The pivotal role of N⁶-methyladenosine (m⁶A) demethylases in regulating plant stress responses has been widely explored; however, the function of apple m⁶A demethylases under heat stress and fixed-carbon starvation is unclear. In this study, the apple RNA demethylase gene family was identified, and the demethylase gene MdALKBH1A was selected for further analysis. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, we demonstrated that MdALKBH1A is the m⁶A demethylase of apple. Moreover, transgenic ‘Micro Tom’ tomato plants overexpressing MdALKBH1A were more sensitive to high temperature, probably due to the decreased antioxidant ability, increased membrane lipid peroxidation and reduced plasma membrane stability. However, these tomato plants overexpressing MdALKBH1A were more resistant to fixed-carbon starvation, as evidenced by the improved plasma membrane stability, enhanced photosynthetic rates and elevated autophagic activity. In summary, our results highlight the crucial role played by MdALKBH1A in the response of apple plants to high-temperature stress and fixed-carbon starvation.

Maize (Zea mays L.) is a crucial global crop, serving as a primary source of food and feed.  However, its kernels are susceptible to infection by Aspergillus flavus, a fungus known for producing aflatoxins- highly carcinogenic compounds harmful to human and animal health. Identifying quantitative trait loci (QTLs) for aflatoxin resistance and developing aflatoxin-resistant maize varieties are essential for mitigating aflatoxin contamination.  In this study, we conducted a genome-wide association study (GWAS) using an enlarged genotypic panel of 311 maize inbred lines to identify genetic loci associated with A. flavus resistance.  Phenotypic data on A. flavus resistance were collected through controlled inoculation experiments conducted under controlled conditions.  The results revealed that the resistance traits to A. flavus follow a normal distribution. Additionally, temperate inbreds exhibited stronger resistance to A. flavus than tropical/subtropical materials.  This study identified 15 novel QTLs encompassing 47 high-expressed genes, with each QTL explaining 8.22-27.71% of the phenotypic variation, indicating that increased marker density improved statistical power.  Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that these genes are related to fatty acid synthesis, glycoside decomposition, and root growth and development.  One specific gene, Zm00001d021197, located on ZmAFR16, displayed clustered peaks and accounted for an average of 10.21% of the phenotypic variation.  This gene was found to play a role in cell membrane formation and possess alpha-L-fucosidase activity, promoting glycoside metabolism and contributing to polysaccharide degradation.  Haplotype analysis showed significant differences in resistance to A. flavus among different haplotypes of the Zm00001d033637 and Zm00001d021197.  Inbreds carrying the favorable haplotype combination of these two genes exhibited strong resistance to A. flavus.  By select sweep analysis, it was found that Zm00001d021197 was selected during the domestication of teosinte (Zea mays ssp. mexicana) to modern maize, as well as during the adaptation from tropical/subtropical maize to temperate maize. Importantly, we developed molecular markers in the promoter region of Zm00001d021197 to efficiently identify maize germplasm with beneficial haplotypes for resistance to A. flavus.  These findings not only enhance our understanding of the genetic factors influencing maize kernel resistance to A. flavus but also offer valuable insights for improving existing germplasm and developing new maize varieties with enhanced resistance to this pathogen.

Pear anthracnose, caused by Colletotrichum fructicola, is a devastating disease that seriously affects most pear varieties, thereby compromising their yield and quality. However, effective control of this pathogen is lacking. Moreover, the critical resistance responses to C. fructicola in pear are unknown. To investigate these resistance mechanisms of pear against Colletotrichum fructicola, transcriptomic and metabolomic were performed and analyzed in pear anthracnose-resistant pear variety ‘Seli’ and the susceptible variety ‘Cuiguan’ after infection with C. fructicola, respectively. The differentially expressed genes and differentially accumulated metabolites (DAMs) were mainly related to metabolism and secondary metabolite synthetic pathways, including alpha-linoleic acid metabolism, phenylalanine biosynthesis metabolism, unsaturated fatty acids biosynthesis, and amino acids and derivatives biosynthesis etc. In particular, the accumulation of unsaturated fatty acids, amino acids and derivatives, such as linoleic acid and derivatives, lauric acid, N-acetyl-L-glutamic acid and L-proline was significantly increased in the resistant pear variety ‘Seli’ upon C. fructicola infection, while the amino acids of oxiglutatione and N-acetyl-L-glutamic acid, as well as the proanthocyanidins were significantly decreased in susceptible pear variety ‘Cuiguan’ upon C. fructicola infection, indicating that these metabolites were responsible for the different levels of resistance to anthracnose in ‘Seli’ and ‘Cuiguan’. Overall, our results provided new insights into pear anthracnose resistance regulation, and this may assist in developing new strategies to control pear anthracnose, as well as in breeding anthracnose-resistant varieties.

