The transcription factor CmERFI-2 represses CmMYB44 expression to increase sucrose levels in oriental melon fruit.

Soluble sugar accumulation in fruit ripening determines fleshy fruit quality. However, the molecular mechanism for this process is not yet understood. Here, we showed a transcriptional repressor, CmMYB44 regulates sucrose accumulation and ethylene synthesis in oriental melon (Cucumis. melo var. makuwa Makino) fruit. Overexpressing CmMYB44 suppressed sucrose accumulation and ethylene production. Furthermore, CmMYB44 repressed the transcriptional activation of CmSPS1 (sucrose phosphate synthase 1) and CmACO1 (ACC oxidase 1), two key genes in sucrose and ethylene accumulation, respectively. During the later stages of fruit ripening, the repressive effect of CmMYB44 on CmSPS1 and CmACO1 could be released by overexpressing CmERFI-2 (ethylene response factor I-2) and exogenous ethylene in "HS" fruit (high sucrose accumulation fruit). CmERFI-2 acted upstream of CmMYB44 as a repressor by directly binding the CmMYB44 promoter region, indirectly stimulating the expression level of CmSPS1 and CmACO1. Taken together, we provided a molecular regulatory pathway mediated by CmMYB44, which determines the degree of sucrose and ethylene accumulation in oriental melon fruit and sheds light on transcriptional responses triggered by ethylene sensing that enable the process of fruit ripening.

CmPIF8-CmERF27-CmACS10-mediated ethylene biosynthesis modulates red light-induced powdery mildew resistance in oriental melon.

Powdery mildew is a serious fungal disease in protected melon cultivation that affects the growth, development and production of melon plants. Previous studies have shown that red light can improve oriental melon seedlings resistance to powdery mildew. Here, after inoculation with Podosphaera xanthii, an obligate fungal pathogen eliciting powdery mildew, we found that red light pretreatment increased ethylene production and this improved the resistance of melon seedlings to powdery mildew, and the ethylene biosynthesis gene CmACS10 played an important role in this process. By analysing the CmACS10 promoter, screening yeast one-hybrid library, it was found that CmERF27 positively regulated the expression of CmACS10, increased powdery mildew resistance and interacted with PHYTOCHROME INTERACTING FACTOR8 (CmPIF8) at the protein level to participate in the regulation of ethylene biosynthesis to respond to the red light-induced resistance to P. xanthii, Furthermore, CmPIF8 also directly targeted the promoter of CmACS10, negatively participated in this process. In summary, this study revealed the specific mechanism by which the CmPIF8-CmERF27-CmACS10 module regulates red light-induced ethylene biosynthesis to resist P. xanthii infection, elucidate the interaction between light and plant hormones under biological stress, provide a reference and genetic resources for breeding of disease-resistant melon plants.

A comparative study on the effectiveness of online and in-class team-based learning on student performance and perceptions in virtual simulation experiments.

BACKGROUND: The swift transition to online teaching in medical education has presented the challenge of replicating in-class engagement and interaction essential for active learning. Despite online team-based learning (TBL) offering potential solutions through structured cooperative activities, its efficacy in virtual simulation experiment courses remains scantily researched. This study investigates the effectiveness of online TBL for teaching virtual patient experiments in a basic medical laboratory course and contrasts it with traditional offline teaching in terms of student performance and perceptions. METHODS: A comparative analysis involved 179 Year 3 medical students using online TBL, face-to-face TBL (FTF-TBL), and the flipped classroom (FC) approach. The learning outcomes were assessed based on experiment reports, IRAT scores, TRAT scores, and final exam performance. Students' perceptions of both online and in-class TBL methodologies were also surveyed. RESULTS: Both online and in-class TBL groups demonstrated comparable academic outcomes and surpassed the FC group in academic performance. Students displayed a marked preference for the TBL format (whether online or in-class), valuing its enhancement of learning interest and practical knowledge application. Nevertheless, refinements in discussion efficiency, platform convenience, and student-instructor interaction were indicated as potential areas of improvement in the online setting. CONCLUSIONS: Online TBL, along with its in-class counterpart, showed superior academic performance and a more positive learning experience compared to the FC group. These findings underscore the potential of online TBL in adapting to modern pedagogical challenges and enriching medical education through virtual simulation experiments.

