Treatment Options for Hepatocellular Carcinoma Using Immunotherapy: Present and Future.

Hepatocellular carcinoma (HCC) is a common cancer, and the body's immune responses greatly affect its progression and the prognosis of patients. Immunological suppression and the maintenance of self-tolerance in the tumor microenvironment are essential responses, and these form part of the theoretical foundations of immunotherapy. In this review, we first discuss the tumor microenvironment of HCC, describe immunosuppression in HCC, and review the major biomarkers used to track HCC progression and response to treatment. We then examine antibody-based therapies, with a focus on immune checkpoint inhibitors (ICIs), monoclonal antibodies that target key proteins in the immune response (programmed cell death protein 1, anti-cytotoxic T-lymphocyte associated protein 4, and programmed death-ligand 1) which have transformed the treatment of HCC and other cancers. ICIs may be used alone or in conjunction with various targeted therapies for patients with advanced HCC who are receiving first-line treatments or subsequent treatments. We also discuss the use of different cellular immunotherapies, including T cell receptor (TCR) T cell therapy and chimeric antigen receptor (CAR) T cell therapy. We then review the use of HCC vaccines, adjuvant immunotherapy, and oncolytic virotherapy, and describe the goals of future research in the development of treatments for HCC.

Phytochrome-interacting factors play shared and distinct roles in regulating shade avoidance responses in Populus trees.

Plants adjust their growth and development in response to changing light caused by canopy shade. The molecular mechanisms underlying shade avoidance responses have been widely studied in Arabidopsis and annual crop species, yet the shade avoidance signalling in woody perennial trees remains poorly understood. Here, we first showed that PtophyB1/2 photoreceptors serve conserved roles in attenuating the shade avoidance syndrome (SAS) in poplars. Next, we conducted a systematic identification and characterization of eight PtoPIF genes in Populus tomentosa. Knocking out different PtoPIFs led to attenuated shade responses to varying extents, whereas overexpression of PtoPIFs, particularly PtoPIF3.1 and PtoPIF3.2, led to constitutive SAS phenotypes under normal light and enhanced SAS responses under simulated shade. Notably, our results revealed that distinct from Arabidopsis PIF4 and PIF5, which are major regulators of SAS, the Populus homologues PtoPIF4.1 and PtoPIF4.2 seem to play a minor role in controlling shade responses. Moreover, we showed that PtoPIF3.1/3.2 could directly activate the expression of the auxin biosynthetic gene PtoYUC8 in response to shade, suggesting a conserved PIF-YUC-auxin pathway in modulating SAS in tree. Overall, our study provides insights into shared and divergent functions of PtoPIF members in regulating various aspects of the SAS in Populus.

SPL16 and SPL23 mediate photoperiodic control of seasonal growth in Populus trees.

Perennial trees in boreal and temperate regions undergo growth cessation and bud set under short photoperiods, which are regulated by phytochrome B (phyB) photoreceptors and PHYTOCHROME INTERACTING FACTOR 8 (PIF8) proteins. However, the direct signaling components downstream of the phyB-PIF8 module remain unclear. We found that short photoperiods suppressed the expression of miR156, while upregulated the expression of miR156-targeted SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE 16 (SPL16) and SPL23 in leaves and shoot apices of Populus trees. Accordingly, either overexpression of MIR156a/c or mutagenesis of SPL16/23 resulted in the attenuation of growth cessation and bud set under short days (SD), whereas overexpression of SPL16 and SPL23 conferred early growth cessation. We further showed that SPL16 and SPL23 directly suppressed FLOWERING LOCUS T2 (FT2) expression while promoted BRANCHED1 (BRC1.1 and BRC1.2) expression. Moreover, we revealed that PIF8.1/8.2, positive regulators of growth cessation, directly bound to promoters of MIR156a and MIR156c and inhibited their expression to modulate downstream pathways. Our results reveal a connection between the phyB-PIF8 module-mediated photoperiod perception and the miR156-SPL16/23-FT2/BRC1 regulatory cascades in SD-induced growth cessation. Our study provides insights into the rewiring of a conserved miR156-SPL module in the regulation of seasonal growth in Populus trees.

The AP2/ERF transcription factor PtoERF15 confers drought tolerance via JA-mediated signaling in Populus.

