Carotenoid sequestration protein FIBRILLIN participates in CmOR-regulated ß-carotene accumulation in melon.

Chromoplasts are plant organelles with a unique ability to sequester and store massive carotenoids. Chromoplasts have been hypothesized to enable high levels of carotenoid accumulation due to enhanced sequestration ability or sequestration substructure formation. However, the regulators that control the substructure component accumulation and substructure formation in chromoplasts remain unknown. In melon (Cucumis melo) fruit, ß-carotene accumulation in chromoplasts is governed by ORANGE (OR), a key regulator for carotenoid accumulation in chromoplasts. By using comparative proteomic analysis of a high ß-carotene melon variety and its isogenic line low-ß mutant that is defective in CmOr with impaired chromoplast formation, we identified carotenoid sequestration protein FIBRILLIN1 (CmFBN1) as differentially expressed. CmFBN1 expresses highly in melon fruit tissue. Overexpression of CmFBN1 in transgenic Arabidopsis (Arabidopsis thaliana) containing ORHis that genetically mimics CmOr significantly enhances carotenoid accumulation, demonstrating its involvement in CmOR-induced carotenoid accumulation. Both in vitro and in vivo evidence showed that CmOR physically interacts with CmFBN1. Such an interaction occurs in plastoglobules and results in promoting CmFBN1 accumulation. CmOR greatly stabilizes CmFBN1, which stimulates plastoglobule proliferation and subsequently carotenoid accumulation in chromoplasts. Our findings show that CmOR directly regulates CmFBN1 protein levels and suggest a fundamental role of CmFBN1 in facilitating plastoglobule proliferation for carotenoid sequestration. This study also reveals an important genetic tool to further enhance OR-induced carotenoid accumulation in chromoplasts in crops.

ORANGE Represses Chloroplast Biogenesis in Etiolated Arabidopsis Cotyledons via Interaction with TCP14.

The conversion of etioplasts into chloroplasts in germinating cotyledons is a crucial transition for higher plants, enabling photoautotrophic growth upon illumination. Tight coordination of chlorophyll biosynthesis and photosynthetic complex assembly is critical for this process. ORANGE (OR), a DnaJ-like zinc finger domain-containing protein, was reported to trigger the biogenesis of carotenoid-accumulating plastids by promoting carotenoid biosynthesis and sequestration. Both nuclear and plastidic localizations of OR have been observed. Here, we show that Arabidopsis (Arabidopsis thaliana) OR physically interacts with the transcription factor TCP14 in the nucleus and represses its transactivation activity. Through this interaction, the nucleus-localized OR negatively regulates expression of EARLY LIGHT-INDUCIBLE PROTEINS (ELIPs), reduces chlorophyll biosynthesis, and delays development of thylakoid membranes in the plastids of germinating cotyledons. Nuclear abundance of OR decreased upon illumination. Together with an accumulation of TCP14 in the nucleus, this derepresses chloroplast biogenesis during de-etiolation. TCP14 is epistatic to OR and expression of ELIPs is directly regulated by the binding of TCP14 to Up1 elements in the ELIP promoter regions. Our results demonstrate that the interaction between OR and TCP14 in the nucleus leads to repression of chloroplast biogenesis in etiolated seedlings and provide new insights into the regulation of early chloroplast development.plantcell;31/12/2996/FX1F1fx1.

Arabidopsis ORANGE protein regulates plastid pre-protein import through interacting with Tic proteins.

Chloroplast-targeted proteins are actively imported into chloroplasts via the machinery spanning the double-layered membranes of chloroplasts. While the key translocons at the outer (TOC) and inner (TIC) membranes of chloroplasts are defined, proteins that interact with the core components to facilitate pre-protein import are continuously being discovered. A DnaJ-like chaperone ORANGE (OR) protein is known to regulate carotenoid biosynthesis as well as plastid biogenesis and development. In this study, we found that OR physically interacts with several Tic proteins including Tic20, Tic40, and Tic110 in the classic TIC core complex of the chloroplast import machinery. Knocking out or and its homolog or-like greatly affects the import efficiency of some photosynthetic and non-photosynthetic pre-proteins. Consistent with the direct interactions of OR with Tic proteins, the binding efficiency assay revealed that the effect of OR occurs at translocation at the inner envelope membrane (i.e. at the TIC complex). OR is able to reduce the Tic40 protein turnover rate through its chaperone activity. Moreover, OR was found to interfere with the interaction between Tic40 and Tic110, and reduces the binding of pre-proteins to Tic110 in aiding their release for translocation and processing. Our findings suggest that OR plays a new and regulatory role in stabilizing key translocons and in facilitating the late stage of plastid pre-protein translocation to regulate plastid pre-protein import.

