Seed size is an important agronomic trait in melons that directly affects seed germination and subsequent seedling growth.  However, the genetic mechanism underlying seed size in melon remains unclear.  In the present study, we employed Bulked-Segregant Analysis sequencing (BSA-seq) to identify a candidate region (~1.35 Mb) on chromosome 6 that corresponds to seed size.  This interval was confirmed by QTL mapping of three seed size-related traits from an F₂ population across three environments.  This mapping region represented nine QTLs that shared an overlapping region on chromosome 6, collectively referred to as qSS6.1.  New InDel markers were developed in the qSS6.1 region, narrowing it down to a 68.35 kb interval that contains eight annotated genes.  Sequence variation analysis of the eight genes identified a SNP with a C to T transition mutation in the promoter region of MELO3C014002, a leucine-rich repeat receptor-like kinase (LRR-RLK) gene.  This mutation affected the promoter activity of the MELO3C014002 gene and was successfully used to differentiate the large-seeded accessions (C-allele) from the small-seeded accessions (T-allele).  qRT-PCR revealed differential expression of MELO3C014002 between the two parental lines.  Its predicted protein has typical LRR-RLK family domains, and phylogenetic analyses reveled its similarity with the homologs in several plant species.  Altogether, these findings suggest MELO3C014002 as the most likely candidate gene involved in melon seed size regulation.  Our results will be helpful for better understanding the genetic mechanism regulating seed size in melons and for genetically improving this important trait through molecular breeding pathways. 

Due to their sessile nature, plants require strong adaptability to complex environments, with stress tolerance often associated with trade-offs in growth and development (Major et al. 2020).  This antagonistic relationship between defense and growth has been interpreted as a competition for limited resources that are allocated to defense at the expense of growth, or vice versa. Recent studies have demonstrated that hormone-based signaling networks trigger transcriptional changes in key genes, leading to trade-offs between growth rates and stress defense (Huot et al. 2014).  Several genes involved in biotic and abiotic stress response have been identified.  These genes contain nonsynonymous variants that show convergent changes in allele frequency across different breeding eras in both China and the United States (Wang et al. 2020), which may reflect the selection of biotic and abiotic stress response genes during modern maize breeding.

Transcription factors (TFs) play vital roles in regulation of gene expression in plant cells, with specific key TFs exhibiting multifunctionality by coordinating various regulatory pathways to promote plant growth (Hufford et al. 2021).  Jasmonates (JAs) are identified among phytohormones for their significant roles in regulating various plant processes, particularly in defense mechanisms against pests. MYC2 is a central transcription factor that orchestrates the JA signaling pathway and defense responses in plants by regulating the expression of numerous genes (Du et al. 2022).  Although MYC2 has been extensively characterized in Arabidopsis, studies in crops have revealed the complexity of MYC2’s function, with reports addressing different aspects, such as growth in wheat (Li et al. 2023) or stress defense in maize (Ma et al. 2023).  However, lack of systematic understanding of the complex regulatory network of MYC2 in crops, particularly in maize constrain the further utilization of MYC2 and its downstream genes in maize genetic modification for breeding elite varieties.  Here, we reported that ZmMYC2 had undergone selection during domestication and modern breeding; it acts as a key regulator of the trade-off between development and defense gene expression in maize, elucidating its regulatory network, which holds significant importance in balancing yield and resistance.

Given that some resistance genes have been selected during modern breeding, we analyzed the history of ZmMYC2 over the processes of maize evolution and artificial selection.  According to maize Haplotype Map v3 (HapMap3) database consisting of 1164 modern maize accessions, 25 landraces, and 21 teosintes (Zea mays. parviglumis) (Bukowski et al. 2018), nucleotide diversity strongly decreased sharply at the promoter region (2000 bp upstream of transcription start site) of ZmMYC2 during breeding, while the coding region and 3’-downstream region of ZmMYC2 showed less dramatic changes in nucleotide diversity changes (Fig. 1-A).  Thus, we hypothesize that the genetic diversity within the promoter region of ZmMYC2 has decreased during the breeding process, with favorable variations being selected.  Moreover, the frequency of three polymorphisms underwent convergent changes during modern breeding in both the United States and China (Fig. 1-B–D).  These three polymorphisms constituted three principal haplotypes: pZmMYC2^Hap1, pZmMYC2^Hap2, and pZmMYC2^Hap3, of which the frequency of pZmMYC2^Hap1 showed an increasing trend during modern maize breeding (Fig. 1-E).  The rare haplotype pZmMYC2^Hap3 (n=4) emerged only during the breeding era of China in 2000.  LUC signal activity for pZmMYC2^Hap3 was significantly lower than that of the other two haplotypes in the promoter region (pZmMYC2^Hap1, pZmMYC2^Hap2) (Fig. 1-F–H), suggesting a differential regulatory potential among the haplotypes.  These data indicate that ZmMYC2 was under-selected during maize evolution and breeding processes of maize.  Next, we investigated the expression level of genome-wide association studies of ZmMYC2 based on 368 maize inbred lines using RNA-seq and genome resequencing data (Fu et al. 2013; Li et al. 2013).  The results showed a strong peak signal containing the genomic region of ZmMYC2 on chromosome 1 (Fig. 1-I).