Evaluating the distinct pleiotropic effects of omega-3 fatty acids on type 2 diabetes mellitus: a mendelian randomization study.

BACKGROUND: Observational studies and conventional Mendelian randomization (MR) studies showed inconclusive evidence to support the association between omega-3 fatty acids and type 2 diabetes. We aim to evaluate the causal effect of omega-3 fatty acids on type 2 diabetes mellitus (T2DM), and the distinct intermediate phenotypes linking the two. METHODS: Two-sample MR was performed using genetic instruments derived from a recent genome-wide association study (GWAS) of omega-3 fatty acids (N = 114,999) from UK Biobank and outcome data obtained from a large-scale T2DM GWAS (62,892 cases and 596,424 controls) in European ancestry. MR-Clust was applied to determine clustered genetic instruments of omega-3 fatty acids that influences T2DM. Two-step MR analysis was used to identify potential intermediate phenotypes (e.g. glycemic traits) that linking omega-3 fatty acids with T2DM. RESULTS: Univariate MR showed heterogenous effect of omega-3 fatty acids on T2DM. At least two pleiotropic effects between omega-3 fatty acids and T2DM were identified using MR-Clust. For cluster 1 with seven instruments, increasing omega-3 fatty acids reduced T2DM risk (OR: 0.52, 95%CI 0.45-0.59), and decreased HOMA-IR (ß = - 0.13, SE = 0.05, P = 0.02). On the contrary, MR analysis using 10 instruments in cluster 2 showed that increasing omega-3 fatty acids increased T2DM risk (OR:1.10; 95%CI 1.06-1.15), and decreased HOMA-B (ß = - 0.04, SE = 0.01, P = 4.52 × 10-5). Two-step MR indicated that increasing omega-3 fatty acid levels decreased T2DM risk via decreasing HOMA-IR in cluster 1, while increased T2DM risk via decreasing HOMA-B in cluster 2. CONCLUSIONS: This study provides evidence to support two distinct pleiotropic effects of omega-3 fatty acids on T2DM risk influenced by different gene clusters, which could be partially explained by distinct effects of omega-3 fatty acids on insulin resistance and beta cell dysfunction. The pleiotropic feature of omega-3 fatty acids variants and its complex relationships with T2DM need to be carefully considered in future genetic and clinical studies.

PIF8-WRKY42-mediated salicylic acid synthesis modulates red light induced powdery mildew resistance in oriental melon.

Light signals and plant hormones are involved in regulating the growth, development and stress resistance of plants; however, it remains unclear whether light affects hormones and thus pathogen resistance in oriental melon. Here, we found that red light promoted salicylic acid (SA) accumulation and powdery mildew resistance by activating the transcription of CmICS, the key gene for SA biosynthesis, and silencing CmICS seriously weakened the induction effect of red light on powdery mildew resistance in oriental melon leaves. Further studies showed that red light induced the expression of CmWRKY42 under powdery mildew stress, and CmWRKY42 directly bound to the CmICS promoter to activate its expression and promote the accumulation of SA under red light. Furthermore, we found that PHYTOCHROME INTERACTING FACTOR 8 (PIF8), as a negative regulator of SA biosynthesis, inhibits CmWRKY42 transcriptional activation by binding to the CmWRKY42 promoter, and thus inhibits transcriptional activation of CmICS by CmWRKY42. Also, CmPIF8 binds to the CmICS promoter and directly inhibits its transcription. In conclusion, our study revealed a new molecular mechanism of the relationship between red light-SA-powdery mildew resistance and provided a theoretical basis for resistance breeding of oriental melon.

