Biology subjects

Fisher, K. E.

Publications and source records attributed to Fisher, K. E..

7 recordsLinked to original sources

bioRxiv2025

Cryptochrome 2 is necessary for promoting a blue light-independent chloroplast signal to control cellular degradation

To thrive in dynamic environments, plants can use their photosynthetic plastid (chloroplast) organelles to sense environmental changes and relay this information to the cell. This signaling can be initiated within chloroplasts by the accumulation of reactive oxygen species (ROS) such as singlet oxygen (1O2), which can accumulate under stress and initiate retrograde (chloroplast-to-nucleus) signaling, chloroplast degradation, and programmed cell death (PCD). However, the mechanisms driving these signals, and the relationship between gene expression and cellular degradation, are unknown. To understand this physiology, here we use the Arabidopsis thaliana plastid ferrochelatase 2 (fc2) mutant, which conditionally produces chloroplast 1O2 and initiates signaling. We show that the blue light photoreceptor cryptochrome 2 (CRY2) is necessary to promote 1O2-induced chloroplast degradation and PCD in the fc2 mutant and that cry2 blocks cellular degradation without affecting bulk 1O2 levels, suggesting that signaling, rather than photo-oxidative stress, has been blocked. Furthermore, a global RNA transcript analysis demonstrated that the impact of the cry2 mutation on retrograde signaling is limited and that the effect of 1O2 on nuclear gene expression and cellular degradation can be uncoupled. Under permissive conditions, fc2 cry2 mutants exhibited constitutive expression of photo-oxidative stress genes, suggesting that cry2 may lead to transcriptional priming to photo-oxidative stress. Finally, we demonstrate that this function of CRY2 is independent of blue light-signaling and that CRY2 isoforms lacking canonical blue light signaling capabilities restore 1O2-induced PCD to fc2 cry2. Together, these results reveal a blue light-independent role for CRY2 in chloroplast signaling and stress acclimation pathways.

plant biology↗

bioRxiv2025

Efficient long-term cryopreservation of Arthrospira platensis NIES-39 and adaptation for the Space Algae-2 spaceflight experiment

Arthrospira platensis, commonly known as spirulina, is a photosynthetically efficient cyanobacterium used for production of nutritional supplements, biofuel, wastewater treatment, and CO2 sequestration. These traits and uses make it a candidate for bioregenerative life support systems for space travel and future habitats. However, A. platensis lacks a cryopreservation method for long-term storage of viable cells, and genetic stocks are currently sub-cultured for maintenance. Here we report an efficient cryopreservation method for A. platensis strain NIES-39 and adapt this method for an upcoming spaceflight experiment. Seven cryoprotective agents were tested to preserve viable A. platensis. We found that a combination of dimethyl sulfoxide and trehalose additives to Zarrouks media protected trichome viability for extended storage periods at -80 {degrees}C. Throughout one year of frozen storage, we observed equivalent viability of cryopreserved cells in comparison to non-frozen cultures. Equivalent cryopreservation was demonstrated with multiple volumes of frozen culture, culture containers, and thawing methods to adapt the method for delivery and return of viable NIES-39 for the Space Algae-2 experiment on the International Space Station. The method requires minimal resources, employs simple freezing and thawing procedures, and could be implemented in commercial production to preserve genetic stocks.

microbiology↗

bioRxiv2021

Singlet oxygen leads to structural changes to chloroplasts during degradation in the Arabidopsis thaliana plastid ferrochelatase two mutant

