The CsDof1.8-CsLIPOXYGENASE09 module regulates C9 aroma production in cucumber.

Nine-carbon aldehydes and their relative alcohols (C9 aromas) are the main aroma compounds of cucumber (Cucumis sativus L.) fruits and provide a unique cucumber-like note. However, the key regulators of C9 aroma accumulation in cucumber fruit are poorly characterized. Based on C9 aroma dynamic analysis and transcriptome analysis during fruit development of two different cucumber inbred lines, Q16 and Q24, Lipoxygenase09 (CsLOX09) was identified as a candidate gene for C9 aroma accumulation. Additionally, Q24 with higher CsLOX09 expression accumulated more C9 aromas than Q16. To verify the function of CsLOX09, Cslox09 homozygote knockout lines were created. C9 aroma content decreased by 80.79% to 99.16% in these mutants compared to the wild type. To further explore the reasons for the difference in CsLOX09 expression between Q16 and Q24 fruits, a co-expression network was constructed by integrating the C9 aroma-associated metabolism and transcriptomic data. Eighteen candidate transcription factors were highly correlated with the expression of CsLOX09. DNA binding with One Finger 1.8 (CsDof1.8) was confirmed to bind directly to the A/TAAAG motif of the CsLOX09 promoter through dual-luciferase, yeast one-hybrid, chromatin immunoprecipitation-qPCR and electrophoretic mobility shift assays. Furthermore, C9 aroma content and CsLOX09 expression were significantly increased in the CsDof1.8 overexpression lines. Overall, these data elucidate the metabolic regulation of C9 aromas in cucumber and provide a foundation for facilitating the regulation of flavor in cucumber breeding.

Identification and characterization of CsERECTA, a major gene controlling stem elongation through regulating GA biosynthesis in cucumber.

Dwarfing is an ideal agronomic trait in crop breeding, which can improve lodging resistance and increase crop productivity. In this study, we identified a dwarf mutant cp-3 from an EMS-mutagenized population, which had extremely short internodes, and the cell length and number of internodes were significantly reduced. Meanwhile, exogenous GA3 treatment partially rescued the plant height of the cp-3. Inheritance analysis showed that the cp-3 mutant was regulated via a recessive nuclear locus. A candidate gene, CsERECTA, encoding an LRR receptor-like serine/threonine-protein kinase, was cloned through a map-based cloning strategy. Sequence analysis showed that a nucleotide mutation (C ~ T) in exon 26 of CsERECTA led to premature termination of the protein. Subsequently, two transgenic lines were generated using the CRISPR/Cas9 system, and they showed plant dwarfing. Plant endogenous hormones quantitative and RNA-sequencing analysis revealed that GA3 content and the expression levels of genes related to GA biosynthesis were significantly reduced in Cser knockout mutants. Meanwhile, exogenous GA3 treatment partially rescued the dwarf phenotype of Cser knockout mutants. These findings revealed that CsERECTA controls stem elongation by regulating GA biosynthesis in cucumber.

Impact of Cucurbita moschata Resistant Rootstocks on Cucumis sativus Fruit and Meloidogyne incognita Development.

Plant grafting can provide resistance to nematodes. There is a distinct need to determine the role of Meloidogyne incognita-resistant rootstocks on the growth and quality of grafted cucumber plants. Cucumber (Cucumis sativus L.) cultivar Jinchun No. 4 (J) was hole grafted onto the pumpkin (Cucurbita moschata) cultivars Xiuli (X), Banzhen No. 3 (B), and its root to generate JX, JB, and JJ plants. The histopathology and M. incognita development associated with JX, JB, and JJ were analyzed under incubator and high plastic tunnel conditions. Under incubator conditions, M. incognita root galls and egg mass indices associated with the JX and JB resistant rootstocks were significantly (P < 0.05) lower than those associated with JJ susceptible rootstocks. In addition, the number of eggs were 73.3 ± 8.8% and 85.3 ± 7.7% less, respectively. The number of second-stage juveniles (J2s) in JX roots decreased by 57.1 ± 9.2% compared with that in JJ, and the giant cell and J2 development were poor in JX and JB roots. In pot experiments under a high plastic tunnel, plant height, stem diameter, leaf area, and yield of M. incognita-infected JX plants were not significantly different from those of noninoculated control. There was no significant difference in fruit weight, length, firmness, soluble solids, and color among the three grafted plants. The yield per JB plant was increased compared with that of JJ, irrespective of nematode presence. In the M. incognita-infested soil experiment in a high plastic tunnel, the yield per JX and JB plant were significantly higher than JJ (P < 0.05). Thus, the pumpkin rootstock Xiuli and Banzhen No. 3 are promising rootstocks for managing M. incognita without affecting cucumber fruit quality. Grafting provides a good basis for studying the defense mechanism of rootstocks against M. incognita.

