Mature Chloroplasts Research Articles

Plastid HSP90C C-terminal extension region plays a regulatory role in chaperone activity and client binding.

HSP90Cs are essential molecular chaperones localized in the plastid stroma that maintain protein homeostasis and assist the import and thylakoid transport of chloroplast proteins. While HSP90C contains all conserved domains as an HSP90 family protein, it also possesses a unique feature in its variable C-terminal extension (CTE) region. This study elucidated the specific function of this HSP90C CTE region. Our phylogenetic analyses revealed that this intrinsically disordered region contains a highly conserved DPW motif in the green lineages. With biochemical assays, we showed that the CTE is required for the chaperone to effectively interact with client proteins PsbO1 and LHCB2 to regulate ATP-independent chaperone activity and to effectuate its ATP hydrolysis. The CTE truncation mutants could support plant growth and development reminiscing the wild type under normal conditions except for a minor phenotype in cotyledon when expressed at a level comparable to wild type. However, higher HSP90C expression was observed to correlate with a stronger response to specific photosystem II inhibitor DCMU, and CTE truncations dampened the response. Additionally, when treated with lincomycin to inhibit chloroplast protein translation, CTE truncation mutants showed a delayed response to PsbO1 expression repression, suggesting its role in chloroplast retrograde signaling. Our study therefore provides insights into the mechanism of HSP90C in client protein binding and the regulation of green chloroplast maturation and function, especially under stress conditions.

Temporal dynamics of chloroplast biogenesis revealed initiation of photosynthesis-related gene expression and protein complexes during alfalfa seed germination

The chloroplast biogenesis occurs in cotyledon during alfalfa seed germination before true leaf formation, and is extremely important for the followed plant development and growth. In this study, we conducted a simulation of alfalfa seed germination in the soil by using tin foil and focused on 10 pivotal time points of chloroplast biogenesis in cotyledons before and after light exposure, which showed significant differences in multispectral images, and covered the whole process of chloroplast biogenesis from proplastid, etioplast to mature chloroplast. We revealed three phases that referred to the programmed involvements of photosynthesis promotion, ultrastructure maturity, transcriptomic expression, and protein complex construction, and observed distinct transcriptional expressions of genes from nuclear and chloroplast genomes. In phase I at dark germination before light exposure, chloroplast-encoded genes showed up-regulated expressions together with the importation of chloroplast proteins. In phase II for the first day after light exposure, nuclear-encoded genes’ expressions were initiated at 2 h after light exposure (E2h), followed by swift assembly of chloroplast thylakoid membrane protein complexes, and roaring Fv/Fm and contents of chlorophyll a, chlorophyll b and carotenoid. The initiation at E2h was pronounced by the observation of gradual accumulation of single lamella, and facilitated the formation of granum stacks (thylakoid) at E8h in phase II. In phase III from the second day after light exposure, chloroplast became gradually complete with the fully established photosynthetic capacity. Altogether, our results layed a theoretical foundation for enhancing potential photosynthetic efficiency in alfalfa and related species.

Regulation of chloroplast biogenesis, development, and signaling by endogenous and exogenous cues.

Chloroplasts are one of the defining features in most plants, primarily known for their unique property to carry out photosynthesis. Besides this, chloroplasts are also associated with hormone and metabolite productions. For this, biogenesis and development of chloroplast are required to be synchronized with the seedling growth to corroborate the maximum rate of photosynthesis following the emergence of seedlings. Chloroplast biogenesis and development are dependent on the signaling to and from the chloroplast, which are in turn regulated by several endogenous and exogenous cues. Light and hormones play a crucial role in chloroplast maturation and development. Chloroplast signaling involves a coordinated two-way connection between the chloroplast and nucleus, termed retrograde and anterograde signaling, respectively. Anterograde and retrograde signaling are involved in regulation at the transcriptional level and downstream modifications and are modulated by several metabolic and external cues. The communication between chloroplast and nucleus is essential for plants to develop strategies to cope with various stresses including high light or high heat. In this review, we have summarized several aspects of chloroplast development and its regulation through the interplay of various external and internal factors. We have also discussed the involvement of chloroplasts as sensors of various external environment stress factors including high light and temperature, and communicate via a series of retrograde signals to the nucleus, thus playing an essential role in plants' abiotic stress response.

