Comparative transcriptomic analysis provides insights into the genetic networks regulating oil differential production in oil crops.

BACKGROUND: Plants differ more than threefold in seed oil contents (SOCs). Soybean (Glycine max), cotton (Gossypium hirsutum), rapeseed (Brassica napus), and sesame (Sesamum indicum) are four important oil crops with markedly different SOCs and fatty acid compositions. RESULTS: Compared to grain crops like maize and rice, expanded acyl-lipid metabolism genes and relatively higher expression levels of genes involved in seed oil synthesis (SOS) in the oil crops contributed to the oil accumulation in seeds. Here, we conducted comparative transcriptomics on oil crops with two different SOC materials. In common, DIHYDROLIPOAMIDE DEHYDROGENASE, STEAROYL-ACYL CARRIER PROTEIN DESATURASE, PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE, and oil-body protein genes were both differentially expressed between the high- and low-oil materials of each crop. By comparing functional components of SOS networks, we found that the strong correlations between genes in "glycolysis/gluconeogenesis" and "fatty acid synthesis" were conserved in both grain and oil crops, with PYRUVATE KINASE being the common factor affecting starch and lipid accumulation. Network alignment also found a conserved clique among oil crops affecting seed oil accumulation, which has been validated in Arabidopsis. Differently, secondary and protein metabolism affected oil synthesis to different degrees in different crops, and high SOC was due to less competition of the same precursors. The comparison of Arabidopsis mutants and wild type showed that CINNAMYL ALCOHOL DEHYDROGENASE 9, the conserved regulator we identified, was a factor resulting in different relative contents of lignins to oil in seeds. The interconnection of lipids and proteins was common but in different ways among crops, which partly led to differential oil production. CONCLUSIONS: This study goes beyond the observations made in studies of individual species to provide new insights into which genes and networks may be fundamental to seed oil accumulation from a multispecies perspective.

GHCU, a Molecular Chaperone, Regulates Leaf Curling by Modulating the Distribution of KNGH1 in Cotton.

Leaf shape is considered to be one of the most significant agronomic traits in crop breeding. However, the molecular basis underlying leaf morphogenesis in cotton is still largely unknown. In this study, through genetic mapping and molecular investigation using a natural cotton mutant cu with leaves curling upward, the causal gene GHCU is successfully identified as the key regulator of leaf flattening. Knockout of GHCU or its homolog in cotton and tobacco using CRISPR results in abnormal leaf shape. It is further discovered that GHCU facilitates the transport of the HD protein KNOTTED1-like (KNGH1) from the adaxial to the abaxial domain. Loss of GHCU function restricts KNGH1 to the adaxial epidermal region, leading to lower auxin response levels in the adaxial boundary compared to the abaxial. This spatial asymmetry in auxin distribution produces the upward-curled leaf phenotype of the cu mutant. By analysis of single-cell RNA sequencing and spatiotemporal transcriptomic data, auxin biosynthesis genes are confirmed to be expressed asymmetrically in the adaxial-abaxial epidermal cells. Overall, these findings suggest that GHCU plays a crucial role in the regulation of leaf flattening through facilitating cell-to-cell trafficking of KNGH1 and hence influencing the auxin response level.

A truncated ETHYLENE INSENSITIVE3-like protein, GhLYI, regulates senescence in cotton.

Numerous endogenous and environmental signals regulate the intricate and highly orchestrated process of plant senescence. Ethylene (ET), which accumulates as senescence progresses, is a major promoter of leaf senescence. The master transcription activator ETHYLENE INSENSITIVE3 (EIN3) activates the expression of a wide range of downstream genes during leaf senescence. Here, we found that a unique EIN3-LIKE 1 (EIL1) gene, cotton LINT YIELD INCREASING (GhLYI), encodes a truncated EIN3 protein in upland cotton (Gossypium hirsutum L.) that functions as an ET signal response factor and a positive regulator of senescence. Ectopic expression or overexpression of GhLYI accelerated leaf senescence in both Arabidopsis (Arabidopsis thaliana) and cotton. Cleavage under targets and tagmentation (CUT&Tag) analyses revealed that SENESCENCE-ASSOCIATED GENE 20 (SAG20) was a target of GhLYI. Electrophoretic mobility shift assay (EMSA), yeast 1-hybrid (Y1H), and dual-luciferase transient expression assay confirmed that GhLYI directly bound the promoter of SAG20 to activate its expression. Transcriptome analysis revealed that transcript levels of a series of senescence-related genes, SAG12, NAC-LIKE, ACTIVATED by APETALA 3/PISTILLATA (NAP/ANAC029), and WRKY53, are substantially induced in GhLYI overexpression plants compared with wild-type (WT) plants. Virus-induced gene silencing (VIGS) preliminarily confirmed that knockdown of GhSAG20 delayed leaf senescence. Collectively, our findings provide a regulatory module involving GhLYI-GhSAG20 in controlling senescence in cotton.