Screening and Identification of Garlic Leaf Blight (Pleospora herbarum)-Resistant Mutants Induced by Ethyl Methane Sulphonate.

Garlic (Allium sativum L.) is a popular condiment used as both medicine and food. Garlic production in China is severely affected by continuous cropping and is especially affected by leaf blight disease. Garlic is sterile, so it is very important to develop specialized genotypes, such as those for disease resistance, nutritional quality, and plant architecture, through genetic modification and innovation. In this experiment, we applied the induction method using EMS to mutate garlic cloves of cultivar G024. From the mutations, 5000 M0 mutants were generated and planted in the field. Then, 199 M1 mutant lines were screened according to growth potential and resistance to leaf blight. From M2 to M3, 169 generational lines were selected that grew well and were resistant to leaf blight in the field. Thereafter, their resistance to leaf blight was further analyzed in the lab; 21 lines resistant to leaf blight that had good growth potential were identified, among which 3 mutants were significantly different, and these were further screened. Also, transcriptome analysis of two mutants infected with Pleospora herbarum, A150 and G024, was performed, and the results revealed 2026 and 4678 differentially expressed genes (DEGs), respectively. These DEGs were highly enriched in hormone signaling pathway, plant-pathogen interaction, and MAPK signaling pathway. Therefore, the results provide a theoretical and technical basis for the creation of garlic germplasm resistant to leaf blight.

Astragaloside IV Overcomes Anlotinib Resistance in Non-small Cell Lung Cancer through miR-181a-3p/UPR-ERAD Axis.

BACKGROUND: Astragaloside IV (AS-IV) has been shown to have a curative effect on non-small cell lung cancer (NSCLC). This study aimed to elucidate the role of AS-IV in NSCLC cell anlotinib resistance (AR). METHODS: The NSCLC/AR cells, resistant to anlotinib, have been produced. The role of AS-IV in the AR of NSCLC cells about the miR-181a-3p/unfolded protein response (UPR)- endoplasmic reticulum associated degradation (ERAD) pathway was then discussed by treating the cells with anlotinib or AS-IV, or by manipulating them with inhibitors or mimics of miR- 181a-3p, HRD1 or Derlin-1 overexpression plasmids. RESULTS: We found that AS-IV could suppress the AR of NSCLC cells. In addition, miR-181a- 3p was elevated in NSCLC/AR cells. Functionally, AS-IV limited the AR of NSCLC cells by reducing miR-181a-3p. Further, activation of the UPR-ERAD pathway was correlated with AR in NSCLC cells. Increased sensitivity of NSCLC cells to anlotinib caused by miR-181a-3p inhibitor could be reversed by overexpression of HRD1 or Derlin-1. CONCLUSION: This research revealed a promising NSCLC/AR treatment approach by showing that AS-IV exposed NSCLC cells to anlotinib by inhibiting the miR-181a-3p/UPR-ERAD axis.

ß-Sitosterol attenuates anlotinib resistance in non-small cell lung cancer cells by inhibiting miR-181a-3p/SHQ1 signaling.