Anthocyanins, the flavonoid pigments responsible for vibrant fruit and flower colors, play key roles in both plant physiology and human health. MYB transcription factors are crucial regulators of anthocyanin biosynthesis and accumulation, the functional differences of homologous MYB transcription factors in regulating anthocyanin content are still unclear. In strawberry (Fragaria × ananassa), FaMYB44.1 and FaMYB44.3 are highly homologous MYB transcription factors localized in the nucleus and significantly induced by weak light. However, they differ in their effects on anthocyanin accumulation in fruits. FaMYB44.1 inhibits anthocyanin synthesis by transcriptionally suppressing FaF3H, essential for anthocyanin regulation, in the ‘BeniHoppe’ and ‘JianDe-Hong’ strawberry varieties. By contrast, FaMYB44.3 does not affect anthocyanin levels. This study provides a comprehensive overview of the roles of FaMYB44.1 and FaMYB44.3 in anthocyanin regulation in strawberry fruits. By elucidating molecular mechanisms underlying their regulation, this study enhances our understanding of how the interaction between genetic and environmental factors controls fruit pigmentation and enhances fruits’ nutritional value.

The postharvest senescence phase of table grapes comprises a series of biological processes. MicroRNAs (miRNAs) regulate downstream genes at the post-transcriptional level; however, whether miRNAs are involved in postharvest grape senescence remains unclear. We used small RNA sequencing to identify postharvest-related miRNAs in ‘Red Globe’ (Vitis vinifera) grapes harvested after 0, 30, and 60 d at 4 °C (RG0, RG30, RG60). In total, 42 known and 219 novel miRNA candidates were obtained. During fruit senescence, the expression of PC-3p-3343_1921, miR2950, miR395k, miR2111, miR159c, miR169q, PC-5p-1112_4500, and miR167b changed significantly (p<0.05). Degradation sequencing identified 218 targets associated with cell wall organization, TCA cycling, pathogen defense, carbon metabolism, hormone signaling, the anthocyanin metabolism pathway, and energy regulation, of which ARF6, GRF3, TCP2, CP1, MYBA2, and WRKY72 were closely related to fruit senescence. We also verified VIT_00s2146g00010, VIT_02s0012g01750, and VIT_03s0038g00160 with unknown functions cleaved by senescence-related PC-5p-1112_4500 via the dual luciferase assay and transient transformation of grape berries and showed that they regulate berry senescence. These results deepen the understanding of the contribution of miRNAs in regulating grape berry senescence and prolonging the shelf life of horticultural products. Based on these results, we propose a new theoretical strategy to delay the postharvest senescence of horticultural products by regulating the expression of key miRNAs (e.g., PC-5p-1112_4500), thereby extending their shelf life.

CRISPR-Cas9 emerged as a powerful tool for gene editing, which has been widely used in plant functional genomics research and crop genetic breeding (Chen et al. 2019). The target specificity of CRISPR-Cas9 relies on the 20-base-pair single guide RNA (sgRNA), making it relatively quick and straightforward to create plant-specific mutant libraries through large-scale synthesis of sgRNAs targeting multiple genes or even the whole genome. Several CRISPR-Cas9 mutant libraries have been developed for crops such as rice (Lu et al. 2017; Meng et al. 2017), soya bean (Bai et al. 2020), Brassica napus (He et al. 2023), and cotton (Sun et al. 2023). However, no CRISPR-Cas9 mutant library has yet been generated in woody crop plants. Grape (Vitis vinifera L.) is one of the oldest and most economically valuable fruit crops worldwide. The MYB family is one of the most abundant and versatile transcription factor families in plant (Wu et al. 2022). Here, we described a strategy for generating a collection of MYB mutant lines in grape using a sgRNA library.