An Interview Study of College Students' Health Emergency Literacy in the Context of COVID-19 Normalized Prevention and Control.

Objective: To explore the changes in college students' awareness of health protection under the normalization of COVID-19, and to seek its connection with the epidemic management in colleges and universities, so as to provide reference information for continuous health education activities and the cultivation of college students' health emergency literacy in colleges and universities. Methods: Qualitative interviews were used to understand the extent of health emergency literacy among college students enrolled in the context of a normalized epidemic and the factors associated with it that cause changes around a question outline. Results: The interviewees generally had a lax mentality in the late stage of the interview, the importance they attached to epidemic prevention and control decreased significantly, and the way to know about epidemic protection measures and other knowledge was mainly through the mass news media. All respondents affirm the importance of social software for outbreak prevention and control. All 17 interviewees were able to mention basic outbreak protection methods, but 15 of them showed inconsistent behavior in words and actions later. Conclusion: The vast majority of respondents' health emergency literacy appears to weaken in the late stages of epidemic normalization, and the effect of traditional approaches used by universities to improve college students' health emergency literacy is weak.

Circ_0099630 knockdown alleviates lipopolysaccharide-induced injuries of human periodontal ligament cells through the inhibition of TLR4 by releasing miR-409-3p.

BACKGROUND: Periodontitis triggers tooth loss and affects the health of population worldwide. Emerging evidence hints that circular RNAs (circRNAs) are involved in various diseases, including periodontitis. This study aimed to investigate the role of circ_0099630 in the progression of periodontitis. METHODS: Periodontitis cell model was constructed by treating human periodontal ligament cells (HPDLCs) with lipopolysaccharide (LPS). Quantitative real-time PCR was used to analyze the expression of circ_0099630, microRNA-409-3p (miR-409-3p) and toll-like receptor 4 (TLR4) mRNA. Western blot was used for detecting protein levels of TLR4, cleaved-caspase 3, Bcl-2, CyclinD1 and NF-κB signaling markers. For function analyses, cell proliferation was assessed by CCK-8 assay and EdU assay. The releases of pro-inflammation factors were monitored by ELISA kits. The potential relationship between miR-409-3p and circ_0099630 or TLR4 was verified by dual-luciferase reporter assay, RIP assay and pull-down assay. RESULTS: The expression of circ_0099630 and TLR4 was elevated in periodontitis patients and LPS-treated HPDLCs. LPS induced HPDLC proliferation inhibition, apoptosis and inflammatory responses, while circ_0099630 knockdown or TLR4 knockdown alleviated these injuries. Besides, TLR4 overexpression reversed the inhibitory effect of circ_0099630 knockdown on LPS-induced HPDLC injuries. Mechanism analysis showed that circ_0099630 positively regulated TLR4 expression by acting as miR-409-3p sponge. MiR-409-3p restoration largely ameliorated LPS-induced HPDLC injuries by depleting TLR4. Moreover, LPS activated the NF-κB signaling pathway, while circ_0099630 knockdown inhibited the activity of NF-κB signaling via the miR-409-3p/TLR4 axis. CONCLUSION: Circ_0099630 knockdown relieved LPS-induced HPDLC injury by miR-409-3p/TLR4 axis, suggesting that circ_0099630 might be a potential target for periodontitis treatment.

Beta-galactosidase gene family genome-wide identification and expression analysis of members related to fruit softening in melon (Cucumis melo L.).