Drought stress is one of the major limiting factors for the growth and development of perennial trees. Xylem vessels act as the center of water conduction in woody species, but the underlying mechanism of its development and morphogenesis under water-deficient conditions remains elucidation. Here, we identified and characterized an osmotic stress-induced ETHYLENE RESPONSE FACTOR 15 (PtoERF15) and its target, PtoMYC2b, which was involved in mediating vessel size, density, and cell wall thickness in response to drought in Populus tomentosa. PtoERF15 is preferentially expressed in differentiating xylem of poplar stems. Overexpression of PtoERF15 contributed to stem water potential maintaining, thus promoting drought tolerance. RNA-Seq and biochemical analysis further revealed that PtoERF15 directly regulated PtoMYC2b, encoding a switch of JA signaling pathway. Additionally, our findings verify that three sets of homologous genes from NAC (NAM, ATAF1/2, and CUC2) gene family: PtoSND1-A1/A2, PtoVND7-1/7-2, and PtoNAC118/120, as the targets of PtoMYC2b, are involved in the regulation of vessel morphology in poplar. Collectively, our study provides molecular evidence for the involvement of the PtoERF15-PtoMYC2b transcription cascade in maintaining stem water potential through the regulation of xylem vessel development, ultimately improving drought tolerance in poplar.

High temperature inhibits vascular development via the PIF4-miR166-HB15 module in Arabidopsis.

The plant vascular system is an elaborate network of conducting and supporting tissues that extends throughout the plant body, and its structure and function must be orchestrated with different environmental conditions. Under high temperature, plants display thin and lodging stems that may lead to decreased yield and quality of crops. However, the molecular mechanism underlying high-temperature-mediated regulation of vascular development is not known. Here, we show that Arabidopsis plants overexpressing the basic-helix-loop-helix (bHLH) transcription factor PHYTOCHROME INTERACTING FACTOR 4 (PIF4), a central regulator of high-temperature signaling, display fewer vascular bundles (VBs) and decreased secondary cell wall (SCW) thickening, mimicking the lodging inflorescence stems of high-temperature-grown wild-type plants. Rising temperature and elevated PIF4 expression reduced the expression of MIR166 and, concomitantly, elevated the expression of the downstream class III homeodomain leucine-zipper (HD-ZIP III) family gene HB15. Consistently, knockdown of miR166 and overexpression of HB15 led to inhibition of vascular development and SCW formation, whereas the hb15 mutant displayed the opposite phenotype in response to high temperature. Moreover, in vitro and in vivo assays verified that PIF4 binds to the promoters of several MIR166 genes and represses their expression. Our study establishes a direct functional link between PIF4 and the miR166-HB15 module in modulating vascular development and SCW thickening and consequently stem-lodging susceptibility at elevated temperatures.

ZmSPL13 and ZmSPL29 act together to promote vegetative and reproductive transition in maize.

Flowering time is a key agronomic trait determining environmental adaptation and yield potential of crops. The regulatory mechanisms of flowering in maize still remain rudimentary. In this study, we combine expressional, genetic, and molecular studies to identify two homologous SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors ZmSPL13 and ZmSPL29 as positive regulators of juvenile-to-adult vegetative transition and floral transition in maize. We show that both ZmSPL13 and ZmSPL29 are preferentially expressed in leaf phloem, vegetative and reproductive meristem. We show that vegetative phase change and flowering time are moderately delayed in the Zmspl13 and Zmspl29 single knockout mutants and more significantly delayed in the Zmspl13/29 double mutants. Consistently, the ZmSPL29 overexpression plants display precocious vegetative phase transition and floral transition, thus early flowering. We demonstrate that ZmSPL13 and ZmSPL29 directly upregulate the expression of ZmMIR172C and ZCN8 in the leaf, and of ZMM3 and ZMM4 in the shoot apical meristem, to induce juvenile-to-adult vegetative transition and floral transition. These findings establish a consecutive signaling cascade of the maize aging pathway by linking the miR156-SPL and the miR172-Gl15 regulatory modules and provide new targets for genetic improvement of flowering time in maize cultivars.

[Analysis on the Distribution Characteristics and Influence Mechanism of Migration and Transformation of Heavy Metals in Mining Wasteland].