Role of Ethylene in the Regulation of Plant Developmental Processes

Ethylene, a gaseous phytohormone, is emerging as a central player in the intricate web of plant developmental processes from germination to senescence under optimal and stressed conditions. The presence of ethylene has been noted in different plant parts, including the stems, leaves, flowers, roots, seeds, and fruits. This review aims to provide a comprehensive overview of the regulatory impact of ethylene on pivotal plant developmental processes, such as cell division and elongation, senescence, abscission, fruit and flower development, root hair formation, chloroplast maturation, and photosynthesis. The review also encompasses ethylene biosynthesis and signaling: a snapshot of the regulatory mechanisms governing ethylene production. Understanding of the impact of ethylene’s regulatory functions on plant developmental processes has significant implications for agriculture, biotechnology, and our fundamental comprehension of plant biology. This review underscores the potential of ethylene to revolutionize plant development and crop management.

RIP-Seq analysis of non-PPR chloroplast editing factors reveals broad RNA interactions and enrichment of less efficiently translated RNAs by OZ1 and ORRM1 complexes.

C-to-U RNA editing in angiosperm chloroplasts requires a large suite of proteins bound together in the editosome. The editosome is comprised of PPR proteins, RIP/MORFs, OZ proteins, and ORRM proteins that physically interact in high molecular weight complexes. The specific functions of non-PPR editing factors in the editosome are unclear, however, specific subsets of editing sites are affected by absence of non-PPR editing factors. Unlike the PPR components of editosomes that have predictable nucleotide specificities, domains present in non-PPR editing factors make RNA associations difficult to predict. In this study, chloroplast extracts were isolated from juvenile maize seedlings. RNAs were immunoprecipitated using polyclonal antibodies targeting non-PPR editing factors RIP9, OZ1, and ORRM1. RNA libraries from duplicate experiments were compared. RIP9 was associated with most of the non-ribosomal RNA content of chloroplasts, consistent with a general binding function to PPR L-motifs and tethering of large ribonucleoprotein complexes. The breadth of RNA associations was greater than predicted and include mRNAs without predicted editing sites, tRNA sequences, and introns. OZ1 and ORRM1 were associated with a highly similar pool of RNAs that have a bias toward lower translational efficiency values in mature chloroplasts. Lower translational efficiency was also associated with the pool of edited RNAs compared to RNAs without editing sites. The unexpected breadth of interactions by non-PPR editing factors suggests the editosome is large, diverse, and associated with RNAs with lower relative translational efficiency in mature chloroplasts.

Probing the in situ volumes of Arabidopsis leaf plastids using three-dimensional confocal and scanning electron microscopy.

Leaf plastids harbor a plethora of biochemical reactions including photosynthesis, one of the most important metabolic pathways on Earth. Scientists are eager to unveil the physiological processes within the organelle but also their interconnection with the rest of the plant cell. An increasingly important feature of this venture is to use experimental data in the design of metabolic models. A remaining obstacle has been the limited in situ volume information of plastids and other cell organelles. To fill this gap for chloroplasts, we established three microscopy protocols delivering in situ volumes based on: (i) chlorophyll fluorescence emerging from the thylakoid membrane, (ii) a CFP marker embedded in the envelope, and (iii) calculations from serial block-face scanning electron microscopy (SBFSEM). The obtained data were corroborated by comparing wild-type data with two mutant lines affected in the plastid division machinery known to produce small and large mesophyll chloroplasts, respectively. Furthermore, we also determined the volume of the much smaller guard cell plastids. Interestingly, their volume is not governed by the same components of the division machinery which defines mesophyll plastid size. Based on our three approaches, the average volume of a mature Col-0 wild-type mesophyll chloroplasts is 93 μm3 . Wild-type guard cell plastids are approximately 18 μm3 . Lastly, our comparative analysis shows that the chlorophyll fluorescence analysis can accurately determine chloroplast volumes, providing an important tool to research groups without access to transgenic marker lines expressing genetically encoded fluorescence proteins or costly SBFSEM equipment.