We obtained 138 grape MYB transcription factor sequences from the Plant Transcription Factor Database (PlantTFDB, http://planttfdb.gao-lab.org/family.php?sp=Vvi&fam=MYB) (Appendix A). Since genes with similar sequences often share similar functions, a phylogenetic tree was constructed and divided all MYBs into 30 sets based on the sequence similarity (Fig. 1-A and Appendix B-a). A total of 127 sgRNAs were designed, each targeting conserved regions shared by two or more MYB transcription factors with fewer than three base-pair differences. This approach aimed to simultaneously mutate multiple MYB members within a cluster using a single sgRNA, addressing the challenge of genetic redundancy (Appendix B-b). These shared target sites were designated as Target1 (T1). In addition, specific sgRNAs targeting individual MYB transcription factors were designed using the  CRISPR-P 2.0 online tool (http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR) (Liu et al. 2017). The design criteria included selecting target sites within exons near the start of open reading frames (ORFs), a GC content of at least 40%, and an off-target efficiency below 0.4. Finally, 138 sgRNAs targeting specific sites for each MYB transcription factor were designed, referred to as Target2 (T2). In total, a comprehensive sgRNA library comprising 265 sgRNA was developed to target 138 MYBs, with an average  of 1.92 sgRNA per MYB (Fig. 1-A and Appendix C).

The sgRNA fragments from the same set were mixed as one sgRNA pool, ligated into the pKSE401 vector using Gibson ligation, and subsequently transformed into the Escherichia coli  TOP10 competence cells (Fig. 1-B and Appendix D-a). To evaluate the ligation efficiency of the sgRNA pool with the vector, 90 E. coli clones from sets #1-3 were randomly selected and sequenced. The results demonstrated that the ligation efficiency exceeded 90% and the sgRNA coverage ratio over 80%, confirming the feasibility of this method (Appendix D-b). Using this approach, approximately 1300 (~5×) positive E. coli clones were obtained across the 30 sets (Fig. 1-D). Plasmids extracted from each set were mixed in equal proportions and transformed into Agrobacterium tumefaciens GV3101 competence cells. Finally, all Agrobacterium colonies were collected and verified with next-generation sequencing (NGS). The results revealed that 95.31% of the sequences in the library were accurate, and 178 of 265 sgRNAs were represented by at least one read, targeting 125 (90.58%) MYB transcription factors. Most (83.93%) sgRNA read counts fell within the range of 2⁸-2¹⁵. These results indicated that the sgRNAs library in Agrobacterium exhibited high accuracy and gene coverage, which is usable for grape transformation (Fig.1-E).

Vitis vinifera L. cv. Cabernet Sauvignon is one of the most renowned red wine grape varieties, widely cultivated worldwide. In this study, pro-embryonic masses of ‘Cabernet Sauvignon’ were used as recipient material for Agrobacterium-mediated transformation (Fig. 1-C and Appendix E). A total of 1354 kanamycin-resistant seedlings were obtained, and which 341 were confirmed as transgenic lines (PCR positive). And, all the lines were determined to harbor a single correct sgRNA, representing 13 unique sgRNAs targeting 18 MYB transcription factors. Target site DNA was amplified and sequenced, revealing only 67 gene-edited lines with mutations in 8 MYB transcription factors (Fig. 1F and Appendix F and G). Among these, 56 lines were chimeric mutants, nine were biallelic mutants, one was a homozygous mutant and one was a heterozygous mutant (Appendix H). Five Target1 type transgenic lines were obtained, three of them did not mutate in all of the targeted genes, in which the sgRNA was targeting two or more completely conserved sites. Additionally, gene-edited lines for GSVIVT01032467001-T1 and GSVIVT01014770001-T1 were producted. However, the sgRNA harbored in these transgenic lines only caused the mutations in GSVIVT01032467001 and GSVIVT01014770001, without off-target effects on genes with similar sequences (Appendix I and J). All MYB-edited lines were subsequently transplanted into a greenhouse for observation (Appendix K). Phenotypic analysisrevealed that the GSVIVT01026481001 edited lines exhibited significantly enhanced tolerance to drought stress (Fig. 1-G).