Identification of genetic modifiers enhancing B7-H3-targeting CAR T cell therapy against glioblastoma through large-scale CRISPRi screening.

BACKGROUND: Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with a poor prognosis. Current treatment options are limited and often ineffective. CAR T cell therapy has shown success in treating hematologic malignancies, and there is growing interest in its potential application in solid tumors, including GBM. However, current CAR T therapy lacks clinical efficacy against GBM due to tumor-related resistance mechanisms and CAR T cell deficiencies. Therefore, there is a need to improve CAR T cell therapy efficacy in GBM. METHODS: We conducted large-scale CRISPR interference (CRISPRi) screens in GBM cell line U87 MG cells co-cultured with B7-H3 targeting CAR T cells to identify genetic modifiers that can enhance CAR T cell-mediated tumor killing. Flow cytometry-based tumor killing assay and CAR T cell activation assay were performed to validate screening hits. Bioinformatic analyses on bulk and single-cell RNA sequencing data and the TCGA database were employed to elucidate the mechanism underlying enhanced CAR T efficacy upon knocking down the selected screening hits in U87 MG cells. RESULTS: We established B7-H3 as a targetable antigen for CAR T therapy in GBM. Through large-scale CRISPRi screening, we discovered genetic modifiers in GBM cells, including ARPC4, PI4KA, ATP6V1A, UBA1, and NDUFV1, that regulated the efficacy of CAR T cell-mediated tumor killing. Furthermore, we discovered that TNFSF15 was upregulated in both ARPC4 and NDUFV1 knockdown GBM cells and revealed an immunostimulatory role of TNFSF15 in modulating tumor-CAR T interaction to enhance CAR T cell efficacy. CONCLUSIONS: Our study highlights the power of CRISPR-based genetic screening in investigating tumor-CAR T interaction and identifies potential druggable targets in tumor cells that confer resistance to CAR T cell killing. Furthermore, we devised targeted strategies that synergize with CAR T therapy against GBM. These findings shed light on the development of novel combinatorial strategies for effective immunotherapy of GBM and other solid tumors.

Co-chaperoning of chlorophyll and carotenoid biosynthesis by ORANGE family proteins in plants.

Chlorophylls and carotenoids are essential photosynthetic pigments. Plants spatiotemporally coordinate the needs of chlorophylls and carotenoids for optimal photosynthesis and fitness in response to diverse environmental and developmental cues. However, how the biosynthesis pathways of these two pigments are coordinated, particularly at posttranslational level to allow rapid control, remains largely unknown. Here, we report that the highly conserved ORANGE (OR) family proteins coordinate both pathways via posttranslationally mediating the first committed enzyme in each pathway. We demonstrate that OR family proteins physically interact with magnesium chelatase subunit I (CHLI) in chlorophyll biosynthesis pathway in addition to phytoene synthase (PSY) in carotenoid biosynthesis pathway and concurrently stabilize CHLI and PSY enzymes. We show that loss of OR genes hinders both chlorophyll and carotenoid biosynthesis, limits light-harvesting complex assembly, and impairs thylakoid grana stacking in chloroplasts. Overexpression of OR safeguards photosynthetic pigment biosynthesis and enhances thermotolerance in both Arabidopsis and tomato plants. Our findings establish a novel mechanism by which plants coordinate chlorophyll and carotenoid biosynthesis and provide a potential genetic target to generate climate-resilient crops.

Plant carotenoids: recent advances and future perspectives.

Carotenoids are isoprenoid metabolites synthesized de novo in all photosynthetic organisms. Carotenoids are essential for plants with diverse functions in photosynthesis, photoprotection, pigmentation, phytohormone synthesis, and signaling. They are also critically important for humans as precursors of vitamin A synthesis and as dietary antioxidants. The vital roles of carotenoids to plants and humans have prompted significant progress toward our understanding of carotenoid metabolism and regulation. New regulators and novel roles of carotenoid metabolites are continuously revealed. This review focuses on current status of carotenoid metabolism and highlights recent advances in comprehension of the intrinsic and multi-dimensional regulation of carotenoid accumulation. We also discuss the functional evolution of carotenoids, the agricultural and horticultural application, and some key areas for future research.

Protein-protein interaction techniques to investigate post-translational regulation of carotenogenesis.