To mine the genes downstream of ZmMYC2, we performed protoplast transient expression-based RNA-sequencing (PER-seq) analysis to facilitate the discovery of new downstream genes utilizing a consistent protoplast system (Zhu et al. 2023).  In total， 281.6 million clean reads were generated, among which an average of approximately 87% of reads were mapped uniquely to the reference genome (Appendices A and B).  The results demonstrated a significant increase in the expression level of ZmMYC2 in each of three replicates of the pRTL2-ZmMYC2-GFP (MYC2-GFP) construct, exceeding a 500-fold increase compared to the pRTL2-GFP-empty (GFP-empty) construct (Fig. 1-J).  Furthermore, upon analyzing differentially expressed genes (DEGs) with a false discovery rate (FDR) <0.05 as the threshold, it was found that 4480 unique DEGs of MYC2-GFP, among which 2,677 were up-regulated compared to GFP-empty (Appendix C).  These up-regulated genes are enriched in circadian rhythm, cell cycles, plant growth, and in response to stress, indicating that these genes are regulated directly or indirectly by ZmMYC2 (Appendix D-A–B).

Several potential candidate genes were selected in an unbiased manner based on their log₂(fold-change) ≥2.5 (Fig. 1-J).  Gene expression profiling analysis of ZmMYC2 and its potential targets revealed essential coincidence (Appendix E).  The interaction between MYC2 and targets observed in the PER-seq system, were further confirmed through expression quantitative trait loci (eQTL) analysis, dual-luciferase reporter assay (DLR), and electrophoretic mobility shift assay (EMSA).  Among the target genes, the members of cytochrome P450 (CYP) gene family are widely distributed in plants involving in various biological processes, such as detoxification of xenobiotics, secondary metabolites production, and terpenoid synthesis (Chakraborty et al. 2023; Sun et al. 2024).  Our results identified an unreported gene of cytochrome P450 family ZmCYP709H1 as a target of ZmMYC2.  Additionally, eQTL analysis of ZmCYP709H1 revealed a strong trans-eQTL signal in the region of chromosome 1, which contains the genomic region of ZmMYC2 (Fig. 1-K).  Subsequent validation through DLR and EMSA confirmed that ZmMYC2 interacts with the promoter region of ZmCYP709H1 and stimulates its expression (Fig. 1-L; Appendices F-A and G-A).  Moreover, the transcriptional activation effect of ZmMYC2 on the promoter of ZmCYP709H1 was suppressed by ZmJAZ8 (Fig. 1-L).  A recent report showed reduced expression of ZmCYP709H1 in three maize dwarf mutants compared to the wild-type, reflecting its potential role in regulating growth, particularly plant height.  This result supports our proposed function of the ZmMYC2-ZmCYP709H1 model (Gao et al. 2024).  Additionally, two other CYP genes, ZmBX5 and ZmBX6, were identified as potential downstream genes of ZmMYC2 that participate in benzoxazinoid synthesis, which is consistent with the findings of a previous study (Ma et al. 2023).  We further confirmed that ZmMYC2 can physically bind to the promoter region of these two genes and activate their expression (Appendix H-A–F).  Besides, the result showed that ZmMYC2 can activate ZmBRD1 expression, which is a member of the CYP gene family and responsible for the final step of brassinosteroid synthesis (Tian et al. 2019) (Fig. 4-A and B; Appendix I-A–D).