HEAT-RESPONSIVE PROTEIN regulates heat stress via fine-tuning ethylene/auxin signaling pathways in cotton.

Plants sense and respond to fluctuating temperature and light conditions during the circadian cycle; however, the molecular mechanism underlying plant adaptability during daytime warm conditions remains poorly understood. In this study, we reveal that the ectopic regulation of a HEAT RESPONSIVE PROTEIN (GhHRP) controls the adaptation and survival of cotton (Gossypium hirsutum) plants in response to warm conditions via modulating phytohormone signaling. Increased ambient temperature promptly enhanced the binding of the phytochrome interacting factor 4 (GhPIF4)/ethylene-insensitive 3 (GhEIN3) complex to the GhHRP promoter to increase its mRNA level. The ectopic expression of GhHRP promoted the temperature-dependent accumulation of GhPIF4 transcripts and hypocotyl elongation by triggering thermoresponsive growth-related genes. Notably, the upregulation of the GhHRP/GhPIF4 complex improved plant growth via modulating the abundance of Arabidopsis thaliana auxin biosynthetic gene YUCCA8 (AtYUC8)/1-aminocyclopropane-1-carboxylate synthase 8 (AtACS8) for fine-tuning the auxin/ethylene interplay, ultimately resulting in decreased ethylene biosynthesis. GhHRP thus protects chloroplasts from photo-oxidative bursts via repressing AtACS8 and AtACS7 and upregulating AtYUC8 and the heat shock transcription factors (HSFA2), heat shock proteins (HSP70 and HSP20). Strikingly, the Δhrp disruption mutant exhibited compromised production of HSP/YUC8 that resulted in an opposite phenotype with the loss of the ability to respond to warm conditions. Our results show that GhHRP is a heat-responsive signaling component that assists plants in confronting the dark phase and modulates auxin signaling to rescue growth under temperature fluctuations.

Retrieving a disrupted gene encoding phospholipase A for fibre enhancement in allotetraploid cultivated cotton.

After polyploidization originated from one interspecific hybridization event in Gossypium, Gossypium barbadense evolved to produce extra-long staple fibres than Gossypium hirsutum (Upland cotton), which produces a higher fibre yield. The genomic diversity between G. barbadense and G. hirsutum thus provides a genetic basis for fibre trait variation. Recently, rapid accumulation of gene disruption or deleterious mutation was reported in allotetraploid cotton genomes, with unknown impacts on fibre traits. Here, we identified gene disruptions in allotetraploid G. hirsutum (18.14%) and G. barbadense (17.38%) through comparison with their presumed diploid progenitors. Relative to conserved genes, these disrupted genes exhibited faster evolution rate, lower expression level and altered gene co-expression networks. Within a module regulating fibre elongation, a hub gene experienced gene disruption in G. hirsutum after polyploidization, with a 2-bp deletion in the coding region of GhNPLA1D introducing early termination of translation. This deletion was observed in all of the 34 G. hirsutum landraces and 36 G. hirsutum cultivars, but not in 96% of 57 G. barbadense accessions. Retrieving the disrupted gene GhNPLA1D using its homoeolog GhNPLA1A achieved longer fibre length in G. hirsutum. Further enzyme activity and lipids analysis confirmed that GhNPLA1A encodes a typical phospholipase A and promotes cotton fibre elongation via elevating intracellular levels of linolenic acid and 34:3 phosphatidylinositol. Our work opens a strategy for identifying disrupted genes and retrieving their functions in ways that can provide valuable resources for accelerating fibre trait enhancement in cotton breeding.