CRISPR-Cas9 has greatly accelerated gene function research and breeding in plants. In this study, we developed a strategy for generating a collection of MYB mutant lines in grape using a CRISPR-Cas9 library. However, the relatively lower transformation efficiency in grape limited the number of mutants obtained. Factors affecting grape transformation efficiency primarily included the regeneration rate of the recipient material and the efficiency of Agrobacterium infection. Numerous studies have demonstrated that plant regeneration efficiency enhanced using developmental regulators such as BABY BOOM (BBM), WUSCHEL (WUS), GROWTH-REGULATING FACTOR (GRF), and REGENERATION FACTOR (REF) (Debernardi et al. 2020; Yang et al. 2022). Additionally, plant transformation and gene editing efficiency improved through optimized genetic transformation methods and gene editing vector designs (Debernardi et al. 2024; Yan et al. 2024). We confirmed that a large number of edited plants could be obtained simultaneously using a sgRNA mixed-pool library, provided that grape transformation efficiency is improved. This strategy holds significant potential for constructing genome-wide mutant libraries in woody crop plants in the future.

PpMYB10 and PpMYB114 have been identified as the key R2R3-MYB transcription factors (TFs) that positively regulate anthocyanin biosynthesis in pear. Our previous study demonstrated that the ethylene-induced PpERF9-PpTPL1 co-repressor complex represses PpMYB114 expression, but not PpMYB10, via histone deacetylation. However, the precise molecular mechanism underlying the ethylene-mediated inhibition of PpMYB10 expression remains to be elucidated. The results of the present study reveal a high correlation between the expression patterns of PpMYB114 and PpMYB10 in response to ethylene signaling. Moreover, PpMYB114 was found to promote the expression of PpMYB10 by directly binding to the MYB-bind site (MBS) element within its promoter region. Transient overexpression or silencing of PpMYB114 resulted in the promotion or inhibition of PpMYB10 expression in mature pear fruit, respectively. The overexpression of PpMYB114 in pear calli significantly induced PpMYB10 expression and anthocyanin biosynthesis. Conversely, transient silencing of PpMYB10 in PpMYB114-OX pear calli hindered the promotive effect of PpMYB114 on anthocyanin biosynthesis, indicating that PpMYB114 induces anthocyanin biosynthesis at least partially, depending on the transcriptionally activating PpMYB10. Collectively, these results indicate that ethylene may inhibit the expression of PpMYB10 by repressing PpMYB114. Our findings provide insights into a possible mechanism involving ethylene-inhibited PpMYB10 in pear and reveal the regulatory relationship between the R2R3-MYBs involved in anthocyanin biosynthesis. 

Insight into carbon turnover in soil aggregates and density fractions is essential to reduce uncertainty in estimating carbon pools on the Tibetan Plateau. Further, how these vary with land-use type is unclear. In this study, the effect of land use type on carbon storage and fractionation based on organic carbon and its ¹³C abundance was quantified at the microscale of soil aggregates and density fractions in Tibetan alpine. The sequence of soil aggregates destruction in plantation (13.1%)<shrubland (32.7%)<grassland (47.9%) farmland (61.8%) shows that plantation strengthen soil structure. Plantation increased light fraction organic carbon (28.3%) but reduced mineral associated organic carbon (40.6%) contribution to carbon stock compared to farmland (13.5%, 70.3%). Interestingly, plantation enhanced aggregational differentiation of organic carbon and ¹³C in each density fraction, whereas no such phenomenon exists in soil organic carbon. Carbon isotope analyses revealed that carbon transfer in the plantation occurred from light fraction in macroaggregate (-24.9‰) to the mineral associated fraction in microaggregate (-19.9‰). When compared to the other three land-use types, the low transferability of carbon in aggregates and density fractions in plantation provides a stable carbon pool for the Tibetan Plateau. This study shows that plantation can mitigate global climate change by slowing carbon transfer and increasing carbon storage at the micro-scale of aggregates and density fractions in alpine regions.