The AUXIN RESPONSE FACTOR (ARF) family consists of plant-specific TFs that are key regulators of gene expression in response to the plant hormone auxin (AUX), and participated in various developmental processes such as vascular tissue differentiation, root and shoot development, and environmental stimuli responses (Hagen and Guilfoyle 2002; Salmon et al. 2008).  However, little evidence has been found to support the regulation of ARF gene expression by the core factor ZmMYC2 in the JA signal transduction pathway in maize.  Our data showed that the expression of ZmARF3 was regulated by a trans-eQTL signal involving the gene region of ZmMYC2 (Appendix F-B).  In addition, ZmMYC2 can bind to the promoter region of the ZmARF3 gene and activate its transcription (Fig. 1-M; Appendix G-B).  Besides, MYC2 can activate expressions of senescence-associated genes in rice and wheat, which could be repressed by physical interactions with TaARF15-A1 (Li et al. 2023).  These data demonstrate the key role of MYC2 in regulating the stress resistance and growth of maize through the synergistic regulation of JA and AUX hormone signaling pathways.

Tonoplast intrinsic proteins (TIPs), a subgroup of the aquaporin family, are integral membrane proteins that are crucial for transporting water and small solutes across cellular membrane to maintain water balance (Chaumont et al. 2001).  We found that ZmTIP3c was activated by ZmMYC2 (Fig. 1-N; Appendices F-C and G-C), which supports the potential role of ZmMYC2 in jointly regulating drought stress and JA signal transduction.  The CER2 gene, which is a member of the ECERIFERUM family, is critical for the synthesis of epicuticular wax (Bourdenx et al. 2011; Zhao et al. 2024).  A recent study demonstrated that wounding-induced wax accumulation was primarily regulated by the JA signaling pathway in Arabidopsis, suggesting the potential of JA signaling in wax synthesis (Huang et al. 2024).  We identified ZmCER2 as a ZmMYC2 target (Fig. 1-O; Appendices F-D and G-D).  Additionally, we confirmed the upregulation of ZmCER2 in response to drought stress in five elite inbred lines representing distinct heterotic groups of maize (Fig. 1-P), as observed by previous studies (Zhang et al. 2018, 2020; Jiang et al. 2023).  The result indicates that the drought-induced trait of ZmCER2 can be observed across different genetic backgrounds, thus supporting the potential role of ZmMYC2 in modulating JA signaling and response to drought stress in maize mediated by ZmCER2.

In summary, our findings support the selection of ZmMYC2 during domestication and breeding, highlighting its critical role in regulating genes involving plant growth and development.  Collectively, our eQTL, DLR, and EMSA data successfully validated several targets (ZmCER2, ZmARF3, ZmBRD1 ZmTIP3c, ZmCYP709H1, ZmBX5, and ZmBX6) of ZmMYC2, that encode diverse proteins and participate in various metabolic pathways (Fig. 1-Q).  Of these, ZmCER2 was confirmed to be induced by drought stress and activated by ZmMYC2, suggesting that ZmMYC2 may play a role in the drought response by regulating synthesizing epicuticular wax.  These findings underscore the diverse functions of ZmMYC2 in maintaining the balance between plant development and defense-response, primarily via the JA signaling pathway.  Our data represent a foundation for the further function and mechanism elucidation of of ZmMYC2 and its “Yin-Yang” roles in regulating plant defense and growth (Fig. 1-Q).  Given the crucial role of ZmMYC2 in balancing development and resistance, further work is needed to confirm to unlock the full potentials of ZmMYC2 in mediating yield and resistance through JA signaling pathway by exploring the function of those downstream targets, which is a significant step toward crop precision breeding. 

Nitrogen is a key nutrient for wheat (Triticum aestivum L.) growth and yield, particularly during the grain-filling stage, where most nitrogen is redistributed from vegetative organs to the grain, significantly influencing yield.  However, the period in which nitrogen translocation from the vegetative phase to grain maturation occurs and its correlation with flag leaf senescence remains unclear.  In this study, a field experiment was conducted using the winter wheat cultivar ‘Xinong 511’ under two nitrogen fertilizer treatments: regular nitrogen supply (240 kg ha^-1 [N₂₄₀]) and no nitrogen supply (0 kg ha^-1 [N₀]).  The results revealed that nitrogen accumulation in wheat flag leaves peaked at 7-14 days, with 4.55% nitrogen content, after which nitrogen was redistributed to the grains.  Nitrogen content in flag leaves decreased by 56% during 21-35 days, while that in the grains increased by 51%.  The Plant Analysis Development value (relative chlorophyll content), photosynthetic rate, free amino acid concentration, and soluble protein content in flag leaves peaked at 7-14 days, indicating nitrogen transportation from the flag leaves to the grains.  Nitrogen application significantly increased the nitrogen remobilization rate in flag leaves by 20% compared with that of N₀, reduced reactive oxygen species accumulation by 21%, and delayed flag leaf senescence.  Under nitrogen deficiency, autophagy was induced earlier, with a 5–7-fold increase in the expression of autophagy-related genes (TaATG8), suggesting that regulating the autophagy pathway and enhancing autophagy activity optimizes nitrogen fertilization.  Our study demonstrates that the remobilization of nitrogen from vegetative parts to grains initiates leaf senescence and is closely correlated with the expression of autophagy-related genes.