Cotton genes GhMML1 and GhMML2 control trichome branching when ectopically expressed in tobacco.

Trichomes exhibit extraordinary diversity in shape, ultrastructure, distribution, secretion capability, biological functions, and morphological differences, which are strongly associated with their multifunction. Previous researches showed MIXTA-like transcription factors involved in regulating trichome initiation and patterning via forming MYB-bHLH-WD40 transcriptional activator complex to induce the expression of downstream genes. Here, we report the characteristics and role of GhMML1 and GhMML2, members of subgroup 9 of the R2R3-type MYB TFs. GhMML1 and GhMML2 were preferentially targeted to the nucleus and prominently expressed in the early stage during fiber development. Ectopic expression of GhMML1 and GhMML2 respectively in the transgenic tobacco plants changed the morphological characteristics of leaf trichomes; that is, the unbranched trichomes turned into multiple branched, and in the meantime, the density of trichomes was reduced on the surface of the leaf. Y2H and LCI assay revealed that both GhMML1 and GhMML2 could physically interact with a bZIP transcription factor family protein (GhbZIP) in vivo and in vitro. It has been reported that GhbZIP's homolog TAG3 in Arabidopsis is involved in the asymmetric growth of leaves and flowers via direct interaction with BOP1. Taken together, our results demonstrated that two MYB MIXTA-like proteins, GhMML1 and GhMML2, together with GhbZIP might form a multimeric complex to involve in trichome development. This study highlights the importance of MIXTA-like genes from TF subgroup 9 and will help to uncover the molecular mechanism underlying differential trichomes and their development.

Identification and characteristics of a novel gland-forming gene in cotton.

The cotton (Gossypium hirsutum) pigment gland is a distinctive structure that functions as the main deposit organ of gossypol and its derivatives. It is also an ideal system in which to study cell differentiation and organogenesis. However, only a few genes that determine the process of gland formation have been reported, including GoPGF, CGP1, and CGFs; the molecular mechanisms underlying gland initiation are still largely unclear. Here, we report the discovery of the novel stem pigment gland-forming gene GoSPGF by map-based cloning; annotated as a GRAS transcription factor, this gene is responsible for the glandless trait specifically on the stem. In the stem glandless mutant T582, a point mutation (C to A) was found to create a premature stop codon and truncate the protein. Similarly, virus-induced gene silencing of GoSPGF resulted in glandless stems and dramatically reduced gossypol content. Comparative transcriptomic data showed that loss of GoSPGF significantly suppressed expression of many genes involved in gossypol biosynthesis and altered expression of genes involved in gibberellic acid signaling/biosynthesis. Overall, these findings provide more insight into the networks regulating glandular structure differentiation and formation in cotton, which will be helpful for understanding other plants bearing special gland structures such as tobacco (Nicotiana benthamiana), artemisia annua, mint (Mentha spp.), and rubber (Hevea brasiliensis).

IL-2 regulates tumor-reactive CD8+ T cell exhaustion by activating the aryl hydrocarbon receptor.

CD8+ T cell exhaustion dampens antitumor immunity. Although several transcription factors have been identified that regulate T cell exhaustion, the molecular mechanisms by which CD8+ T cells are triggered to enter an exhausted state remain unclear. Here, we show that interleukin-2 (IL-2) acts as an environmental cue to induce CD8+ T cell exhaustion within tumor microenvironments. We find that a continuously high level of IL-2 leads to the persistent activation of STAT5 in CD8+ T cells, which in turn induces strong expression of tryptophan hydroxylase 1, thus catalyzing the conversion to tryptophan to 5-hydroxytryptophan (5-HTP). 5-HTP subsequently activates AhR nuclear translocation, causing a coordinated upregulation of inhibitory receptors and downregulation of cytokine and effector-molecule production, thereby rendering T cells dysfunctional in the tumor microenvironment. This molecular pathway is not only present in mouse tumor models but is also observed in people with cancer, identifying IL-2 as a novel inducer of T cell exhaustion.

Cell softness regulates tumorigenicity and stemness of cancer cells.

