes were cultivated on sorghum ( Sorghum bicolor L. Moench) from compost inocula. Biomass loss and biochemical assays indicated that these microbiomes diverged in their ability to deconstruct biomass. Network reconstructions from gene expression dynamics identified key groups and potential interactions within the adapted sorghum-degrading communities, including Actinotalea , Filomicrobium , and Gemmatimonadetes populations. Functional analysis demonstrated that the microbiomes proceeded through successive stages that are linked to enzymes that deconstruct plant cell wall polymers. The combination of network and functional analysis highlighted the importance of cellulose-degrading Actinobacteria in differentiating the performance of these microbiomes. Conclusions The two-tier cultivation of compost-derived microbiomes on sorghum led to the establishment of microbiomes for which community structure and performance could be assessed. The work reinforces the observation that subtle differences in community composition and the genomic content of strains may lead to significant differences in community performance.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Feed status and skin injury modulate immunopathology, global gene expression, and survival in channel catfish during virulent Aeromonas hydrophila infection

Introduction VirulentAeromonas hydrophilais a major pathogen in channel catfish (Ictalurus punctatus), that causes motileAeromonassepticemia and significant economic losses. We investigated the effect of feeding status and skin integrity on the host immune response, disease survival, and gastrointestinal pathology following a vAh challenge. Methods Using a bath immersion model, channel catfish were divided into four treatment groups: fin clipped and fed (FCF), fin clipped but not fed (FCN), not fin clipped but fed (NCF), and not fin clipped nor fed (NCN) alongside non-challenged control groups The FCF and NCF groups were fed 2 h prior to the challenge, but the FCN and NCN groups were not. Survival analysis, histopathological assessment, and RNA sequencing were conducted across groups at different time intervals throughout the vAh challenge. Results Survival rates were lowest in the FCF and FCN groups (30% and 23% survival, respectively), suggesting that both feeding and skin damage contributed to disease severity. Histopathological analyses revealed more severe intestinal and gastric lesions in fed groups, characterized by epithelial necrosis, hemorrhage, and edema. Transcriptomic analysis among the groups identified significant differentially expressed genes associated with inflammation, apoptosis, and metabolic stress, with notable upregulation of interleukin 1-beta (il-1β), and complement C3 (c3). Gene ontology enrichment highlighted distinct immune activation patterns between fed and unfed groups, with enhanced pathogen recognition and pro-inflammatory responses in unfed fish. Discussion These findings suggest feeding prior to infection may exacerbate disease pathology, potentially by creating a physiological state conducive to facilitate pathogen proliferation and dampened early immune responses, whereas short-term fasting appears to promote early immune activation. This study provides novel insights into the complex interplay between feed status, physical injury, and immune response to vAh infection.

Immunology↗

DOE OSTI2023

A spatio-temporally constrained gene regulatory network directed by PBX1/2 acquires limb patterning specificity via HAND2

A lingering question in developmental biology has centered on how transcription factors with widespread distribution in vertebrate embryos can perform tissue-specific functions. Here, using the murine hindlimb as a model, we investigate the elusive mechanisms whereby PBX TALE homeoproteins, viewed primarily as HOX cofactors, attain context-specific developmental roles despite ubiquitous presence in the embryo. We first demonstrate that mesenchymal-specific loss of PBX1/2 or the transcriptional regulator HAND2 generates similar limb phenotypes. By combining tissue-specific and temporally controlled mutagenesis with multi-omics approaches, we reconstruct a gene regulatory network (GRN) at organismal-level resolution that is collaboratively directed by PBX1/2 and HAND2 interactions in subsets of posterior hindlimb mesenchymal cells. Genome-wide profiling of PBX1 binding across multiple embryonic tissues further reveals that HAND2 interacts with subsets of PBX-bound regions to regulate limb-specific GRNs. Our research elucidates fundamental principles by which promiscuous transcription factors cooperate with cofactors that display domain-restricted localization to instruct tissue-specific developmental programs.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

An IRE1-proteasome system signalling cohort controls cell fate determination in unresolved proteotoxic stress of the plant endoplasmic reticulum

Excessive accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress, which is an underlying cause of major crop losses and devastating human conditions. Here, ER proteostasis surveillance is mediated by the conserved master regulator of the unfolded protein response (UPR), Inositol Requiring Enzyme 1 (IRE1), which determines cell fate by controlling pro-life and pro-death outcomes through as yet largely unknown mechanisms. Here we report that Arabidopsis IRE1 determines cell fate in ER stress by balancing the ubiquitin–proteasome system (UPS) and UPR through the plant-unique E3 ligase, PHOSPHATASE TYPE 2CA (PP2CA)-INTERACTING RING FINGER PROTEIN 1 (PIR1). Indeed, PIR1 loss leads to suppression of pro-death UPS and the lethal phenotype of an IRE1 loss-of-function mutant in unresolved ER stress in addition to activating pro-survival UPR. Specifically, in ER stress, PIR1 loss stabilizes ABI5, a basic leucine zipper (bZIP) transcription factor, that directly activates expression of the critical UPR regulator gene, bZIP60, triggering transcriptional cascades enhancing pro-survival UPR. Collectively, our results identify new cell fate effectors in plant ER stress by showing that IRE1’s coordination of cell death and survival hinges on PIR1, a key pro-death component of the UPS, which controls ABI5, a pro-survival transcriptional activator of bZIP60.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

Engineered Production of Hydroxycinnamoyl Tyramine Conjugates Limits the Growth of the Pathogen Pseudomonas syringae in Arabidopsis

Hydroxycinnamoyl tyramine conjugates are phenolamides produced by plants in response to pathogen attack and biotic stresses. Their proposed mechanisms of action include cytotoxicity towards pathogens, cell wall reinforcement to restrict pathogen proliferation, and signaling activity to trigger general stress responses. Here, we engineered the production of the tyramine conjugates p-coumaroyltyramine (CT) and feruloyltyramine (FT) in Arabidopsis to gain insight into their mode of action. Co-expression of feedback-insensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and tyrosine decarboxylase increased tyramine content. Additional expression of tyramine hydroxycinnamoyltransferase led to de-novo production of CT and FT, which were found as soluble and cell-wall-bound forms. FT was associated with lignin in stems. The growth of pathogenic Pseudomonas syringae was reduced in rosettes of the Arabidopsis CT- and FT-producing lines compared to wild type. These lines also exhibited increased transpirational water loss in excised rosettes. Transcriptomic analysis of transgenic lines grown under normal conditions revealed alterations in the expression of genes associated with the biological circadian clock. These changes led to a reduction in flavonoids and an early flowering phenotype. Important changes in the expression of genes related to abiotic stress such as drought, cold, heat, and hypoxia potentially contribute to reduced growth of P. syringae in engineered Arabidopsis.

Phenolamides↗