Stilbenes, natural plant phytoalexin, are involved in the response of various biotic and abiotic stresses in plant environment. STILBENE SYNTHASE (STS) is the key enzyme regulating resveratrol synthesis in grapevine. However, the regulatory mechanism of STS genes expression remains unclear. In this study, we reported a NAC transcription factor, VqNAC17, in Vitis quinquangularis, which can improve plant resistance to salt stress, drought stress and Pseudomonas syringae pv. Tomato DC3000 (Pst DC3000) in transgenic Arabidopsis thaliana. Besides, the interaction between the transcription factor VqNAC17 and VqMYB15 was confirmed using yeast two-hybrid and BiFC. In transgenic A. thaliana, VqNAC17 participates in plant immunity through interaction with VqMYB15 to affect the stilbene synthesis. Furthermore, the experimental results of yeast one-hybrid assay and LUC transient expression assay found that VqNAC17 can also bind to the promoter of VqMYB15. These results indicate that VqNAC17 is a key regulator that can promote the expression of STS by interacting with VqMYB15.

Japanese encephalitis (JE) is a zoonotic mosquito-borne viral disease caused by the Japanese encephalitis virus (JEV). The virus is transmitted among adult pigs, causing abortion in sows and orchitis in boars. Vaccination remains the most effective strategy for the prevention and control of this disease. Studies have shown that genotype I (GI) JEV has replaced GIII JEV as the dominant strain in many Asian countries. However, all currently licensed JE vaccines, including the widely used SA14-14-2 live attenuated vaccine, are derived from the GIII strain. It has been reported that GIII-based vaccines do not provide complete protection against the GI strain. In this study, we conducted vaccination-challenge protection assays in mice and boars to evaluate the protective efficacy of live attenuated GI (SD12-F120) derived vaccines against challenge by a homologous genotype. In mice, immunisation with the vaccine induced a potent viral-neutralising response against the homologous GI JEV SD12 strain. The SD12-F120 vaccine provided complete protection against lethal challenge by SD12, whilst also attenuating viraemia. JEV was not detected in the blood, oronasal swabs, or testicles of boars receiving the SD12-F120 vaccine. Vaccination induced high levels of neutralising antibodies against the homologous GI strain in boars, with titers as high as 64. Histopathological analysis showed that interstitial cells of the boar testis and spermatogonia at all levels were well preserved in the vaccine-immunised group, effectively suppressing the occurrence of orchitis. These results showed that the SD12-F120 vaccine provides boars complete protection against challenge by SD12, whilst also protecting against viraemia and testicular damage. Our findings indicate that SD12-F120 is a promising live-attenuated vaccine candidate for controlling the spread of GI JEV.

Innate immunity is the primary defense against viral infections, with Toll-like receptors (TLRs) playing a crucial role in this process. This study aims to highlight the effectiveness of a pyrrolo[3,2-d]pyrimidine derivative (named TLR713), a potential TLR7 agonist, in inhibiting pseudorabies virus (PRV) replication both in vitro and in vivo. Tests on PK-15 cells demonstrated that TLR713 had no significant impact on cell viability, cell cycle progression, or apoptosis at concentrations of 0 – 3 μmol L^-1. TLR713 could promote the phosphorylation of IκBα, p38, and JNK through TLR7, and increase the expression of inflammatory cytokines. In vitro, when cells were treated with TLR713, PRV proliferation was inhibited via TLR7 pathway. Analysis of the viral life cycle indicated that TLR713 could inhibit the replication of PRV, but not affect viral attachment, entry, assembly, or release. In vivo, TLR713 showed no side effects on mice at a concentration of 25 mg kg^-1. It improved the survival rate of PRV-infected mice, reduced tissue viral load, and alleviated the inflammatory response. In summary, this study highlights the potential of TLR713 as a novel TLR7 agonist capable of inhibiting PRV replication and may offer new opportunities for developing antiviral therapies.