Identifying and sorting highly tumorigenic and metastatic tumor cells from a heterogeneous cell population is a daunting challenge. Here, we show that microfluidic devices can be used to sort marker-based heterogeneous cancer stem cells (CSC) into mechanically stiff and soft subpopulations. The isolated soft tumor cells (< 400 Pa) but not the stiff ones (> 700 Pa) can form a tumor in immunocompetent mice with 100 cells per inoculation. Notably, only the soft, but not the stiff cells, isolated from CD133+ , ALDH+ , or side population CSCs, are able to form a tumor with only 100 cells in NOD-SCID or immunocompetent mice. The Wnt signaling protein BCL9L is upregulated in soft tumor cells and regulates their stemness and tumorigenicity. Clinically, BCL9L expression is correlated with a worse prognosis. Our findings suggest that the intrinsic softness is a unique marker of highly tumorigenic and metastatic tumor cells.

Novel Insights Into the Role of N6-Methyladenosine RNA Modification in Bone Pathophysiology.

Thus far, there are more than known 150 modifications to RNA, in which common internal modifications of mRNA include N6-methyladenosine (m6A), N1-methyladenosine, and 5-methylcytosine. Among them, m6A RNA modification is one of the highest abundance modifications in eukaryotes, regulating mechanisms controlling gene expression at the post-transcription level. As an invertible and dynamic epigenetic marker, m6A base modification influences almost all vital biological processes, cellular components, and molecular functions. Once the m6A modification process is abnormal, a series of diseases-including cancer, neurological diseases, and growth disorders-will be caused. Besides, several base modification activities also have been created by noncoding RNAs (ncRNAs), for instance, microRNAs, and circular RNAs, long ncRNAs, which were dynamically regulated during bone and cartilage pathophysiology processes. Therefore, it has now been clear that dynamic modification on coding RNAs and ncRNAs represents a completely new way to modulate genetic information. In this review, we highlight up-to-date progress and applications of m6A RNA modification in bone and cartilage pathophysiology, and we discuss the pathological roles and underlying molecular mechanism of m6A modifications in osteoarthritis and osteoporosis and osteosarcoma pathogenesis.

Cell Softness Prevents Cytolytic T-cell Killing of Tumor-Repopulating Cells.

Biomechanics is a fundamental feature of a cell. However, the manner by which actomysin tension affects tumor immune evasion remains unclear. Here we show that although cytotoxic T lymphocytes (CTL) can effectively destroy stiff differentiated tumor cells, they fail to kill soft tumor-repopulating cells (TRC). TRC softness prevented membrane pore formation caused by CTL-released perforin. Perforin interacting with nonmuscle myosin heavy-chain 9 transmitted forces to less F-actins in soft TRC, thus generating an inadequate contractile force for perforin pore formation. Stiffening TRC allowed perforin the ability to drill through the membrane, leading to CTL-mediated killing of TRC. Importantly, overcoming mechanical softness in human TRC also enhanced TRC cell death caused by human CTL, potentiating a mechanics-based immunotherapeutic strategy. These findings reveal a mechanics-mediated tumor immune evasion, thus potentially providing an alternative approach for tumor immunotherapy. SIGNIFICANCE: Tumor-repopulating cells evade CD8+ cytolytic T-cell killing through a mechanical softness mechanism, underlying the impediment of perforin pore formation at the immune synapse site.

Factors Influencing Administration, Recognition, and Compliance of Medicine among Community Residents from Jilin Province, China: A Questionnaire Study.

INTRODUCTION: To identify and analyze factors that influence administration, recognition, and compliance of medicine among community residents in Jilin Province, China. METHODS: A survey was carried out among 2417 community residents in Jilin Province, China, to study their administration (CRA), recognition (CRR), and compliance (CRC) of medicine. Multivariate logistic regression analyses and chi-squared tests were performed to assess factors influencing CRA, CRR, and CRC. RESULTS: Logistic analyses showed that gender, educational level, and occupation were influencing factors on CRA; age, educational level, smoking status, and health condition were influencing factors on CRR; and gender, age, occupation, and health condition were influencing factors on CRC. CONCLUSIONS: CRA, CRR, and CRC are associated with specific lifestyles and social economic statuses of community residents. Attention should be paid to influencing factors in order to facilitate community pharmaceutical care, promote the rational use of drugs, and ensure the safe use of medications. This study explores the type and extent of professional services provided through community pharmacies in Jilin Province, China, and provides evidence for optimizing the quality of community pharmacy services.