BACKGROUND: Texture quality is impotent for melon (Cucumis melo L.) fruit. ß-galactosidase (ß-Gal, EC 3.2.1.23) is an important cell wall glycosyl hydrolase involved in fruit softening, However, the ß-Gal gene (BGALs) family hasn't been identified genome-wide in melon. Thus, it's necessary to conduct an in-depth bioinformatic analysis on melon BGALs family and to seek out the key members who participated in melon fruit softening. RESULTS: A total of 21 BGALs members designated as CmBGAL1-CmBGAL21 were identified genome-wide in melon, clustered into A-G seven clades. Among them, three duplications CmBGAL1:CmBGAL3, CmBGAL19:CmBGAL21, and CmBGAL20:CmBGAL21 happened. For conserved domains, besides the Glyco_hydro_35 domain (PF01301), all the members also contained the GHD domain (PF17834) except for CmBGAL12, and the Gal_Lectin (PF02140) domain existed in most CmBGALs at the C-termini. Motifs, protein secondary and tertiary structure analysis showed that the CmBGAL12 is a unique member. Moreover, protein-protein association network analysis showed that the CmBGAL12 is the only node protein. Furthermore, spatiotemporal expression pattern analysis by quantitative real-time PCR (qRT-PCR) suggested that most of CmBGALs expressed in tissues with vigorous cell wall remodeling/disassembly. In addition, cis-acting regulatory elements analysis in promoters inferred that CmBGALs might participate in diverse responsiveness to phytohormone, biotic and abiotic signaling. CONCLUSIONS: A novel clade of CmBGAL members (Clade F) related to melon fruit softening was discovered, since their expression showed a specific surge in the mature fruit of 'HPM' with mealy texture (softening sharply), but not in 'HDB' with crisp texture (softening bluntly). The homologous CmBGAL7-11 in Clade F exhibited identical spatiotemporal expression patterns may multiple genes leading to melon fruit softening.

SlFHY3 and SlHY5 act compliantly to enhance cold tolerance through the integration of myo-inositol and light signaling in tomato.

Plants have evolved sophisticated regulatory networks to cope with dynamically changing light and temperature environments during day-night and seasonal cycles. However, the integration mechanisms of light and low temperature remain largely unclear. Here, we show that low red : far-red ratio (LR : FR) induces FAR-RED ELONGATED HYPOCOTYL3 (SlFHY3) transcription under cold stress in tomato (Solanum lycopersicum). Reverse genetic approaches revealed that knocking out SlFHY3 decreases myo-inositol accumulation and increases cold susceptibility, whereas overexpressing SlFHY3 induces myo-inositol accumulation and enhances cold tolerance in tomato plants. SlFHY3 physically interacts with ELONGATED HYPOCOTYL5 (SlHY5) to promote the transcriptional activity of SlHY5 on MYO-INOSITOL-1-PHOSPHATE SYNTHASE 3 (SlMIPS3) and induce myo-inositol accumulation in tomato plants under cold stress. Disruption of SlHY5 and SlMIPS3 largely suppresses the cold tolerance of SlFHY3-overexpressing plants and myo-inositol accumulation in tomato. Furthermore, silencing of SlMIPS3 drastically reduces myo-inositol accumulation and compromises LR : FR-induced cold tolerance in tomato. Together, our results reveal a crucial role of SlFHY3 in LR : FR-induced cold tolerance in tomato and unravel a novel regulatory mechanism whereby plants integrate dynamic environmental light signals and internal cues (inositol biosynthesis) to induce and control cold tolerance in tomato plants.

Emerging role of exosomal long non-coding RNAs in lung cancer.

BACKGROUND: Lung cancer is one of the most common malignancies worldwide. Also, it is the leading cause of cancer morbidity and mortality in men. Despite advances in lung cancer diagnosis and treatment, novel approaches are strongly needed to promote early diagnosis and effective treatment of lung cancer. Presently, accumulating data reveal that long noncoding RNAs (lncRNAs) are differentially enriched in exosomes and mediate multiple biological processes in lung cancer, suggesting the potential application of exosomal lncRNAs as diagnostic biomarkers and therapeutic targets. CONCLUSION: In this review, we described the emerging roles of lncRNAs specifically sorted into exosomes in lung cancer. We discussed the current knowledge of the exosomal lncRNA sorting mechanism and highlighted opportunities for exosome-derived lncRNAs as biomarkers in clinical practice. In particular, we systematically summarized the biological functions of exosomal lncRNAs in lung cancer.