Aminoacyl-tRNA Synthetase Evolution within the Dynamic Tripartite Translation System of Plant Cells.

Eukaryotes maintain separate protein translation systems for nuclear and organellar genes, including distinct sets of tRNAs and aminoacyl-tRNA synthetases (aaRSs). In animals, mitochondrial-targeted aaRSs are expressed at lower levels and are less conserved in sequence than cytosolic aaRSs involved in translation of nuclear mRNAs, likely reflecting lower translational demands in mitochondria. In plants, translation is further complicated by the presence of plastids, which share most aaRSs with mitochondria. In addition, plant mitochondrial tRNA pools have a dynamic history of gene loss and functional replacement by tRNAs from other compartments. To investigate the consequences of these distinctive features of translation in plants, we analyzed sequence evolution in angiosperm aaRSs. In contrast to previously studied eukaryotic systems, we found that plant organellar and cytosolic aaRSs exhibit only a small difference in expression levels, and organellar aaRSs are slightly more conserved than cytosolic aaRSs. We hypothesize that these patterns result from high translational demands associated with photosynthesis in mature chloroplasts. We also investigated aaRS evolution in Sileneae, an angiosperm lineage with extensive mitochondrial tRNA replacement and aaRS retargeting. We predicted positive selection for changes in aaRS sequence resulting from these recent changes in subcellular localization and tRNA substrates but found little evidence for accelerated sequence divergence. Overall, the complex tripartite translation system in plant cells appears to have imposed more constraints on the long-term evolutionary rates of organellar aaRSs compared with other eukaryotic lineages, and plant aaRS protein sequences appear largely robust to more recent perturbations in subcellular localization and tRNA interactions.

Pseudouridylation of chloroplast ribosomal RNA contributes to low temperature acclimation in rice.

Ribosomal RNAs (rRNAs) undergo many modifications during transcription and maturation; homeostasis of rRNA modifications is essential for chloroplast biogenesis in plants. The chloroplast acts as a hub to sense environmental signals, such as cold temperature. However, how RNA modifications contribute to low temperature responses remains unknown. Here we reveal that pseudouridine (Ψ) modification of rice chloroplast rRNAs mediated by the pseudouridine synthase (OsPUS1) contributes to cold tolerance at seedling stage. Loss-function of OsPUS1 leads to abnormal chloroplast development and albino seedling phenotype at low temperature. We find that OsPUS1 is accumulated upon cold and binds to chloroplast precursor rRNAs (pre-rRNAs) to catalyse the pseudouridylation on rRNA. These modifications on chloroplast rRNAs could be required for their processing, as the reduction of mature chloroplast rRNAs and accumulation of pre-rRNAs are observed in ospus1-1 at low temperature. Therefore, the ribosome activity and translation in chloroplasts is disturbed in ospus1-1. Furthermore, transcriptome and translatome analysis reveals that OsPUS1 balances growth and stress-responsive state, preventing excess reactive oxygen species accumulation. Taken together, our findings unveil a crucial function of Ψ in chloroplast ribosome biogenesis and cold tolerance in rice, with potential applications in crop improvement.

Role of HSP90 Family Molecular Chaperone in Regulating Chloroplast Protein Homeostasis and Plant Development in Arabidopsis

Chloroplasts carry out photosynthesis in plants and provide most food sources for living organisms. Either divided or differentiated from proplastids and etioplasts, the biogenesis of thylakoid membranes is the hallmark of chloroplast maturation and functionality, and this process requires a large set of photosynthetic proteins that must be synthesized on cytosolic ribosomes and then imported into the chloroplast stroma. Thylakoid lumen proteins travel further through the stroma and then cross the thylakoid membrane through one of the ancestral translocases. Like the cytosolic environment and the other major organelles, a cohort of molecular chaperones and proteases reside within the chloroplast to ensure proper protein homeostasis. We have identified and confirmed the interaction between the essential plastid HSP90 family heat shock protein HSP90C and a thylakoid lumen located photosystem II subunit PsbO1. To understand the biological significance of the interaction, we adopted transgenic approaches, specific HSP90 inhibitors, biochemical and in organello protein transport assays to study how the HSP90 family protein facilitates the representative lumen proteins transport across the thylakoid membrane. Specifically, we showed that the expression of the molecular chaperone is developmentally regulated, and its expression is highly correlated with some photosynthetic proteins while not with the others. Additionally, we demonstrated that HSP90C physically interacts with the thylakoid SEC translocon subunit, and that its chaperone activity is required for efficient PsbO1 transport. We also investigated the signature sequences of different HSP90 subfamily proteins and provided evidence that the chloroplast located HSP90C contains features specialized for some photosynthetic proteins interaction, and thus the chloroplast maturation and functionality.