Anlotinib is used for the treatment of advanced non-small cell lung cancer; however, the emergence of drug resistance limits its clinical application. ß-sitosterol may also be used to treat lung cancer, but there have been no studies evaluating ß-sitosterol against anlotinib-resistant lung cancer. The purpose of this study was to determine the mechanism by which ß-sitosterol enhances the sensitivity of lung cancer cells to anlotinib. A549 cells were treated with different concentrations of anlotinib to generate anlotinib-resistant cells (A549/anlotinib cells). miR-181a-3p mimics were transfected into A549/anlotinib cells. A549 and A549/anlotinib cells were treated with ß-sitosterol at various concentrations. The Cell Counting Kit-8 (CCK-8) assay was used to measure cell proliferation. Apoptosis was assessed by flow cytometry. Real-time quantitative PCR was used to measure the expression of miR-181a-3p. The interaction of miR-181a-3p with the H/ACA ribonucleoprotein assembly factor (SHQ1) was predicted using the miRDB and TargetScan Human databases and verified with a luciferase reporter assay. The expression of SHQ1, activating transcription factor 6 (ATF6), and glucose-regulated protein 78 (GRP78) were measured by western blot analysis. ß-Sitosterol effectively suppressed A549/anlotinib cell proliferation and promoted apoptosis. SHQ1 is a downstream target of miR-181a-3p. The expression of miR-181a-3p was inhibited; however, SHQ1 expression was increased by ß-sitosterol treatment of A549/anlotinib cells. The inhibition of SHQ1, ATF6, and GRP78 protein expression by ß-sitosterol in A549/anlotinib cells was rescued by increased miR-181a-3p. ß-Sitosterol markedly promotes anlotinib-resistant A549 cell apoptosis and inhibits cell proliferation by activating SHQ1/UPR signaling through miR-181a-3p inhibition.

Knockdown of NUPR1 inhibits angiogenesis in lung cancer through IRE1/XBP1 and PERK/eIF2α/ATF4 signaling pathways.

The stress response molecule nuclear protein­1 (NUPR1) is essential for the growth of multiple types of human malignant tumor cells. However, the significance of NUPR1 in lung cancer is still not entirely elucidated. Therefore, this study is aimed to explore the function and underlying mechanisms of NUPR1 in lung cancer. NUPR1 mRNA and protein levels in lung cancer cell lines (A549 or H1299 cells) were silenced through siRNA transfection and western blot observed its successful infection efficiency. Then, using tube formation and wound healing experiments, the effects of si-NUPR1 on angiogenesis and migration of human umbilical vein endothelial cells (HUVEC) were examined, respectively, which indicated inhibitory effects on the angiogenesis and migration of HUVEC. Vascular endothelial growth factor A (VEGFA), a vital molecule in angiogenesis, was detected by PCR and western blot assays, manifesting NUPR1 knockdown represses VEGFA expression. Furthermore, the knockdown of NUPR1 may reduce angiogenesis by lowering VEGFA expression through inositol-requiring enzyme 1 (IRE1)/X box binding protein 1 (XBP1) and protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic translation initiation factor 2 A (eIF2α)/recombinant activating transcription factor 4 (ATF4) signaling pathways in A549 or H1299 cells. In conclusion, these findings demonstrated that NUPR1 knockdown inhibits angiogenesis in A549 and H1299 cells through IRE1/XBP1 and PERK/eIF2α/ATF4 signaling pathways, indicating that NUPR1 could represent a novel lung cancer therapeutic target.

COL3A1 Overexpression Associates with Poor Prognosis and Cisplatin Resistance in Lung Cancer

Background: Collagen type III alpha 1 chain (COL3A1) is reported to mediate drug resistance in various cancers, and public database analysis indicated its overexpression in lung cancer. Aims: To investigate the effects of COL3A1 on modulating cisplatin (DDP) resistance in lung carcinoma. Study Design: A cell study. Methods: Gene Expression Omnibus datasets were used to determine the differentially expressed genes between H460 and H460/DDP cell lines using bioinformatics analysis. COL3A1 expression and its clinical value in lung cancer prognosis were analyzed using GEPIA and UALCAN databases. Its roles in modulating the growth, viability, apoptosis, and drug resistance were also assessed in vitro. Results: In H460/DDP cells, the CLO3A1 was among the up-regulated genes compared to H460 cells.COL3A1 overexpression and its association with poor survival in patients with adenocarcinoma were detected by public database analysis. In A549 and H1299 cells, COL3A1 overexpression was associated with increased cell growth and clone formation but decreased cell apoptosis, whereas its reduced expression led to decreased cell growth and clone formation and increased cell apoptosis. Conclusion: COL3A1 is upregulated in lung cancer cells with DDP resistance, and its downregulation sensitizes the cells to DDP.