The association between early-life famine exposure and adulthood obesity on the risk of dyslipidemia.

BACKGROUND AND AIMS: The joint effect of famine exposure and adulthood obesity on risk of dyslipidemia remains unclear. Thus, we aim to explore the joint effect of famine exposure and adulthood obesity on the risk of dyslipidemia, and the potential effect of adult general or abdominal obesity on the association between famine exposure and dyslipidemia. METHODS AND RESULTS: We conducted a community-based cohort study in 8880 subjects aged 40 years or older. Participants were divided into nonexposed, fetal-exposed, childhood-exposed, adolescent-exposed according to birth date. General obesity and abdominal obesity were defined according to body mass index (BMI: overweight≥24.0 kg/m2, obesity≥28.0 kg/m2) and waist-to-hip ratio (WHR, men/women: moderate≥0.90/0.85, high≥0.95/0.90). Dyslipidemia was defined using the National Cholesterol Education Program Adult Treatment Panel III criteria. Compared with nonexposed participants, fetal-exposed individuals had significantly increased risk of dyslipidemia (OR:1.24, 95%CI: 1.03-1.50) in the whole study. Significant increased risk of dyslipidemia related to famine exposure was observed in women [ORs (95%CIs) were 1.36 (1.05-1.76) and 1.70 (1.22-2.37) for the fetal and childhood-exposed group, respectively] but not in men. Moreover, both general and central obesity had significant multiplicative interactions with famine exposure for the risk of dyslipidemia (P for interaction = 0.0001 and < 0.0001, respectively). Significant additive interaction was found between famine exposure and WHR on risk of dyslipidemia in women, with the relative excess risk due to interaction (RERI) and 95% CI of 0.43 (0.10-0.76). CONCLUSION: Coexistence of early-life undernutrition and adulthood obesity was associated with a higher risk of dyslipidemia in later life.

Treatment of scarring alopecia in children using follicular unit hair transplantation.

BACKGROUND: Scarring alopecia can significantly affect children emotionally. Follicular unit excision (FUE) and follicular unit transplantation (FUT) have been applied for scar treatment. OBJECTIVE: This study aimed to evaluate the safety and feasibility of follicular unit hair transplantation in treating scarring alopecia in children. PATIENTS AND METHODS: A total of nine children (seven males and two females) with cicatricial alopecia, ranging in age from 5 years, 2 months to 12 years, 10 months were included in this study. Scar formation time ranged from 7 months to 5 years. Sites were vertex (2), eyebrow (3), frontal hairline (3), and temporal regions (2). RESULTS: Nine children in this group were followed up for 6-34 months with the following treatment options: FUE (5 cases), FUT (3 cases), and FUT combined with FUE (1 case). No significant complications were observed during the treatment. The transplanted hair grew well, the direction and shape were satisfactory, and the survival rate was >90%. CONCLUSION: For children with burn trauma and cicatricial alopecia after surgery, hair transplantation can significantly improve their appearance with low surgical risk and high patient satisfaction rate.

Incorporating modified team-based learning into a flipped basic medical laboratory course: impact on student performance and perceptions.

BACKGROUND: Basic medical laboratory courses (BMLCs) play an essential role in medical education and offer several benefits to students. Although various student-centered and active learning strategies have been increasingly incorporated into medical education, their applications in BMLCs are limited. This paper aimed to explore the educational effects of a flipped classroom (FC) combined with team-based learning (TBL) strategy in BMLCs at Zhejiang University School of Medicine. METHODS: Four hundred eight 3rd-Year medical students were assigned to either the FC-TBL group (n = 235) or the FC group (n = 173) to complete three experiments on the respiration block of BMLCs. The two groups' immediate and long-term academic performance were compared, and the FC-TBL students' perceptions of different instructional strategies were surveyed. RESULTS: Students in the FC-TBL group scored higher on the immediate post-tests after class and higher on the final exams in two of the three experiment sessions. They preferred FC-TBL to FC for its higher engagement, more feedback, and better learning environment. Students felt the FC with TBL blended instructional strategy stimulated their interest in learning and deep thinking. CONCLUSIONS: Compared with the FC group, students in the FC-TBL group improved academic performance and had a more positive experience overall. Our findings support the feasibility and advantage of the flipped classroom with team-based learning as a blended learning strategy in the BMLC curriculum.