Proteomic Responses of Dark-Adapted Euglena gracilis and Bleached Mutant Against Light Stimuli.

Euglena gracilis (E. gracilis) has secondary endosymbiotic chloroplasts derived from ancient green algae. Its chloroplasts are easily lost under numerous conditions to become permanently bleached mutants. Green cells adapted in the dark contain undeveloped proplastids and they will develop into mature chloroplasts after 3 days of light exposure. Thus, E. gracilis is an ideal model species for a chloroplast development study. Previous studies about chloroplast development in E. gracilis focused on morphology and physiology, whereas few studies have addressed the regulatory processes induced by light in the proteome. In this study, the whole-genome proteome of dark-adapted E. gracilis (WT) and permanently ofloxacin-bleached mutant (B2) was compared under the light exposure after 0, 12, and 72 h. The results showed that the photosynthesis-related proteins were up-regulated over time in both WT and B2. The B2 strain, with losing functional chloroplasts, seemed to possess a complete photosynthetic function system. Both WT and B2 exhibited significant light responses with similar alternation patterns, suggesting the sensitive responses to light in proteomic levels. The main metabolic activities for the utilization of carbon and energy in WT were up-regulated, while the proteins with calcium ion binding, cell cycle, and non-photosynthetic carbon fixation were down-regulated in B2. This study confirmed light-induced chloroplast development in WT from dark, and also for the first time investigates the light responses of a bleached mutant B2, providing more information about the unknown functions of residual plastids in Euglena bleached mutants.

Leaf color formation mechanisms in Alternanthera bettzickiana elucidated by metabolite and transcriptome analyses.

The difference in leaf color among the three cultivars of A. bettzickiana is due to different chloroplast morphology and chlorophyll-to-anthocyanin ratios. Alternanthera bettzickiana is one of the most important ornamental plants in modern flower beds because of its colorful leaves. The present study examined the mechanism of leaf color formation in A. bettzickiana. Three cultivars of A. bettzickiana (red, green, and mixed red and green) were selected for comprehensive analyses of leaf color formation by examining cellular and subcellular structures and pigment biosynthesis and metabolism. The difference in leaf colors between the three cultivars of A. bettzickiana was due to different chlorophyll-to-anthocyanin ratios. A. bettzickiana 'Green' showed very low expression of CHS, F3H, and DFR, the key genes of the anthocyanin biosynthesis pathway, and a low anthocyanin content but had mature chloroplasts and a green color. A. bettzickiana 'Red' exhibited a low chlorophyll content and deformed chloroplasts but a high cyanidin content and, thus, a red color. A. bettzickiana 'Variegated' presented high anthocyanin and chlorophyll contents and exhibited red and green variegation, indicating a balance between coloration and photosynthetic efficiency. These data provide a good explanation for the coloration of different cultivars of A. bettzickiana and an important reference for better explaining the color formation mechanisms of plant leaves.

Fatty acid composition and cpDNA content in Arabidopsis thaliana mutants deprived of EGY1 protease

EGY1 (ethylene-dependent gravitropism-deficient and yellow-green 1) is an intramembrane metalloprotease located in chloroplasts, involved in many diverse processes including chloroplast development, chlorophyll biosynthesis, and the ethylene-dependent gravitropic response. Plants deprived of this protease display pleiotropic effects such as the yellow-green early senescence phenotype and a poorly developed thylakoid system membrane in the mature chloroplasts. We applied the GC/MS technique to analyze the changes in fatty acid composition in two egy1 mutant lines. We used DAPI staining and transmission electron microscopy methods to establish the number of nucleoids and the amount of chloroplast DNA. Our results indicated that the lack of EGY1 protease led to a dramatic overaccumulation and a dramatic decrease in the content of linolenic acid C18:3 and hexadecatrienoic acid C16:3, respectively. The amount of chloroplast DNA and the number of nucleoids were severely reduced in egy1 mutant lines. Similarly, a reduced correlation between DAPI and autofluorescence signal was observed, which may indicate some perturbations in nucleoid anchoring.