In order to explore the distribution characteristics and the influence mechanism of migration and transformation of heavy metals in mining wasteland, soil and tailings samples were collected from the mining wasteland in the Dabaoshan Mining area, Guangdong Province, and the morphological characteristics of heavy metals were analyzed. At the same time, the pollution sources of the mining area were analyzed using Pb stable isotope analysis, and the characteristics and influencing factors of heavy metal migration and transformation in the mining area were expounded by combining X-ray diffraction analysis, transmission electron microscope-energy spectrum analysis (TEM-EDS), and Raman analysis of typical minerals in the mining area, as well as laboratory-simulated leaching experiments. Morphological analysis showed that the forms of Cd, Pb, and As in the soil and tailings samples in the mining area were mainly the residual phase, accounting for 85%-95% of the total, followed by the iron and manganese oxide-bound form (1%-15%). The main mineral types in the soil and tailings in the Dabaoshan Mining area were pyrite (FeS2), chalcopyrite (CuFeS2), and metal oxides, as well as a small amount of sphalerite (ZnS) and galena (PbS). Acidic conditions (pH=3.0) were beneficial to the release and migration of Cd and Pb from soil, tailings, and minerals (pyrite, chalcopyrite) and from the residual phase to the non-residual phase. Lead isotope analysis showed that the lead in the soil and tailings mainly came from the release of metal minerals in the mining area, and the contribution of diesel in the mining area was less than 30%. Multivariate statistical analysis showed that Pyrite, Chalcopyrite, Sphalerite, and Metal oxide were the main sources of heavy metals in the soil and tailings in the mining area, in which Cd, As, and Pb were mainly contributed by sphalerite and metal oxide. The form change in heavy metals in the mining wasteland was easily affected by environmental factors. The form characteristics and migration and transformation factors of heavy metals should be considered in the source control of heavy metal pollution in mining wasteland.

De novo genome assembly and analyses of 12 founder inbred lines provide insights into maize heterosis.

Hybrid maize displays superior heterosis and contributes over 30% of total worldwide cereal production. However, the molecular mechanisms of heterosis remain obscure. Here we show that structural variants (SVs) between the parental lines have a predominant role underpinning maize heterosis. De novo assembly and analyses of 12 maize founder inbred lines (FILs) reveal abundant genetic variations among these FILs and, through expression quantitative trait loci and association analyses, we identify several SVs contributing to genomic and phenotypic differentiations of various heterotic groups. Using a set of 91 diallel-cross F1 hybrids, we found strong positive correlations between better-parent heterosis of the F1 hybrids and the numbers of SVs between the parental lines, providing concrete genomic support for a prevalent role of genetic complementation underlying heterosis. Further, we document evidence that SVs in both ZAR1 and ZmACO2 contribute to yield heterosis in an overdominance fashion. Our results should promote genomics-based breeding of hybrid maize.

UB2/UB3/TSH4-anchored transcriptional networks regulate early maize inflorescence development in response to simulated shade.

Increasing planting density has been adopted as an effective means to increase maize (Zea mays) yield. Competition for light from neighbors can trigger plant shade avoidance syndrome, which includes accelerated flowering. However, the regulatory networks of maize inflorescence development in response to high-density planting remain poorly understood. In this study, we showed that shade-mimicking treatments cause precocious development of the tassels and ears. Comparative transcriptome profiling analyses revealed the enrichment of phytohormone-related genes and transcriptional regulators among the genes co-regulated by developmental progression and simulated shade. Network analysis showed that three homologous Squamosa promoter binding protein (SBP)-like (SPL) transcription factors, Unbranched2 (UB2), Unbranched3 (UB3), and Tasselsheath4 (TSH4), individually exhibited connectivity to over 2,400 genes across the V3-to-V9 stages of tassel development. In addition, we showed that the ub2 ub3 double mutant and tsh4 single mutant were almost insensitive to simulated shade treatments. Moreover, we demonstrated that UB2/UB3/TSH4 could directly regulate the expression of Barren inflorescence2 (BIF2) and Zea mays teosinte branched1/cycloidea/proliferating cell factor30 (ZmTCP30). Furthermore, we functionally verified a role of ZmTCP30 in regulating tassel branching and ear development. Our results reveal a UB2/UB3/TSH4-anchored transcriptional regulatory network of maize inflorescence development and provide valuable targets for breeding shade-tolerant maize cultivars.