ClPIF3-ClHY5 Module Regulates ClPSY1 to Promote Watermelon Fruit Lycopene Accumulation Earlier under Supplementary Red Lighting.

Lycopene content is one of the important factors for determining watermelon fruit quality. In this study, a small-type watermelon was grown in a greenhouse with supplementary red lighting for 10 h per day. The results showed that the content of lycopene in the flesh was increased 6.3-fold after 25 days of supplementary red lighting. qRT-PCR analysis showed that PHYTOENE SYNTHASE 1(ClPSY1) is the major gene that responds to red light within the lycopene synthesis pathway. Moreover, we identified two key transcription factors that were involved in light signal transduction PHYTOCHROME INTERACTING FACTORS 3 (ClPIF3) and LONG HYPOCOTYL 5 (ClHY5) in watermelon flesh. The interaction experiments showed that ClHY5, a potent ClPIF3 antagonist, regulated ClPSY1 expression by directly targeting a common promoter cis-element (G-box). Collectively, our findings identified that ClHY5 and ClPIF3 formed an activation-suppression transcriptional module that is responsive to red light and, through this model, regulated watermelon lycopene accumulation in greenhouse winter cultivation.

Transcription Factor CmNAC34 Regulated CmLCYB-Mediated ß-Carotene Accumulation during Oriental Melon Fruit Ripening.

Ripened oriental melon (Cucumis melo) with orange-colored flesh is rich in ß-carotene. Lycopene ß-cyclase (LCYB) is the synthetic enzyme that directly controls the massive accumulation of ß-carotene. However, the regulatory mechanism underlying the CmLCYB-mediated ß-carotene accumulation in oriental melon is fairly unknown. Here, we screened and identified a transcription factor, CmNAC34, by combining bioinformatics analysis and yeast one-hybrid screen with CmLCYB promoter. CmNAC34 was located in the nucleus and acted as a transcriptional activator. The expression profile of CmNAC34 was consistent with that of CmLCYB during the fruit ripening. Additionally, the transient overexpression of CmNAC34 in oriental melon fruit promoted the expression of CmLCYB and enhanced ß-carotene concentration, while transient silence of CmNAC34 in fruit was an opposite trend, which indicated CmNAC34 could modulate CmLCYB-mediated ß-carotene biosynthesis in oriental melon. Finally, the yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), ß-glucuronidase (GUS) analysis assay, and luciferase reporter (LUC) assay indicated that CmNAC34 could bind to the promoter of CmLCYB and positively regulated the CmLCYB transcription level. These findings suggested that CmNAC34 acted as an activator to regulate ß-carotene accumulation by directly binding the promoter of CmLCYB, which provides new insight into the regulatory mechanism of carotenoid metabolism during the development and ripening of oriental melon.

Physiological and Transcriptomic Analyses Reveal the Effects of Elevated Root-Zone CO2 on the Metabolism of Sugars and Starch in the Roots of Oriental Melon Seedlings.