Gasdermin E-mediated target cell pyroptosis by CAR T cells triggers cytokine release syndrome.

Cytokine release syndrome (CRS) counteracts the effectiveness of chimeric antigen receptor (CAR) T cell therapy in cancer patients, but the mechanism underlying CRS remains unclear. Here, we show that tumor cell pyroptosis triggers CRS during CAR T cell therapy. We find that CAR T cells rapidly activate caspase 3 in target cells through release of granzyme B. The latter cleaves gasdermin E (GSDME), a pore-forming protein highly expressed in B leukemic and other target cells, which results in extensive pyroptosis. Consequently, pyroptosis-released factors activate caspase 1 for GSDMD cleavage in macrophages, which results in the release of cytokines and subsequent CRS. Knocking out GSDME, depleting macrophages, or inhibiting caspase 1 eliminates CRS occurrence in mouse models. In patients, GSDME and lactate dehydrogenase levels are correlated with the severity of CRS. Notably, we find that the quantity of perforin/granzyme B used by CAR T cells rather than existing CD8+ T cells is critical for CAR T cells to induce target cell pyroptosis.

Gossypium barbadense and Gossypium hirsutum genomes provide insights into the origin and evolution of allotetraploid cotton.

Allotetraploid cotton is an economically important natural-fiber-producing crop worldwide. After polyploidization, Gossypium hirsutum L. evolved to produce a higher fiber yield and to better survive harsh environments than Gossypium barbadense, which produces superior-quality fibers. The global genetic and molecular bases for these interspecies divergences were unknown. Here we report high-quality de novo-assembled genomes for these two cultivated allotetraploid species with pronounced improvement in repetitive-DNA-enriched centromeric regions. Whole-genome comparative analyses revealed that species-specific alterations in gene expression, structural variations and expanded gene families were responsible for speciation and the evolutionary history of these species. These findings help to elucidate the evolution of cotton genomes and their domestication history. The information generated not only should enable breeders to improve fiber quality and resilience to ever-changing environmental conditions but also can be translated to other crops for better understanding of their domestication history and use in improvement.

Visualization of perforin/gasdermin/complement-formed pores in real cell membranes using atomic force microscopy.

Different types of pores ubiquitously form in cell membranes, leading to various types of cell death that profoundly influence the fate of inflammation and the disease status. However, these pores have never truly been visualized to date. Atomic force microscopy (AFM), which is emerging as a powerful tool to analyze the mechanical properties of biomolecules and cells, is actually an excellent imaging platform that allows biological samples to be visualized by probing surface roughness at the level of atomic resolution. Here, membrane pore structures were clearly visualized using AFM. This visualization not only describes the aperture and depth of the pore complexes but also highlights differences among the pores formed by perforin and gasdermins in tumor cell membranes and by complement in immune cell membranes. Additionally, this type of visualization also reveals the dynamic process of pore formation, fusion, and repair.

Mechanisms by Which Dendritic Cells Present Tumor Microparticle Antigens to CD8+ T Cells.

Tumor cell-derived microparticles (T-MP) contain tumor antigen profiles as well as innate signals, endowing them with vaccine potential; however, the precise mechanism by which DCs present T-MP antigens to T cells remains unclear. Here, we show that T-MPs activate a lysosomal pathway that is required for DCs presenting tumor antigens of T-MPs. DCs endocytose T-MPs to lysosomes, where T-MPs increase lysosomal pH from 5.0 to a peak of 8.5 via NOX2-catalyzed reactive oxygen species (ROS) production. This increased pH, coupled with T-MP-driven lysosomal centripetal migration, promotes the formation of MHC class I-tumor antigen peptide complexes. Concurrently, endocytosis of T-MPs results in the upregulation of CD80 and CD86. T-MP-increased ROS activate lysosomal Ca2+ channel Mcoln2, leading to Ca2+ release. Released Ca2+ activates transcription factor EB (TFEB), a lysosomal master regulator that directly binds to CD80 and CD86 promoters, promoting gene expression. These findings elucidate a pathway through which DCs efficiently present tumor antigen from T-MPs to CD8+ T cells, potentiating T-MPs as a novel tumor cell-free vaccine with clinical applications. Cancer Immunol Res; 6(9); 1057-68. ©2018 AACR.