Carotenoids are indispensable to plants. The regulatory mechanisms underlying carotenoid metabolism have been subjected to intensive investigation. Post-translational regulation is critically important to rapidly modulate enzyme protein level and activity in fine-tuning carotenoid production in living organisms. However, the regulatory controls at the post-translational level are poorly understood. This chapter highlights the recent advances in this area of research and presents the protein-protein interaction protocols to study the post-translational regulation of carotenogenesis.

Phytoene Synthase: The Key Rate-Limiting Enzyme of Carotenoid Biosynthesis in Plants.

Phytoene synthase (PSY) catalyzes the first committed step in the carotenoid biosynthesis pathway and is a major rate-limiting enzyme of carotenogenesis. PSY is highly regulated by various regulators and factors to modulate carotenoid biosynthesis in response to diverse developmental and environmental cues. Because of its critical role in controlling the total amount of synthesized carotenoids, PSY has been extensively investigated and engineered in plant species. However, much remains to be learned on its multifaceted regulatory control and its catalytic efficiency for carotenoid enrichment in crops. Here, we present current knowledge on the basic biology, the functional evolution, the dynamic regulation, and the metabolic engineering of PSY. We also discuss the open questions and gaps to stimulate additional research on this most studied gene/enzyme in the carotenogenic pathway.

Involvement of cytokinins in STOP1-mediated resistance to proton toxicity.

STOP1 (sensitive to proton rhizotoxicity1) is a master transcription factor that governs the expression of a set of regulatory and structural genes involved in resistance to aluminum and low pH (i.e., proton) stresses in Arabidopsis. However, the mechanisms and regulatory networks underlying STOP1-mediated resistance to proton stresses are largely unclear. Here, we report that low-pH stresses severely inhibited root growth of the stop1 plants by suppressing root meristem activities. Interestingly, the stop1 plants were less sensitive to exogenous cytokinins at normal and low pHs than the wild type. Significantly, low concentrations of cytokinins promoted root growth of the stop1 mutant under low-pH stresses. Moreover, lateral and adventitious root formation was stimulated in stop1 and by low-pH stresses but suppressed by cytokinins. Further studies of the expression patterns of a cytokinin signaling reporter suggest that both the loss-of-function mutation of STOP1 and low-pH stresses suppressed cytokinin signaling outputs in the root. Furthermore, the expression of critical genes involved in cytokinin biosynthesis, biodegradation, and signaling is altered in the stop1 mutant in response to low-pH stresses. In conclusion, our results reveal a complex network of resistance to low-pH stresses, which involves coordinated actions of STOP1, cytokinins, and an additional low-pH-resistant mechanism for controlling root meristem activities and root growth upon proton stresses.

Characterization of cassava ORANGE proteins and their capability to increase provitamin A carotenoids accumulation.

Cassava (Manihot esculenta Crantz) biofortification with provitamin A carotenoids is an ongoing process that aims to alleviate vitamin A deficiency. The moderate content of provitamin A carotenoids achieved so far limits the contribution to providing adequate dietary vitamin A levels. Strategies to increase carotenoid content focused on genes from the carotenoids biosynthesis pathway. In recent years, special emphasis was given to ORANGE protein (OR), which promotes the accumulation of carotenoids and their stability in several plants. The aim of this work was to identify, characterize and investigate the role of OR in the biosynthesis and stabilization of carotenoids in cassava and its relationship with phytoene synthase (PSY), the rate-limiting enzyme of the carotenoids biosynthesis pathway. Gene and protein characterization of OR, expression levels, protein amounts and carotenoids levels were evaluated in roots of one white (60444) and two yellow cassava cultivars (GM5309-57 and GM3736-37). Four OR variants were found in yellow cassava roots. Although comparable expression was found for three variants, significantly higher OR protein amounts were observed in the yellow varieties. In contrast, cassava PSY1 expression was significantly higher in the yellow cultivars, but PSY protein amount did not vary. Furthermore, we evaluated whether expression of one of the variants, MeOR_X1, affected carotenoid accumulation in cassava Friable Embryogenic Callus (FEC). Overexpression of maize PSY1 alone resulted in carotenoids accumulation and induced crystal formation. Co-expression with MeOR_X1 led to greatly increase of carotenoids although PSY1 expression was high in the co-expressed FEC. Our data suggest that posttranslational mechanisms controlling OR and PSY protein stability contribute to higher carotenoid levels in yellow cassava. Moreover, we showed that cassava FEC can be used to study the efficiency of single and combinatorial gene expression in increasing the carotenoid content prior to its application for the generation of biofortified cassava with enhanced carotenoids levels.

The cauliflower Orange gene enhances petiole elongation by suppressing expression of eukaryotic release factor 1.