Apple fruit firmness is a crucial index for measuring the internal quality of apples, influencing palatability and determining storage and transportation capacity. The primary cause of decreased firmness during fruit development is the hydrolysis of cell wall polysaccharides. Xyloglucan endotransglycosylase/hydrolase (XTH) is a key enzyme involved in the depolymerization of cell wall polysaccharides, but its mechanism in the formation of fruit firmness remains unclear. Here, we identified the gene MdXTH2 by integrating metabolomic and transcriptomic data, and further analyzed its function and molecular mechanism in the formation of apple fruit firmness. The results showed a downward trend in both fruit firmness and cell wall components throughout fruit development. The contents of cell wall material, cellulose, and hemicellulose in various apple varieties exhibited significant positive correlations with firmness, with total correlation coefficients of 0.862, 0.884, and 0.891, respectively. Overexpression of MdXTH2 significantly increased fruit firmness in apple and tomato, inhibited fruit ripening, and significantly suppressed calli growth. The upstream transcription factor MdNAC72 of the MdXTH2 gene can promote the expression of fruit ripening-related genes. Furthermore, dual-luciferase, yeast one-hybrid, and electrophoretic mobility shift assay assays demonstrated that MdNAC72 down-regulated the transcription of MdXTH2 by binding to its promoter. In summary, these results provide a strategy for studying fruit quality regulation and a theoretical basis for breeding apple varieties with moderate firmness through genetic improvement.

Cold stress widely impairs the quality and yield of tea plants. The miR164 family and its target NAC transcription factor have been identified as crucial regulators in response to cold stress. However, the role of miR164 and CsNAC in cold tolerance in tea plants was little understood. In our study, the expression level of csn-miR164a was significantly reduced under cold stress, and was significantly negative correlation with that of CsNAC1. 5’ RACE and GUS histochemical assays clearly showed that CsNAC1 was specifically cleaved by csn-miR164a. The csn-miR164a-silenced tea leaves promoted expression level of CsNAC1 and CsCBFs, and exhibited greater cold tolerance, also overexpression of CsNAC1 enhanced cold tolerance in transgenic Arabidopsis plants by promoting the expression levels of AtCBFs. In contrast, the heterologous overexpression of csn-miR164a in Arabidopsis decreased the expression level of AtNACs and AtCBFs, and thus impaired cold tolerance. Additionally, silencing of CsNAC1-impaired the expression levels of CsCBFs resulted in greater cold sensitivity in tea leaves. Taken together, our present study demonstrated that the miR164a-CsNAC1 module may play a negative role in cold tolerance of tea plants via CsCBF-dependent pathway.

Stone fruits, also known as drupes, have evolved an extremely hard wood-like shell called a stone to protect seeds. Currently, the market value of stoneless cultivars has risen dramatically, which highlights the need to cultivate stoneless fruit. Therefore, the underlying mechanism of fruit stones is urgently needed. By employing the stone-containing jujube cultivar ‘Youhe’ and two stoneless Chinese jujube cultivars, ‘Wuhefeng’ and ‘Jinsixiaozao’, we carried out a comprehensive study on the mechanism of fruit stone development in jujube. Anatomical analysis and lignin staining revealed that the stone cultivar ‘Youhe’ jujube exhibited much greater lignin accumulation in the endocarp than did the other two stoneless cultivars. Lignin accumulation may be the key reason for the formation of fruit stone. By analysing the transcriptome data and identifying differentially expressed genes (DEGs), 49 overlapping DEGs between ‘Youhe’ jujube vs ‘Wuhefeng’ jujube and ‘Youhe’ jujube vs ‘Daguowuhe’ jujube were identified. Among these DEGs, ZjF6H1-3 and ZjPOD, which are involved in lignin synthesis, were identified. Overexpression and silencing of ZjF6H1-3 and ZjPOD in wild jujube seedlings further confirmed their role in lignin synthesis. In addition, two bHLH transcription factors were also included in these 49 overlapping DEGs, and bHLH transcription factor motifs were found in the promoters of ZjF6H1-3 and ZjPOD, indicating that bHLH transcription factors are also involved in lignin synthesis and stone formation in Chinese jujube. This study provides new insight into the molecular networks underlying fruit stone formation and provides an important reference for the molecular design and breeding of stoneless fruit cultivars for jujube and fruit trees.