During photosynthesis, chloroplasts can produce large amounts of reactive oxygen species (ROS), particularly under stressful conditions. Along with other nutrients, chloroplasts also contain 80% of a leafs nitrogen supply. For these reasons, chloroplasts are prime targets for cellular degradation to protect cells from photo-oxidative damage and to redistribute nutrients to sink tissues. Multiple chloroplast degradation pathways have been described and are induced by photo-oxidative stress and nutrient starvation. However, the mechanisms by which damaged or senescing chloroplasts are identified, transported to the central vacuole, and ultimately degraded are not well characterized. Here, we investigated the subcellular structures involved with degrading chloroplasts induced by the ROS singlet oxygen (1O2) in the Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant. Using a three-dimensional serial-block face electron microscopy analysis, we show up to 35% of degrading chloroplasts in fc2 mutants protrude into the central vacuole. While the location of a chloroplast within a cell had no effect on the likelihood of its degradation, chloroplasts in spongy mesophyll cells were degraded at a higher rate than those in palisade mesophyll cells. To determine if degrading chloroplasts have unique structural characteristics allowing them to be distinguished from healthy chloroplasts, we analyzed fc2 seedlings grown under different levels of photo-oxidative stress. A clear correlation was observed between chloroplast swelling, 1O2-signaling, and the state of degradation. Finally, plastoglobule enzymes involved in chloroplast disassembly were shown to be upregulated while plastoglobules increased their association with the thylakoid grana, implicating an interaction between 1O2-induced chloroplast degradation and senescence pathways.

plant biology↗

bioRxiv2021

HepT1-derived murine models of high-risk hepatoblastoma display vascular invasion, metastasis, and circulating tumor cells

Hepatoblastoma (HB) is the most common pediatric primary liver malignancy, and survival for high-risk disease approaches 50%. Mouse models of HB fail to recapitulate hallmarks of high-risk disease. The aim of this work was to generate murine models that show high-risk features including multifocal tumors, vascular invasion, metastasis, and circulating tumor cells (CTCs). HepT1 cells were injected into the livers or tail veins of mice, and tumor growth was monitored with magnetic resonance and bioluminescent imaging. Blood was analyzed with fluorescence activated cell sorting to identify CTCs. Intra- and extra-hepatic tumor samples were harvested for immunohistochemistry and RNA and DNA sequencing. Cell lines were grown from tumor samples and profiled with RNA sequencing. With intrahepatic injection of HepT1 cells, 100% of animals grew liver tumors and showed vascular invasion, metastasis, and CTCs. Mutation profiling revealed genetic alterations in seven cancer-related genes, while transcriptomic analyses showed changes in gene expression with cells that invade vessels. Tail vein injection of HepT1 cells resulted in multifocal, metastatic disease. These unique models will facilitate further meaningful studies of high-risk HB. Summary StatementIn this work, we developed and thoroughly characterized several unique models of hepatoblastoma derived from the HepT1 cell line that show high-risk features.

cancer biology↗

bioRxiv2021

Singlet oxygen-dependent chloroplast degradation is independent of macroautophagy in the Arabidopsis ferrochelatase two mutant

BackgroundChloroplasts respond to stress and changes in the environment by producing reactive oxygen species (ROS) that have specific signaling abilities. The ROS singlet oxygen (1O2) is unique in that it can signal to initiate selective degradation of damaged chloroplasts and then cell death. This chloroplast quality control pathway can be monitored in the Arabidopsis mutant plastid ferrochelatase two (fc2) that conditionally accumulates chloroplast 1O2 under diurnal light cycling conditions leading to rapid chloroplast degradation and eventual cell death. The cellular machinery involved in such degradation, however, remains unknown. Recently it has been demonstrated that whole damaged chloroplasts can be transported to the central vacuole via a process requiring autophagosomes and core components of the autophagy machinery. The relationship between this process, referred to as chlorophagy, and the degradation of 1O2-stressed chloroplasts and cells has remained unexplored. ResultsTo further understand 1O2-induced cellular degradation and determine what role autophagy may play, the expression of autophagy-related genes were monitored in 1O2-stressed fc2 seedlings and found to be induced. Although autophagosomes were present in fc2 cells, they did not associate with chloroplasts during 1O2 stress. Mutations blocking the core autophagy machinery (atg5, atg7, and atg10) were unable to suppress 1O2-induced chloroplast degradation or cell death in the fc2 mutant, suggesting autophagosome formation and macroautophagy are dispensable for 1O2-mediated cellular degradation. However, both atg5 and atg7 led to specific defects in chloroplast ultrastructure and photosynthetic efficiencies, suggesting macroautophagy may be involved in protecting chloroplasts from photo-oxidative damage. Finally, genes predicted to be involved in microautophagy were shown to be induced in stressed fc2 seedlings, indicating a possible role for an alternate form of autophagy in the dismantling of 1O2-damaged chloroplasts. ConclusionsOur results support the hypothesis that 1O2-dependent chloroplast degradation is independent from autophagosome formation, canonical macroautophagy, and chlorophagy. Instead, ATG-independent microautophagy may be involved in such degradation. However, canonical macroautophagy may still play a role in protecting chloroplasts from 1O2-induced photo-oxidative stress. Together, this suggests chloroplast function and degradation is a complex process that utilizes multiple autophagy and degradation machineries, possibly depending on the type of stress or damage incurred.