Chlorophyllase, a Common Plant Hydrolase Enzyme with a Long History, Is Still a Puzzle.

Chlorophyllase (Chlase, CLH) is one of the earliest discovered enzymes present in plants and green algae. It was long considered to be the first enzyme involved in chlorophyll (Chl) degradation, while strong evidence showed that it is not involved in Chl breakdown during leaf senescence. On the other hand, it is possible that CLH is involved in Chl breakdown during fruit ripening. Recently, it was discovered that Arabidopsis CLH1 is located in developing chloroplasts but not in mature chloroplasts, and it plays a role in protecting young leaves from long-term photodamage by catalysing Chl turnover in the photosystem II (PSII) repair cycle. However, there remain other important questions related to CLH. In this article, we briefly reviewed the research progress on CLH and listed the main unanswered questions related to CLH for further study.

Molecular landscape of etioplast inner membranes in higher plants

Etioplasts are photosynthetically inactive plastids that accumulate when light levels are too low for chloroplast maturation. The etioplast inner membrane consists of a paracrystalline tubular lattice and peripheral, disk-shaped membranes, respectively known as the prolamellar body and prothylakoids. These distinct membrane regions are connected into one continuous compartment. To date, no structures of protein complexes in or at etioplast membranes have been reported. Here, we used electron cryo-tomography to explore the molecular membrane landscape of pea and maize etioplasts. Our tomographic reconstructions show that ATP synthase monomers are enriched in the prothylakoids, and plastid ribosomes in the tubular lattice. The entire tubular lattice is covered by regular helical arrays of a membrane-associated protein, which we identified as the 37-kDa enzyme, light-dependent protochlorophyllide oxidoreductase (LPOR). LPOR is the most abundant protein in the etioplast, where it is responsible for chlorophyll biosynthesis, photoprotection and defining the membrane geometry of the prolamellar body. Based on the 9-Å-resolution volume of the subtomogram average, we propose a structural model of membrane-associated LPOR.

Bn.YCO affects chloroplast development in Brassica napus L.

Mature chloroplasts, as the main sites of photosynthesis, are essential for seedling growth in higher plants. Loss of function of genes involved in chloroplast development changes plant phenotype. We obtained a YELLOW COTYLEDON (YCO) mutant in rapeseed (Brassica napus L.) using CRISPR-Cas9. Bn.YCO, a gene of unknown function, has two homologous copies (BnaA01.YCO and BnaC01.YCO) in B. napus. Homozygous mutation of these two homologs resulted in yellow cotyledons and chlorotic true leaves. Transmission electron microscopy revealed that the formation of thylakoid membranes was inhibited in yellow cotyledons. Sequence similarity search revealed that YCO was conserved in different species, and a subcellular location assay verified that Bn.YCO was located in the chloroplast. Bn.YCO was expressed in multiple tissues, most highly in cotyledons. Knockout of Bn.YCO blocked the transcription of plastid genes, especially those of photosystem genes transcribed by plastid-encoded polymerase. Transcriptome sequencing showed that the majority of genes involved in ribosome assembly and photosynthesis were down-regulated in Bn.yco mutants. These results suggested that loss of function of Bn.YCO affected plastid gene transcription, which influenced chloroplast biogenesis in rapeseed seedlings.