Lysophosphatidic acid acyltransferases (LPATs) are enzymes widely expressed in various plant species, contributing to growth, development, and stress responses.  Currently, little information regarding the LPAT gene family is available in soybeans.  In this study, genome-wide analyses identified 15 soybean LPATs, which were then evaluated for the conserved protein motifs.  These genes were grouped into three clusters based on their phylogenetic relationships.  Confocal microscopy was used to visualize the localization of six GmLPATs within Arabidopsis mesophyll protoplasts.  cis-Acting regulatory element analyses and qRT-PCR experiments revealed that these GmLPATs were upregulated in response to hormone stimulation or exposure to abiotic stressors, including drought, alkaline conditions, and salt stress.  The expression patterns of these GmLPATs varied across different soybean tissue types.  One member of the solLPAT1 subtype (GmLPAT11) was found to be upregulated in response to a range of treatments, highlighting its role in soybean salt stress responses. GmLPAT11 expression in Escherichia coli confirmed the LPAT activity of this recombinant enzyme, and overexpressing this LPAT reduced reactive oxygen species production in transgenic soybean plants, enhancing their salt stress tolerance.  Gene association analyses indicated that GmLPAT11 variants are closely associated with seedling salt tolerance, and a polymorphism in the GmLPAT11 CDS region was potentially associated with salt tolerance.  These results provide new insight into the nature of the LPAT gene family in soybeans while also suggesting promising candidate genes for future research efforts aimed at enhancing the overall salt tolerance of soybean crops. 

Excellent nitrogen (N) management techniques can improve crop yields while mitigating reactive N (Nr) losses. The synergistic effects of applying paired N management techniques have important implications for designing excellent N management strategies, but the interaction effects remain poorly known. Here, a meta-analysis was conducted to quantify the effects of optimized N management techniques (optimized N application rate, optimized topdressing, and applying enhanced-efficiency fertilizers) on wheat yield, N use efficiency (NUE), and Nr losses, as well as the interactive effects of paired N management techniques (combining an optimized N rate with topdressing or enhanced-efficiency fertilizers). The results demonstrated that an optimized N fertilizer rate reduced Nr losses by 28–31% while the wheat yield declined by 2%; however, the wheat yield increased by 2% when the reduction of N fertilizer was less than 20%. The adoption of topdressing and enhanced-efficiency fertilizers significantly increased wheat yields by 4–8% and NUE by 8–14%, while reducing Nr losses by 28–40%, and high topdressing frequency and nitrification inhibitors showed stronger positive effects on wheat yield. Paired N management techniques increased wheat yields by 3–4% and NUE by 37–38%, with additive or synergistic effects; and they also reduced Nr losses by 5–66% but showed an antagonistic effect. Such non-additive interactions amplified the positive effects on wheat production, but the benefits in terms of environmental risk reduction were weakened. Overall, this study highlights the importance of synergistic effects in innovative N management to address the trade-off between crop yield and Nr losses.

Wheat (Triticum aestivum L.) quality is a major focus of wheat breeding, which is influenced by multiple factors. The Huang-Huai wheat region, one of the main wheat-producing areas in China, provides favourable conditions for cultivating wheat cultivars with strong-gluten and medium-strong-gluten. In this study, a systematic assessment of seven crucial quality traits and important genetic loci (Glu-1 and Sec-1) in 436 wheat cultivars in the Huang-Huai wheat region of China by principal component analysis (PCA) and fuzzy comprehensive evaluation (FCE) methods showed that the stability time (ST), stretch area (SA), and maximum resistance (MAXR) were identified as three key factors, which significantly influenced wheat quality. Glu-1 and Sec-1 primarily impacted these three traits and subsequently influenced wheat quality. Compared to Glu-A1 and Glu-B1, Glu-D1 has a more significant impact on the comprehensive evaluation value D, principal components PC1-PC3, and the main traits ST, SA and MAXR of PC1. Wheat cultivars carrying the high-molecular-weight glutenin subunit (HMW-GS) Dx5+Dy10 exhibited a notable improvement in ST, SA, and MAXR traits compared with those carrying HMW-GS Dx2+Dy12, suggesting that Dx5+Dy10 may enhance wheat quality by improving ST, SA, and MAXR. By combining the results of D value, GYT (genotype by yield×trait) index, and HMW-GS score, 20 high-quality and high yield wheat cultivars were identified, which can be used as elite parents for wheat quality breeding.

