LeafNet: a tool for segmenting and quantifying stomata and pavement cells.

Stomata play important roles in gas and water exchange in leaves. The morphological features of stomata and pavement cells are highly plastic and are regulated during development. However, it is very laborious and time-consuming to collect accurate quantitative data from the leaf surface by manual phenotyping. Here, we introduce LeafNet, a tool that automatically localizes stomata, segments pavement cells (to prepare them for quantification), and reports multiple morphological parameters for a variety of leaf epidermal images, especially bright-field microscopy images. LeafNet employs a hierarchical strategy to identify stomata using a deep convolutional network and then segments pavement cells on stomata-masked images using a region merging method. LeafNet achieved promising performance on test images for quantifying different phenotypes of individual stomata and pavement cells compared with six currently available tools, including StomataCounter, Cellpose, PlantSeg, and PaCeQuant. LeafNet shows great flexibility, and we improved its ability to analyze bright-field images from a broad range of species as well as confocal images using transfer learning. Large-scale images of leaves can be efficiently processed in batch mode and interactively inspected with a graphic user interface or a web server (https://leafnet.whu.edu.cn/). The functionalities of LeafNet could easily be extended and will enhance the efficiency and productivity of leaf phenotyping for many plant biologists.

Diurnal Regulation of Plant Epidermal Wax Synthesis through Antagonistic Roles of the Transcription Factors SPL9 and DEWAX.

Plant surface waxes form an outer barrier that protects the plant from many forms of environmental stress. The deposition of cuticular waxes on the plant surface is regulated by external environmental changes, including light and dark cycles. However, the underlying molecular mechanisms controlling light regulation of wax production are still poorly understood, especially at the posttranscriptional level. In this paper, we report the regulation of cuticular wax production by the miR156-SPL9 (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9) module in Arabidopsis (Arabidopsis thaliana). When compared with wild-type plants, miR156 and SPL9 mutants showed significantly altered cuticular wax amounts in both stems and leaves. Furthermore, it was found that SPL9 positively regulates gene expression of the alkane-forming enzyme ECERIFERUM1 (CER1), as well as the primary (1-) alcohol-forming enzyme ECERIFERUM4 (CER4), to enhance alkane and 1-alcohol synthesis, respectively. Our results indicate that complex formation of SPL9 with a negative regulator of wax synthesis, DEWAX, will hamper SPL9 DNA binding ability, possibly by interfering with SPL9 homodimerization. Combined with their diurnal gene and protein expressions, this dynamic repression-activation transcriptional module defines a dynamic mechanism that may allow plants to optimize wax synthesis during daily cycles. These findings provide a regulatory framework for environmental signal integration in the regulation of wax synthesis.

The Arabidopsis endoplasmic reticulum associated degradation pathways are involved in the regulation of heat stress response.

The Cytosolic Protein Response (CPR) in the cytosol and the Unfolded Protein Response (UPR) and ER-associated degradation (ERAD) in the endoplasmic reticulum are major pathways of the cellular proteostasis network. However, despite years of effort, how these protein quality control systems coordinated in vivo remains largely unknown, particularly in plants. In this study, the roles of two evolutionarily conserved ERAD pathways (DOA10 and HRD1) in heat stress response were investigated through reverse genetic approaches in Arabidopsis. Phenotypic analysis of the mutants showed that the two ERAD pathways additively play negative roles in heat tolerance, which was demonstrated by higher survival rate and lower electrolyte leakage in the loss of function mutants compared to the wild type plants. Importantly, gene expression analysis revealed that the mutant plants showed elevated transcriptional regulation of several downstream genes, including those encoding CPR and UPR marker genes, under both basal and heat stress conditions. Finally, multiple components of ERAD genes exhibited rapid response to increasing temperature. Taken together, our data not only unravels key insights into the crosstalk between different protein quality control processes, but also provides candidate genes to genetically improve plant heat tolerance in the future.

Dendrobine Suppresses Tumor Growth by Regulating the PD-1/PD-L1 Checkpoint Pathway in Lung Cancer.