Root-zone CO2 is a major factor that affects crop growth, development, nutrient uptake, and metabolism. Oriental melon is affected by root-zone gases during growth, the microstructure, sugar and starch contents, enzymatic activities related to sugar and starch metabolism, and gene expression in the roots of oriental melon seedlings were investigated under three root-zone CO2 concentrations (CK: 0.2%, T1: 0.4%, T2: 1.1%). Elevated root-zone CO2 altered the cellular microstructure, accelerated the accumulation and release of starch grains, disrupted organelle formation, and accelerated root senescence. The sugar and starch contents and metabolic activity in the roots increased within a short duration following treatment. Compared to the control, 232 and 1492 differentially expressed genes (DEGs) were identified on the 6th day of treatment in T1 and T2 plants, respectively. The DEGs were enriched in three metabolic pathways. The majority of genes related to sucrose and starch hydrolysis were upregulated, while the genes related to sucrose metabolism were downregulated. The study revealed that oriental melon seedlings adapt to elevated root-zone CO2 stress by adjusting sugar and starch metabolism at the transcriptome level and provides new insights into the molecular mechanism underlying the response to elevated root-zone CO2 stress.

Root-Zone CO2 Concentration Affects Partitioning and Assimilation of Carbon in Oriental Melon Seedlings.

Root-zone CO2 is essential for plant growth and metabolism. However, the partitioning and assimilation processes of CO2 absorbed by roots remain unclear in various parts of the oriental melon. We investigated the time at which root-zone CO2 enters the oriental melon root system, and its distribution in different parts of the plant, using 13C stable isotopic tracer experiments, as well as the effects of high root-zone CO2 on leaf carbon assimilation-related enzyme activities and gene expressions under 0.2%, 0.5% and 1% root-zone CO2 concentrations. The results showed that oriental melon roots could absorb CO2 and transport it quickly to the stems and leaves. The distribution of 13C in roots, stems and leaves increased with an increase in the labeled root-zone CO2 concentration, and the δ13C values in roots, stems and leaves increased initially, and then decreased with an increase in feeding time, reaching a peak at 24 h after 13C isotope labeling. The total accumulation of 13C in plants under the 0.5% and 1% 13CO2 concentrations was lower than that in the 0.2% 13CO2 treatment. However, the distributional proportion of 13C in leaves under 0.5% and 1% 13CO2 was significantly higher than that under the 0.2% CO2 concentration. Photosynthetic carbon assimilation-related enzyme activities and gene expressions in the leaves of oriental melon seedlings were inhibited after 9 days of high root-zone CO2 treatment. According to these results, oriental melon plants' carbon distribution was affected by long-term high root-zone CO2, and reduced the carbon assimilation ability of the leaves. These findings provide a basis for the further quantification of the contribution of root-zone CO2 to plant communities in natural field conditions.

Drought-induced ABA, H2O2 and JA positively regulate CmCAD genes and lignin synthesis in melon stems.

BACKGROUND: Cinnamyl alcohol dehydrogenase (CAD) is an important enzyme functions at the last step in lignin monomer synthesis pathway. Our previous work found that drought induced the expressions of CmCAD genes and promoted lignin biosynthesis in melon stems. RESULTS: Here we studied the effects of abscisic acid (ABA), hydrogen peroxide (H2O2) and jasmonic acid (JA) to CmCADs under drought stress. Results discovered that drought-induced ABA, H2O2 and MeJA were prevented efficiently from increasing in melon stems pretreated with fluridone (Flu, ABA inhibitor), imidazole (Imi, H2O2 scavenger) and ibuprofen (Ibu, JA inhibitor). ABA and H2O2 are involved in the positive regulations to CmCAD1, 2, 3, and 5, and JA is involved in the positive regulations to CmCAD2, 3, and 5. According to the expression profiles of lignin biosynthesis genes, ABA, H2O2 and MeJA all showed positive regulations to CmPAL2-like, CmPOD1-like, CmPOD2-like and CmLAC4-like. In addition, positive regulations were also observed with ABA to CmPAL1-like, CmC4H and CmCOMT, with H2O2 to CmPAL1-like, CmC4H, CmCCR and CmLAC17-like, and with JA to CmCCR, CmCOMT, CmLAC11-like and CmLAC17-like. As expected, the signal molecules positively regulated CAD activity and lignin biosynthesis under drought stress. Promoter::GUS assays not only further confirmed the regulations of the signal molecules to CmCAD1~3, but also revealed the important role of CmCAD3 in lignin synthesis due to the strongest staining of CmCAD3 promoter::GUS. CONCLUSIONS: CmCADs but CmCAD4 are positively regulated by ABA, H2O2 and JA under drought stress and participate in lignin synthesis.