AUXIN RESPONSE FACTOR7 integrates gibberellin and auxin signaling via interactions between DELLA and AUX/IAA proteins to regulate cambial activity in poplar.

Cambial development in the stems of perennial woody species is rigorously regulated by phytohormones. Auxin and gibberellin (GA) play crucial roles in stimulating cambial activity in poplar (Populus spp.). In this study, we show that the DELLA protein REPRESSOR of ga1-3 Like 1 (RGL1), AUXIN RESPONSE FACTOR 7 (ARF7), and Aux/INDOLE-3-ACETIC ACID 9 (IAA9) form a ternary complex that mediates crosstalk between the auxin and GA signaling pathways in poplar stems during cambial development. Biochemical analysis revealed that ARF7 physically interacts with RGL1 and IAA9 through distinct domains. The arf7 loss-of-function mutant showed markedly attenuated responses to auxin and GA, whereas transgenic poplar plants overexpressing ARF7 displayed strongly improved cambial activity. ARF7 directly binds to the promoter region of the cambial stem cell regulator WOX4 to modulate its expression, thus integrating auxin and GA signaling to regulate cambial activity. Furthermore, the direct activation of PIN-FORMED 1 expression by ARF7 in the RGL1-ARF7-IAA9 module increased GA-dependent cambial activity via polar auxin transport. Collectively, these findings reveal that the crosstalk between auxin and GA signaling mediated by the RGL1-ARF7-IAA9 module is crucial for the precise regulation of cambial development in poplar.

[Characteristics of Plant Diversity and Heavy Metal Enrichment and Migration Under Different Ecological Restoration Modes in Abandoned Mining Areas].

In order to select plant materials suitable for the ecological restoration of abandoned mining land, ecological restoration experiments were set up in landfills. The effects of different ecological restoration measures on plant diversity and heavy metal enrichment and migration characteristics were studied. The results showed that under different ecological restoration measures, a total of eight families and 10 species of surviving plants appeared, most of which were herbs. The restoration effect of the arbor shrub and grass plots was the best; the plant coverage reached 100%, and the survival rate was over 69%. The diversity index of the plant community was as follows:arbor shrub grass > shrub grass > joe grass > grass. The dominant plants under different ecological restoration measures were Pinus elliottii, Ligustrum lucidum, Boehmeria nivea, Lagerstroemia indica, and Plantago asiatica. The contents of most heavy metals in plants were higher than the normal values. Among them, the enrichment and transfer coefficients of the leaf stems of P. elliottii and L. indica were close to or greater than 1, showing a strong ability of enrichment and transfer to Cd; these two plants belong to Cd-enrichment type plants. The roots of L. lucidum Ait and P. asiatica, which are root-hoarding plants, had a high retention rate of heavy metals. Boehmeria nivea was a heavy metal-avoidance plant with a low content of heavy metals in each part and had the ability to fix and repair potential soil heavy metal pollution. Considering the diversity of plants and the ability of plant enrichment and migration, the combination of trees, shrubs, and grasses was the best restoration mode. Pinus elliottii, L. indica, and P. asiatica can be the target plants for ecological restoration in the abandoned land of mining areas.

Arabidopsis FHY3 and FAR1 Function in Age Gating of Leaf Senescence.

Leaf senescence is the terminal stage of leaf development. Both light and the plant hormone ethylene play important roles in regulating leaf senescence. However, how they coordinately regulate leaf senescence during leaf development remains largely unclear. In this study, we show that FHY3 and FAR1, two homologous proteins essential for phytochrome A-mediated light signaling, physically interact with and repress the DNA binding activity of EIN3 (a key transcription factor essential for ethylene signaling) and PIF5 (a bHLH transcription factor negatively regulating light signaling), and interfere with their DNA binding to the promoter of ORE1, which encodes a key NAC transcription factor promoting leaf senescence. In addition, we show that FHY3, PIF5, and EIN3 form a tri-protein complex(es) and that they coordinately regulate the progression of leaf senescence. We show that during aging or under dark conditions, accumulation of FHY3 protein decreases, thus lifting its repression on DNA binding of EIN3 and PIF5, leading to the increase of ORE1 expression and onset of leaf senescence. Our combined results suggest that FHY3 and FAR1 act in an age gating mechanism to prevent precocious leaf senescence by integrating light and ethylene signaling with developmental aging.