Characterizing the N uptake and utilization of different maize hybrids is essential for optimizing N application and increasing the profits from maize production.  Research trials were conducted with controlled-release urea (CRU) as a base fertilizer (TC) and urea split application in one (T1), two (T2), and three (T3) stages to evaluate the effects on N uptake, NUE, and yield using the ¹⁵N tracer technique between two maize hybrids; DH518 (an mid-early-maturing hybrid) and DH605 (a late-maturing hybrid).  According to the results, compared with urea, CRU as a base fertilizer and urea split application in two and three stages significantly increased grain yield and NUE while reducing environmental N loss.  Compared with T1, the grain yields of the TC, T2, and T3 treatments were, respectively, increased by 11.1, 9.8, and 11.7% in DH518 and by 16.4, 15.7, and 22.9% in DH605.  Regression analysis showed that the grain yield of DH518 displayed a bilinear trend of an initial rapid increase and then a slow increase with the increase in post-anthesis N accumulation, total N accumulation, N recovery efficiency, and N nutrition index (NNI).  By contrast, DH605 consistently showed a linear regression relationship with a rapid increase.  The crop recovery N efficiency (CRN) values in the T3 treatment for urea applied at the sowing stage and topdressing at the V9 stage in DH518 were 60.0 and 62.4% higher than under topdressing at the VT stage, respectively, while the CRN values of urea topdressing at the V9 and VT stages in DH605 were 37.7 and 37.1% higher than when applied at the sowing stage, respectively.  The higher pre-anthesis N demand and shorter growth period of DH518 maintained the N supply–demand balance, resulting in NNI (NNI≥0.988) falling within the range of slow yield increase under the T2 and TC treatments, while the N status of DH605 plants only reached optimal levels in the T3 treatment.  Therefore, a split three-stage application of urea or applying CRU as a base fertilizer and topdressing with urea in the later growth stages is recommended for mid-late-maturing hybrids to obtain an optimal yield.  In addition, for mid-early-maturing hybrids, applying CRU or reducing the number of times of split application, e.g., a split two-stage application, can ensure an adequate N supply in the later growth stages and increase production and thus profits.

Maize is a cornerstone of global food security, but it faces increasing challenges from corn aphids, particularly with the widespread adoption of genetically modified Bt maize. This trend suggests a growing need for sustainable pest control strategies. Methyl salicylate has been proposed as a volatile compound with the potential for managing aphids. In this study, Y-tube olfactometer and Petri dish dispersal assays showed that methyl salicylate can repel wingless and winged aphids at 0.1 to 1,000 ng μL^-1. Moreover, at concentrations of 100 and 1,000 ng μL^-1, it was found to attract beneficial insects such as adults and larvae of Harmonia axyridis. Exposing maize plants to methyl salicylate resulted in a prominent reduction in the number of aphids compared to the control. In addition, clip cage experiment assays showed that the nymphal development duration was increased, while the adult duration and generation time were reduced, and the reproductive duration and total number of aphid offspring in plants treated with methyl salicylate were dramatically lower than in the control. Over two years of field trials, methyl salicylate-impregnated alginate beads provided significant reductions in the populations of key aphid species, including Rhopalosiphum padi, Rhopalosiphum maidis, and Aphis gossypii. Concurrently, there were marked increases in the presence of natural predators such as H. axyridis, Propylaea japonica, Syrphus corollae, and Chrysoperla sinica. These compelling results underscore the potential of methyl salicylate as a key component in integrated pest management strategies for maize, offering a green alternative to traditional chemical control.