Circulating Tumor Microparticles Promote Lung Metastasis by Reprogramming Inflammatory and Mechanical Niches via a Macrophage-Dependent Pathway.

Despite the frequency of lung metastasis and its associated mortality, the mechanisms behind metastatic tumor cell survival and colonization in the lungs remain elusive. Here, we show that tumor cell-released microparticles (T-MPs) from the primary tumor site play a critical role in the metastatic process. The T-MPs remodeled the lung parenchyma via a macrophage-dependent pathway to create an altered inflammatory and mechanical response to tumor cell invasion. Mechanistically, we show that circulating T-MPs readily enter the lung parenchyma where they are taken up by local macrophages and induce CCL2 production. CCL2 recruits CD11b+Ly6Chigh inflammatory monocytes to the lungs where they mature into F4/80+CD11b+Ly6C- macrophages that not only produce IL6 but also trigger fibrin deposition. IL6 and the deposited fibrin facilitate the survival and growth of tumor-repopulating cells in the lungs by providing chemical and mechanical signals, respectively, thus setting the stage for lung metastasis. These data illustrate that T-MPs reprogram the lung microenvironment promoting metastasis. Cancer Immunol Res; 6(9); 1046-56. ©2018 AACR.

Characterization of gossypol biosynthetic pathway.

Gossypol and related sesquiterpene aldehydes in cotton function as defense compounds but are antinutritional in cottonseed products. By transcriptome comparison and coexpression analyses, we identified 146 candidates linked to gossypol biosynthesis. Analysis of metabolites accumulated in plants subjected to virus-induced gene silencing (VIGS) led to the identification of four enzymes and their supposed substrates. In vitro enzymatic assay and reconstitution in tobacco leaves elucidated a series of oxidative reactions of the gossypol biosynthesis pathway. The four functionally characterized enzymes, together with (+)-δ-cadinene synthase and the P450 involved in 7-hydroxy-(+)-δ-cadinene formation, convert farnesyl diphosphate (FPP) to hemigossypol, with two gaps left that each involves aromatization. Of six intermediates identified from the VIGS-treated leaves, 8-hydroxy-7-keto-δ-cadinene exerted a deleterious effect in dampening plant disease resistance if accumulated. Notably, CYP71BE79, the enzyme responsible for converting this phytotoxic intermediate, exhibited the highest catalytic activity among the five enzymes of the pathway assayed. In addition, despite their dispersed distribution in the cotton genome, all of the enzyme genes identified show a tight correlation of expression. Our data suggest that the enzymatic steps in the gossypol pathway are highly coordinated to ensure efficient substrate conversion.

Diurnal down-regulation of ethylene biosynthesis mediates biomass heterosis.

Heterosis is widely applied in agriculture; however, the underlying molecular mechanisms for superior performance are not well understood. Ethylene biosynthesis and signaling genes are shown to be down-regulated in Arabidopsis interspecific hybrids. Ethylene is a plant hormone that promotes fruit ripening and maturation but inhibits hypocotyl elongation. Here we report that application of exogenous ethylene could eliminate biomass vigor in Arabidopsis thaliana F1 hybrids, suggesting a negative role of ethylene in heterosis. Ethylene biosynthesis is mediated by the rate-limiting enzyme, 1-aminocyclopropane-1-carboxylate synthase (ACS). Down-regulation of ACS genes led to the decrease of ethylene production, which was associated with the high-vigor F1 hybrids, but not with the low-vigor ones. At the mechanistic level, expression of ACS genes was down-regulated diurnally and indirectly by Circadian Clock Associated 1 (CCA1) during the day and directly by Phyotochrome-Interacting Factor 5 (PIF5) at night. Consistent with the negative role of ethylene in plant growth, biomass vigor was higher in the acs mutants than in wild-type plants, while increasing endogenous ethylene production in the hybridizing parents reduced growth vigor in the hybrids. Thus, integrating circadian rhythms and light signaling into ethylene production is another regulatory module of complex biological networks, leading to biomass heterosis in plants.

Publisher Correction: Chloroquine modulates antitumor immune response by resetting tumor-associated macrophages toward M1 phenotype.

In the originally published version of this Article, images in Fig. 5n were inadvertently replaced with duplicates of images in Fig. 5o during the production process. This has now been corrected in both the PDF and HTML versions of the Article.