DWARF53 interacts with transcription factors UB2/UB3/TSH4 to regulate maize tillering and tassel branching.

Strigolactones (SLs) are a recently identified class of phytohormones that regulate diverse developmental processes in land plants. However, the signaling mechanism of SLs in maize (Zea mays) remains largely unexplored. Here, we identified the maize gene DWARF 53 (ZmD53) and demonstrated that ZmD53 interacts with the SL receptors DWARF 14A/B (ZmD14A/B) in a rac-GR24-dependent manner. Transgenic maize plants expressing a gain-of-function mutant version of Zmd53 exhibited insensitivity to exogenous rac-GR24 treatment and a highly pleiotropic phenotype, including excess tillering and reduced tassel branching, indicating that ZmD53 functions as an authentic SL signaling repressor in maize. In addition, we showed that ZmD53 interacts with two homologous maize SPL transcription factors, UB3 and TSH4, and suppresses their transcriptional activation activity on TB1 to promote tillering. We also showed that UB2, UB3, and TSH4 can physically interact with each other and themselves, and that they can directly regulate the expression of TSH4, thus forming a positive feedback loop. Furthermore, we demonstrated that ZmD53 can repress the transcriptional activation activity of UB3 and TSH4 on their own promoters, thus decreasing tassel branch number. Our results reveal new insights into the integration of SL signaling and the miR156/SPL molecular module to coordinately regulate maize development.

ZmSPL10/14/26 are required for epidermal hair cell fate specification on maize leaf.

The epidermal hair and stomata are two types of specialized structures on the surface of plant leaves. On mature maize leaves, stomatal complexes and three types of hairs are distributed in a stereotyped pattern on the adaxial epidermis. However, the spatiotemporal relationship between epidermal hair and stomata development and the regulatory mechanisms governing their formation in maize remain largely unknown. Here, we report that three homologous ZmSPL transcription factors, ZmSPL10, ZmSPL14 and ZmSPL26, act in concert to promote epidermal hair fate on maize leaf. Cytological analyses revealed that Zmspl10/14/26 triple mutants are completely glabrous, but possess ectopic stomatal files. Strikingly, the precursor cells for prickle and bicellular hairs are transdifferentiated into ectopic stomatal complexes in the Zmspl10/14/26 mutants. Molecular analyses demonstrated that ZmSPL10/14/26 bind directly to the promoter of a WUSCHEL-related homeobox gene, ZmWOX3A, and upregulate its expression in the hair precursor cells. Moreover, several auxin-related genes are downregulated in the Zmspl10/14/26 triple mutants. Our results suggest that ZmSPL10/14/26 play a key role in promoting epidermal hair fate on maize leaves, possibly through regulating ZmWOX3A and auxin-related gene expression, and that the fates of epidermal hairs and stomata are switchable.

Phytohormones and their crosstalk in regulating stomatal development and patterning.

Phytohormones play important roles in regulating various aspects of plant growth and development as well as in biotic and abiotic stress responses. Stomata are openings on the surface of land plants that control gas exchange with the environment. Accumulating evidence shows that various phytohormones, including abscisic acid, jasmonic acid, brassinosteroids, auxin, cytokinin, ethylene, and gibberellic acid, play many roles in the regulation of stomatal development and patterning, and that the cotyledons/leaves and hypocotyls/stems of Arabidopsis exhibit differential responsiveness to phytohormones. In this review, we first discuss the shared regulatory mechanisms controlling stomatal development and patterning in Arabidopsis cotyledons and hypocotyls and those that are distinct. We then summarize current knowledge of how distinct hormonal signaling circuits are integrated into the core stomatal development pathways and how different phytohormones crosstalk to tailor stomatal density and spacing patterns. Knowledge obtained from Arabidopsis may pave the way for future research to elucidate the effects of phytohormones in regulating stomatal development and patterning in cereal grasses for the purpose of increasing crop adaptive responses.

Light Regulation of Stomatal Development and Patterning: Shifting the Paradigm from Arabidopsis to Grasses.