The cauliflower (Brassica oleracea var. botrytis) Orange (Or) gene affects plant growth and development in addition to conferring ß-carotene accumulation. This study was undertaken to investigate the molecular basis for the effects of the Or gene mutation in on plant growth. The OR protein was found to interact with cauliflower and Arabidopsis eukaryotic release factor 1-2 (eRF1-2), a member of the eRF1 family, by yeast two-hybrid analysis and by bimolecular fluorescence complementation (BiFC) assay. Concomitantly, the Or mutant showed reduced expression of the BoeRF1 family genes. Transgenic cauliflower plants with suppressed expression of BoeRF1-2 and BoeRF1-3 were generated by RNA interference. Like the Or mutant, the BoeRF1 RNAi lines showed increased elongation of the leaf petiole. This long-petiole phenotype was largely caused by enhanced cell elongation, which resulted from increased cell length and elevated expression of genes involved in cell-wall loosening. These findings demonstrate that the cauliflower Or gene controls petiole elongation by suppressing the expression of eRF1 genes, and provide new insights into the molecular mechanism of leaf petiole regulation.

Multi-strategy engineering greatly enhances provitamin A carotenoid accumulation and stability in Arabidopsis seeds.

Staple grains with low levels of provitamin A carotenoids contribute to the global prevalence of vitamin A deficiency and therefore are the main targets for provitamin A biofortification. However, carotenoid stability during both seed maturation and postharvest storage is a serious concern for the full benefits of carotenoid biofortified grains. In this study, we utilized Arabidopsis as a model to establish carotenoid biofortification strategies in seeds. We discovered that manipulation of carotenoid biosynthetic activity by seed-specific expression of Phytoene synthase (PSY) increases both provitamin A and total carotenoid levels but the increased carotenoids are prone to degradation during seed maturation and storage, consistent with previous studies of provitamin A biofortified grains. In contrast, stacking with Orange (OR His ), a gene that initiates chromoplast biogenesis, dramatically enhances provitamin A and total carotenoid content and stability. Up to 65- and 10-fold increases of ß-carotene and total carotenoids, respectively, with provitamin A carotenoids composing over 63% were observed in the seeds containing OR His and PSY. Co-expression of Homogentisate geranylgeranyl transferase (HGGT) with OR His and PSY further increases carotenoid accumulation and stability during seed maturation and storage. Moreover, knocking-out of ß-carotene hydroxylase 2 (BCH2) by CRISPR/Cas9 not only potentially facilitates ß-carotene accumulation but also minimizes the negative effect of carotenoid over production on seed germination. Our findings provide new insights into various processes on carotenoid accumulation and stability in seeds and establish a multiplexed strategy to simultaneously target carotenoid biosynthesis, turnover, and stable storage for carotenoid biofortification in crop seeds. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-021-00046-1.

Comparative transcriptome analyses shed light on carotenoid production and plastid development in melon fruit.

Carotenoids, such as ß-carotene, accumulate in chromoplasts of various fleshy fruits, awarding them with colors, aromas, and nutrients. The Orange (CmOr) gene controls ß-carotene accumulation in melon fruit by posttranslationally enhancing carotenogenesis and repressing ß-carotene turnover in chromoplasts. Carotenoid isomerase (CRTISO) isomerizes yellow prolycopene into red lycopene, a prerequisite for further metabolism into ß-carotene. We comparatively analyzed the developing fruit transcriptomes of orange-colored melon and its two isogenic EMS-induced mutants, low-ß (Cmor) and yofi (Cmcrtiso). The Cmor mutation in low-ß caused a major transcriptomic change in the mature fruit. In contrast, the Cmcrtiso mutation in yofi significantly changed the transcriptome only in early fruit developmental stages. These findings indicate that melon fruit transcriptome is primarily altered by changes in carotenoid metabolic flux and plastid conversion, but minimally by carotenoid composition in the ripe fruit. Clustering of the differentially expressed genes into functional groups revealed an association between fruit carotenoid metabolic flux with the maintenance of the photosynthetic apparatus in fruit chloroplasts. Moreover, large numbers of thylakoid localized photosynthetic genes were differentially expressed in low-ß. CmOR family proteins were found to physically interact with light-harvesting chlorophyll a-b binding proteins, suggesting a new role of CmOR for chloroplast maintenance in melon fruit. This study brings more insights into the cellular and metabolic processes associated with fruit carotenoid accumulation in melon fruit and reveals a new maintenance mechanism of the photosynthetic apparatus for plastid development.