Editing miR482b and miR482c Simultaneously by CRISPR/Cas9 Enhanced Tomato Resistance to Phytophthora infestans.

Late blight, caused by Phytophthora infestans, is severely damaging to the global tomato industry. Micro-RNAs (miRNAs) have been widely demonstrated to play vital roles in plant resistance by repressing their target genes. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) method has been continuously improved and extensively applied to edit plant genomes. However, editing multiplex miRNAs by CRISPR/Cas9 in tomato has not been studied yet. We knocked out miR482b and miR482c simultaneously in tomato through the multiplex CRISPR/Cas9 system. Two transgenic plants with silenced miR482b and miR482c simultaneously and one transgenic line with silenced miR482b alone were obtained. Compared with wild-type plants, the disease symptoms of three transgenic plants upon infection were reduced, accompanied by increased expression of their common target nucleotide binding site-leucine-rich repeat genes and decreased levels of reactive oxygen species. Furthermore, silencing miR482b and miR482c simultaneously was more resistant than silencing miR482b alone in tomato. More importantly, we found that knocking out miR482b and miR482c can elicit expression perturbation of other miRNAs, suggesting cross-regulation between miRNAs. Our study demonstrated that editing miR482b and miR482c simultaneously with CRISPR/Cas9 is an efficient strategy for generating pathogen-resistant tomatoes, and cross-regulation between miRNAs may reveal the novel mechanism in tomato-P. infestans interactions.

Lignin synthesized by CmCAD2 and CmCAD3 in oriental melon (Cucumis melo L.) seedlings contributes to drought tolerance.

KEY MESSAGE: CmCAD2 and CmCAD3 function more positively than CmCAD1 in oriental melon for lignin synthesis which is important to ensure internal water status and thus for drought tolerance. Well-lignification may be the guarantee of efficient axial water transport and barrier of lateral water flow in oriental melon tolerating drought stress, however remains to be verified. As an important enzyme in monolignol synthesis pathway, five cinnamyl alcohol dehydrogenase (CAD) genes were generally induced in melon seedlings by drought. Here we further revealed the roles of CmCAD1, 2, and 3 in lignin synthesis and for drought tolerance. Results found that overexpressing CmCAD2 or 3 strongly recovered CAD activities, lignin synthesis and composition in Arabidopsis cadc cadd, whose lignin synthesis is disrupted, while CmCAD1 functioned modestly. In melon seedlings, silenced CmCAD2 and 3 individually or collectively decreased CAD activities and lignin depositions drastically, resulting in dwarfed phenotypes. Reduced lignin, mainly composed by guaiacyl units catalyzed by CmCAD3, is mainly due to the limited lignification in tracheary elements and development of Casparion strip. While CmCAD1 and 2 exhibited catalysis to p-coumaraldehyde and sinapaldehyde, respectively. Compared with CmCAD1, drought treatments revealed higher sensitivity of CmCAD2 and/or 3 silenced melon seedlings, accompanying with lower relative water contents, water potentials and relatively higher total soluble sugar contents. Slightly up-regulated expressions of aquaporin genes together with limited lignification might imply higher lateral water loss in stems of silenced lines. In Arabidopsis, CmCAD2 and 3 transgenic lines enhanced cadc cadd drought tolerance through recovering lignin synthesis and root development, accompanying with decreased electrolyte leakage ratios and increased RWCs, thus improved survival rates. Briefly, lignin synthesized by CmCAD2 and 3 functions importantly for drought tolerance in melon.