The stomatal pores of plant leaves control gas exchange with the environment. Stomatal development is prevised regulated by both internal genetic programs and environmental cues. Among various environmental factors, light regulation of stomata formation has been extensively studied in Arabidopsis. In this review, we summarize recent advances in the genetic control of stomata development and its regulation by light. We also present a comparative analysis of the conserved and diverged stomatal regulatory networks between Arabidopsis and cereal grasses. Lastly, we provide our perspectives on manipulation of the stomata density on plant leaves for the purpose of breeding crops that are better adapted to the adverse environment and high-density planting conditions.

Light-Mediated Signaling and Metabolic Changes Coordinate Stomatal Opening and Closure.

Stomata are valves on the leaf surface controlling carbon dioxide (CO2) influx for photosynthesis and water loss by transpiration. Thus, plants have to evolve elaborate mechanisms controlling stomatal aperture to allow efficient photosynthesis while avoid excessive water loss. Light is not only the energy source for photosynthesis but also an important signal regulating stomatal movement during dark-to-light transition. Our knowledge concerning blue and red light signaling and light-induced metabolite changes that contribute to stomatal opening are accumulating. This review summarizes recent advances on the signaling components that lie between the perception of blue/red light and activation of the PM H+-ATPases, and on the negative regulation of stomatal opening by red light-activated phyB signaling and ultraviolet (UV-B and UV-A) irradiation. Besides, light-regulated guard cell (GC)-specific metabolic levels, mesophyll-derived sucrose, and CO2 concentration within GCs also play dual roles in stomatal opening. Thus, light-induced stomatal opening is tightly accompanied by brake mechanisms, allowing plants to coordinate carbon gain and water loss. Knowledge on the mechanisms regulating the trade-off between stomatal opening and closure may have potential applications toward generating superior crops with improved water use efficiency (CO2 gain vs. water loss).

CRISPR/Cas9-mediated knockout and overexpression studies reveal a role of maize phytochrome C in regulating flowering time and plant height.

Maize is a major staple crop widely used for food, feedstocks and industrial products. Shade-avoidance syndrome (SAS), which is triggered when plants sense competition of light from neighbouring vegetation, is detrimental for maize yield production under high-density planting conditions. Previous studies have shown that the red and far-red photoreceptor phytochromes are responsible for perceiving the shading signals and triggering SAS in Arabidopsis; however, their roles in maize are less clear. In this study, we examined the expression patterns of ZmPHYC1 and ZmPHYC2 and found that ZmPHYC1, but not ZmPHYC2, is highly expressed in leaves and is regulated by the circadian clock. Both ZmPHYC1 and ZmPHYC2 proteins are localized to both the nucleus and cytoplasm under light conditions and both of them can interact with themselves or with ZmPHYBs. Heterologous expression of ZmPHYCs can complement the Arabidopsis phyC-2 mutant under constant red light conditions and confer an attenuated SAS in Arabidopsis in response to shading. Double knockout mutants of ZmPHYC1 and ZmPHYC2 created using the CRISPR/Cas9 technology display a moderate early-flowering phenotype under long-day conditions, whereas ZmPHYC2 overexpression plants exhibit a moderately reduced plant height and ear height. Together, these results provided new insight into the function of ZmPHYCs and guidance for breeding high-density tolerant maize cultivars.

Arabidopsis FHY3 and FAR1 integrate light and strigolactone signaling to regulate branching.

Branching/tillering is an important parameter of plant architecture and is tightly regulated by both internal factors (such as plant hormones) and external factors (such as light conditions). How the various signaling pathways converge to coordinately regulate branching is not well understood. Here, we report that in Arabidopsis, FHY3 and FAR1, two homologous transcription factors essential for phytochrome A-mediated light signaling, and SMXL6/SMXL7/SMXL8, three key repressors of the strigolactone (SL) signaling pathway, directly interact with SPL9 and SPL15 and suppress their transcriptional activation of BRC1, a key repressor of branching, thus promoting branching. In addition, FHY3 and FAR1 also directly up-regulate the expression of SMXL6 and SMXL7 to promote branching. Simulated shade treatment reduces the accumulation of FHY3 protein, leading to increased expression of BRC1 and reduced branching. Our results establish an integrated model of light and SL coordinately regulating BRC1 expression and branching through converging at the BRC1 promoter.