Light and ripening-regulated BBX protein-encoding genes in Solanum lycopersicum

Light controls several aspects of plant development through a complex signalling cascade. Several B-box domain containing proteins (BBX) were identified as regulators of Arabidopsis thaliana seedling photomorphogenesis. However, the knowledge about the role of this protein family in other physiological processes and species remains scarce. To fill this gap, here BBX protein encoding genes in tomato genome were characterised. The robust phylogeny obtained revealed how the domain diversity in this protein family evolved in Viridiplantae and allowed the precise identification of 31 tomato SlBBX proteins. The mRNA profiling in different organs revealed that SlBBX genes are regulated by light and their transcripts accumulation is directly affected by the chloroplast maturation status in both vegetative and fruit tissues. As tomato fruits develops, three SlBBXs were found to be upregulated in the early stages, controlled by the proper chloroplast differentiation and by the PHYTOCHROME (PHY)-dependent light perception. Upon ripening, other three SlBBXs were transcriptionally induced by RIPENING INHIBITOR master transcriptional factor, as well as by PHY-mediated signalling and proper plastid biogenesis. Altogether, the results obtained revealed a conserved role of SlBBX gene family in the light signalling cascade and identified putative members affecting tomato fruit development and ripening.

The Transfer of the Ferredoxin Gene From the Chloroplast to the Nuclear Genome Is Ancient Within the Paraphyletic Genus Thalassiosira.

Ferredoxins are iron–sulfur proteins essential for a wide range of organisms because they are an electron transfer mediator involved in multiple metabolic pathways. In phytoplankton, these proteins are active in the mature chloroplasts, but the petF gene, encoding for ferredoxin, has been found either to be in the chloroplast genome or transferred to the nuclear genome as observed in the green algae and higher plant lineage. We experimentally determined the location of the petF gene in 12 strains of Thalassiosira covering three species using DNA sequencing and qPCR assays. The results showed that petF gene is located in the nuclear genome of all confirmed Thalassiosira oceanica strains (CCMP0999, 1001, 1005, and 1006) tested. In contrast, all Thalassiosira pseudonana (CCMP1012, 1013, 1014, and 1335) and Thalassiosira weissflogii (CCMP1010, 1049, and 1052) strains studied retained the gene in the chloroplast genome, as generally observed for Bacillariophyceae. Our evolutionary analyses further extend the dataset on the localization of the petF gene in the Thalassiosirales. The realization that the petF gene is nuclear-encoded in the Skeletonema genus allowed us to trace the petF gene transfer back to a single event that occurred within the paraphyletic genus Thalassiosira. Phylogenetic analyses revealed the need to reassess the taxonomic assignment of the Thalassiosira strain CCMP1616, since the genes used in our study did not cluster within the T. oceanica lineage. Our results suggest that this strains’ diversification occurred prior to the ferredoxin gene transfer event. The functional transfer of petF genes provides insight into the evolutionary processes leading to chloroplast genome reduction and suggests ecological adaptation as a driving force for such chloroplast to nuclear gene transfer.

Light-induced psbA translation in plants is triggered by photosystem II damage via an assembly-linked autoregulatory circuit

The D1 reaction center protein of photosystem II (PSII) is subject to light-induced damage. Degradation of damaged D1 and its replacement by nascent D1 are at the heart of a PSII repair cycle, without which photosynthesis is inhibited. In mature plant chloroplasts, light stimulates the recruitment of ribosomes specifically to psbA mRNA to provide nascent D1 for PSII repair and also triggers a global increase in translation elongation rate. The light-induced signals that initiate these responses are unclear. We present action spectrum and genetic data indicating that the light-induced recruitment of ribosomes to psbA mRNA is triggered by D1 photodamage, whereas the global stimulation of translation elongation is triggered by photosynthetic electron transport. Furthermore, mutants lacking HCF136, which mediates an early step in D1 assembly, exhibit constitutively high psbA ribosome occupancy in the dark and differ in this way from mutants lacking PSII for other reasons. These results, together with the recent elucidation of a thylakoid membrane complex that functions in PSII assembly, PSII repair, and psbA translation, suggest an autoregulatory mechanism in which the light-induced degradation of D1 relieves repressive interactions between D1 and translational activators in the complex. We suggest that the presence of D1 in this complex coordinates D1 synthesis with the need for nascent D1 during both PSII biogenesis and PSII repair in plant chloroplasts.