plant biology↗

bioRxiv2020

Cytidine triphosphate synthase two provides nucleotides for chloroplast development and stress responses

O_LIReactive oxygen species (ROS) produced in chloroplasts cause oxidative damage, but also signal to initiate chloroplast quality control pathways, cell death, and gene expression. The mechanisms behind these signals are largely unknown. C_LIO_LIThe Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant produces the ROS singlet oxygen in chloroplasts that activates such signaling pathways. Here we mapped one fc2 suppressor mutation to CYTIDINE TRIPHOSPHATE SYNTHASE TWO (CTPS2), which encodes one of five enzymes in Arabidopsis necessary for de novo cytoplasmic CTP (and dCTP) synthesis. C_LIO_LIThe ctps2 mutation reduces chloroplast transcripts and DNA content without similarly affecting mitochondria. Chloroplast nucleic acid content and singlet oxygen signaling are restored by exogenous feeding of the dCTP precursor deoxycytidine, suggesting ctps2 blocks signaling by limiting nucleotides for chloroplast genome maintenance. C_LIO_LIAn investigation of CTPS orthologs in Brassicaceae showed CTPS2 is a member of an ancient lineage distinct from CTPS3. Complementation studies confirmed this analysis; CTPS3 was unable to compensate for CTPS2 function in providing nucleotides for chloroplast DNA and signaling. C_LIO_LIOur studies link cytoplasmic nucleotide metabolism with chloroplast quality control pathways. Such a connection is achieved by a conserved clade of CTPS enzymes that may have evolved specialized functions in providing nucleotides to specific subcellular compartments. C_LI

plant biology↗

bioRxiv2020

Plastid gene expression is required for singlet oxygen-induced chloroplast degradation and cell death

Chloroplasts constantly experience photo-oxidative stress while performing photosynthesis. This is particularly true under abiotic stresses that lead to the accumulation of reactive oxygen species (ROS). While ROS leads to the oxidation of DNA, proteins, and lipids, it can also act as a signal to induce acclimation through chloroplast degradation, cell death, and nuclear gene expression. Although the mechanisms behind ROS signaling from chloroplasts remain mostly unknown, several genetic systems have been devised in the model plant Arabidopsis to understand their signaling properties. One system uses the plastid ferrochelatase two (fc2) mutant that conditionally accumulates the ROS singlet oxygen (1O2) leading to chloroplast degradation and eventually cell death. Here we have mapped three mutations that suppress chloroplast degradation in the fc2 mutant and demonstrate that they affect two independent loci (PPR30 and mTERF9) encoding chloroplast proteins predicted to be involved in post-transcriptional gene expression. Mutations in either gene were shown to lead to broadly reduced chloroplast gene expression, impaired chloroplast development, and reduced chloroplast stress signaling. In these mutants, however, 1O2 levels were uncoupled to chloroplast degradation suggesting that PPR30 and mTERF9 are involved in ROS signaling pathways. In the wild type background, ppr30 and mTERF9 mutants were also observed to be less susceptible to cell death induced by excess light stress. Together these results suggest that plastid gene expression (or the expression of specific plastid genes) is a necessary prerequisite for chloroplasts to activate 1O2 signaling pathways to induce chloroplast degradation and/or cell death. Significance summaryReactive oxygen species accumulate in the chloroplast (photosynthetic plastids) and signal for stress acclimation by inducing chloroplast degradation, cell death, and changes in nuclear gene expression. We have identified two chloroplast-localized proteins involved in gene regulation that are required to transmit these signals, suggesting that proper plastid gene expression and chloroplast development is necessary to activate chloroplast controlled cellular degradation and nuclear gene expression pathways.

plant biology↗