Quantitative trait locus mapping of fruit aroma compounds in cucumber (Cucumber sativus L.) based on a recombinant inbred line population.

The fresh and unique flavor of cucumber fruits, mainly composed of aldehydes and alcohols, is one of its most important fruit qualities. However, little is known about the genetic basis of aroma compounds in cucumber fruit and the related quantitative trait loci (QTLs). In this study, genomic screening of QTLs underlying aroma compounds was performed based on the genetic linkage map constructed using 1301 single-nucleotide polymorphism (SNP) markers from genotyping-by-sequencing of a recombinant inbred line (RIL) population developed from Q16 × Q24. Significant genetic variations of aroma compounds in the RIL population were observed, and a total of 28 QTLs were screened. A major QTL (qol8-2.1) related to (E,Z)-2,6-nonadien-1-ol was detected with a markedly high LOD score (10.97 in 2020 and 3.56 in 2019) between mk190 and mk204 on chromosome 2. Genome scans identified a cluster of nine lipoxygenase genes in this region. A significant positive correlation was detected between CsaV3_2G005360 (CsLOX08) and (E,Z)-2,6-nonadien-1-ol, and five amino acid variations were detected between the CsLOX08 protein sequences of the two parental lines. Based on the genome variation of CsLOX08, we developed an InDel marker. Genotyping of InDel markers was consistent with the content of (E,Z)-2,6-nonadien-1-ol in RILs, which were also verified in nine cucumber inbred lines. The results will give breeders guidance for obtaining better flavor in cucumber.

Transcriptomic and Histological Analysis of the Response of Susceptible and Resistant Cucumber to Meloidogyne incognita Infection Revealing Complex Resistance via Multiple Signaling Pathways.

The root-knot nematode (RKN), Meloidogyne incognita, is a devastating pathogen for cucumber (Cucumis sativus L.) specially in production under protected environments or continuous cropping. High level RKN resistance has been identified in African horned melon Cucumis metuliferus (CM). However, the resistance mechanism remains unclear. In this study, the comparative analysis on phenotypic and transcriptomic responses in the susceptible cucumber inbred line Q24 and the resistant CM, after M. incognita infection, was performed. The results showed that, in comparison with Q24, the CM was able to significantly reduce penetration numbers of second stage juveniles (J2), slow its development in the roots resulting in fewer galls and smaller giant cells suggesting the presence of host resistance in CM. Comparative transcriptomes analysis of Q24 and CM before and after M. incognita infection was conducted and differentially expressed genes (DEGs) associated with host resistance were identified in CM. Enrichment analyses revealed most enriched DEGs in Ca2+ signaling, salicylic acid (SA)/jamonate signaling (JA), as well as auxin (IAA) signaling pathways. In particular, in CM, DEGs in the Ca2+ signaling pathway such as those for the calmodulin and calcium-binding proteins were upregulated at the early stage of M. incognita infection; genes for SA/JA synthesis/signal transduction were markedly activated, whereas the IAA signaling pathway genes were inhibited upon infection suggesting the importance of SA/JA signaling pathways in mediating M. incognita resistance in CM. A model was established to explain the different molecular mechanisms on M. incognita susceptibility in cucumber and resistance to M. incognita infection in CM.

Relationship between key environmental factors and profiling of volatile compounds during cucumber fruit development under protected cultivation.

To provide precision management of cucumber under protected cultivation, the relationships between environmental factors and daily increment of aroma in two lines were analyzed during development, using Pearson correlation and stepwise regression. Irrespective of the line tested, the daily increment of volatile compounds and C6 aldehydes were positively correlated with average daily light intensity (X1), while the daily increment of C9 aldehydes was negatively correlated with average daily relative humidity (X4) and average daytime relative humidity (X7). X1 was considered as the most significant environmental factor which affected the daily increment of volatile compounds in both lines, X7 was the environmental factor which affected the daily increment of C9 aldehydes most in No.14-1, and X1 and X4 were the factors which affected the daily increment of C6 aldehydes and C9 aldehydes most in No.26. A range of environmental factors was forecast for the maximal daily increment of volatile compounds.