BACKGROUND: Dendrobine is a bioactive alkaloid isolated from Dendrobium nobile. Studies have evaluated the anti-tumor effect of dendrobine in cancers, including lung cancer. However, the mechanism of dendrobine inhibiting tumors requires further study. METHODS: Bioinformatics was performed to screen the potential targets of dendrobine. The in-tersection of dendrobine and lung cancer targets was performed for KEGG analysis. CCK-8 was used to detect cell viability after dendrobine treatment. A xenograft mouse model was es-tablished to explore the effect of dendrobine on lung cancer. The percentages of PD-L1+, CD4+, CD8+, CD11b+, CD25+FOXP3+ cells, the expression of Ki-67 and caspase-3, the ex-pression of pathway-related proteins, the levels of IL-2, IFN-γ, and TGF-ß, and the changes of indicators of liver and renal function were measured. RESULTS: Dendrobine regulated the PD1/PD-L1 checkpoint signaling pathway and affected the occurrence and development of lung cancer. Dendrobine decreased the cell viability of lung cancer. Dendrobine and anti-PD-L1 decreased tumor growth, increased caspase-3 expression, and reduced Ki-67 expression in tumor tissues. Dendrobine and anti-PD-L1 suppressed pro-tein expression of PD-L1, p-JAK1/JAK1, and p-JAK2/JAK2 in tumor tissues. Greatly, den-drobine and anti-PD-L1 decreased the percentages of PD-L1+, CD11b+, and CD25+FOXP3+ cells, increased the percentages of CD4+ and CD8+cells, and enhanced the levels of IL-2, IFN-γ, and TGF-ß in tumor tissues. Dendrobine demonstrated no hepatorenal toxicity to the tumor mice. The combination of dendrobine and anti-PD-L1 greatly strengthened the effects of dendrobine on tumors. CONCLUSION: Dendrobine inhibited tumor immune escape by suppressing the PD-1/PD-L1 checkpoint pathway, thus restricting tumor growth of lung cancer.

Comprehensive bioinformatics analysis on exportins in lung adenocarcinoma and lung squamous cell carcinoma.

Background: Lung cancer is one of the most common malignant tumors in the world. Exportins are closely associated with the cellular activity and disease progression in a variety of different tumors. However, the expression level, genetic variation, immune infiltration, and biological function of different exportins in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), as well as their relationship with the prognosis of patients with LUAD and LUSC have not been fully clarified. Methods: To analyze the differential expression, prognostic value, genetic variation, biological function, and immune cell infiltration of exportins in patients with LUAD and LUSC, the ONCOMINE; UALCAN; Human Protein Atlas (HPA); Kaplan-Meier plotter; cBioPortal; Search Tool for the Retrieval of Interacting Genes/Proteins (STRING); Database for Annotation, Visualization, and Integrated Discovery (DAVID); Tumor Immune Estimation Resource (TIMER); and LinkedOmics databases were used in this study. Results: The transcriptional and protein expression levels of CSE1L and XPO1/5/6/7 were increased in patients with LUAD and LUSC, and the increased transcriptional levels of CSE1L and XPO5/6/7 were related to worse prognosis. An increased transcriptional level of XPO1 was associated with a better prognosis. These results indicated that CSE1L and XPO1/5/6/7 may be potential prognostic biomarkers for the survival of patients with LUAD and LUSC. Moreover, the high mutation rate of exportins in non-small cell lung cancer was 50.48%, and the largest proportion of mutations included high messenger RNA expression. The expression of exportins was significantly correlated with the infiltration of various immune cells. Differentially expressed exportins could regulate the occurrence and development of LUAD and LUSC by involving a variety of microRNAs and transcription factor E2F1. Conclusions: Our study provides novel insights into the selection of prognostic biomarkers of exportins in LUAD and LUSC.

De novo Assembly and Characterization of the Fruit Transcriptome of Idesia polycarpa Reveals Candidate Genes for Lipid Biosynthesis.

Idesia polycarpa, is a valuable oilseed-producing tree of the Flacourtiaceae family that has the potential to fulfill edible oil production and is also a possible biofuel feedstock. The fruit is unique in that it contains both saturated and unsaturated lipids present in pericarp and seed, respectively. However, triglyceride synthesis and storage in tissues outside of the seeds has been poorly studied in previous researches. To gain insight into the unique properties of I. polycarpa fruit lipid synthesis, biochemical, and transcriptomic approaches were used to compare the lipid accumulation between pericarp and seed of the fruit. Lipid accumulation rates, final lipid content and composition were significantly different between two tissues. Furthermore, we described the annotated transcriptome assembly and differential gene expression analysis generated from the pericarp and seed tissues. The data allowed the identification of distinct candidate genes and reconstruction of lipid pathways, which may explain the differences of oil synthesis between the two tissues. The results may be useful for engineering alternative pathways for lipid production in non-seed or vegetative tissues.