Abstract 3459: Water extract from Euglena gracilis prevents lung carcinoma growth in mice via attenuation of myeloid-derived suppressor cells and granulocytes

Abstract Phytochemicals have been developed into treatments against many diseases, including various cancers. Euglena is a genus of single-cell flagellate eukaryotes having features of both animals and plants. These organisms, which are found in both fresh and salt water, have been used as a dietary supplement for their rich nutrient content. Although recent studies suggest that Euglena extracts have anti-tumor properties, the mechanism is yet to be clarified. Accordingly, the anti-tumor and immunomodulatory effects of partially purified water extract from Euglena gracilis (EWE) was evaluated in cell culture and a mouse orthotopic lung carcinoma allograft model. The EWE was prepared by a PBS extraction at 37°C for 30 min, centrifugation at 2,000 g for 20 min, and filtration through a membrane filter (0.22 μm pore size). Primary components of this extract are water soluble materials and exclude mature paramylons, intact chloroplasts, and oil droplets. In two-dimensional cell culture, the EWE treatment inhibited cell growth of both murine Lewis lung carcinoma (LLC) and human lung carcinoma cells (A549 and H1299) in a dose (1-100 μg/ml) and time (24-72 h)-dependent manner. In contrast, the growth of mouse bone marrow cells (BMCs), but not mouse splenocytes (SPLs) were dose (1-100 μg/ml) and time (24-72 h)-dependently stimulated by the treatment with EWE. In three-dimensional spheroid culture, spheroid growth of LLC cells was significantly attenuated by EWE treatment. Flow cytometry revealed that the EWE treatment attenuated granulocytic myeloid-derived suppressor cells in BMCs. In a mouse LLC orthotopic allograft model, pretreatment with EWE (100-200 mg/kg/day, via drinking water) three weeks prior to LLC cell inoculation significantly attenuated the growth of LLC tumors in the lungs of immunocompetent syngeneic mice. However, this attenuation was not seen for EWE treatment initiated after LLC cell inoculation. The tumor growth attenuation by EWE pretreatment coincided with a significant decrease of myeloid-derived cells, primarily neutrophils. EWE pretreatment appears to inhibit lung carcinoma growth mainly by stimulating host anti-tumor immunity via an attenuation of the host myeloid-derived immune suppressor cells. Hence, the present study suggests that the partially purified extract derived from Euglena gracilis contains bioactive materials that prevent lung carcinoma growth. This study was supported by 2016EUGLENA-RC1 (MT), 2017EUGLENA-RC2 (MT and JC), a Kansas State University College of Veterinary Medicine SMILE award (MT and JC), and NIH grant P20 RR017686 (MT). Citation Format: Susumu Ishiguro, Deepa Upreti, Nicole Robben, Page Cote, Riley Burghart, Damilola Ogun, Tran Le, Jennifer Delzeit, Jayson Carswell, Arashi Nakashima, Ayaka Nakashima, Kengo Suzuki, Jeffrey Comer, Masaaki Tamura. Water extract from Euglena gracilis prevents lung carcinoma growth in mice via attenuation of myeloid-derived suppressor cells and granulocytes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3459.

The cold response regulator CBF1 promotes Arabidopsis hypocotyl growth at ambient temperatures.

Light and temperature are two core environmental factors that coordinately regulate plant growth and survival throughout their entire life cycle. However, the mechanisms integrating light and temperature signaling pathways in plants remain poorly understood. Here, we report that CBF1, an AP2/ERF-family transcription factor essential for plant cold acclimation, promotes hypocotyl growth under ambient temperatures in Arabidopsis. We show that CBF1 increases the protein abundance of PIF4 and PIF5, two phytochrome-interacting bHLH-family transcription factors that play pivotal roles in modulating plant growth and development, by directly binding to their promoters to induce their gene expression, and by inhibiting their interaction with phyB in the light. Moreover, our data demonstrate that CBF1 promotes PIF4/PIF5 protein accumulation and hypocotyl growth at both 22°C and 17°C, but not at 4°C, with a more prominent role at 17°C than at 22°C. Together, our study reveals that CBF1 integrates light and temperature control of hypocotyl growth by promoting PIF4 and PIF5 protein abundance in the light, thus providing insights into the integration mechanisms of light and temperature signaling pathways in plants.