Magnesium (Mg) deficiency is becoming a limiting factor for citrus production in acid soils of subtropical and tropical zones. It is speculated that soil Mg leaching and thereby its imbalance may be a major cause of yield decline, yet Mg deficiency in citrus receives little attention. A two-year field experiment was therefore conducted to quantify soil Mg leaching in a typical citrus orchard in China fertilized with varying levels of Mg (0 (Mg₀), 45 (Mg₄₅), 90 (Mg₉₀) and 180 (Mg₁₈₀) kg MgO ha^-1 yr^-1). Results showed that Mg application significantly increased citrus fruit yield by 4.1-16.4% compared with where MgO was not added. The average amount of soil Mg leaching was 65.7 kg ha^-1 yr^-1 where no Mg fertilizer was added, while it reached up to 91.3 kg Mg ha^-1 yr^-1 where MgO was added at the rate of 180 kg ha^-1. Over the 4 treatments, Mg leaching accounted for 12.1-42.4% of the applied Mg fertilizer. Mg leaching and its removal through harvested fruits resulted in an orchard soil Mg balance of -69.9, -51.1, -27.4 and 10.9 kg ha^-1 in the Mg_0, Mg_45, Mg₉₀ and Mg₁₈₀, treatments, respectively. The pH values of leachate from the acid soil were alkaline and it contained higher amounts of calcium and potassium than that of Mg. Considering the high leaching of Mg from the acid soils of citrus orchards, applications of Mg fertilizer or Mg-fortified soil conditioner are vital to sustain soil Mg balance, high fruit yield and fruit quality in citrus production systems in humid subtropical regions.

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.

The mammary glands of dairy cows with ketosis face unique challenges, including the supraphysiological circulating concentrations of free fatty acids (FFA) and apoptosis of epithelial cells. The fact that endoplasmic reticulum (ER) stress and apoptosis are closely related processes (at least in nonruminants) suggests that mechanisms of metabolic stress-induced apoptosis in bovine mammary epithelial cells may involve the ER stress pathway. The objective of this study was to investigate (1) the status of the ER stress pathway in mammary gland of dairy cows with ketosis, and (2) the role of ER stress in the apoptosis of bovine mammary epithelial cells challenged with high concentrations of FFA. Ketosis or exogenous FFA activated the ER stress pathway in the mammary gland of dairy cows or MAC-T cells. Pretreatment with the ER stress activator Tunicamycin (Tun) aggravated ER stress and apoptosis in MAC-T cells induced by FFA. However, ER stress inhibitor Tauroursodeoxycholate (TUDCA) attenuated ER stress induced by FFA and also attenuated the apoptosis in MAC-T cells. In conclusion, the data confirmed that FFA induced apoptosis of bovine mammary epithelial cells in dairy cows with ketosis via ER stress signaling. Thus, timely resolution of ER stress may help counteract the negative effects of ketosis on the mammary gland.

Chloroplast gene expression relies on nucleus-encoded factors for RNA metabolism processing, but the mechanisms under cold stress remain poorly understood.  In this study, we isolated and characterized a foxtail millet (Setaria italica) mutant, temperature-sensitive chlorophyll-deficient (sitcd1), which exhibited reduced chlorophyll content and abnormal chloroplasts, resulting in an albino phenotype during early leaf development at low temperatures (20°C during the day and 18°C at night).  Map-based cloning revealed that SiTCD1 encoded a P-type PPR protein localized in chloroplasts.  In sitcd1 background, transgenic lines of SiTCD1 overexpression appeared nearly normal green leaves under L20/D18 condition.  SiTCD1 was especially expressed in earlier development of leaves under low temperature.  Additionally, SiTCD1 directly bound to the plastid gene atpF in vitro, which might participate in the splicing of plastid gene atpF under low temperature.  RNA-seq indicated that the expression of genes related to metabolism (such as porphyrin, chlorophyll and glutathione metabolism), which required ATP for energy, was down-regulated in sitcd1, resulting in decreased chlorophyll content, GSH, and its redox couple (GSH/GSSG) at low temperature.  As sitcd1 exhibited more sensitive at the bud bursting stage than germination and seedling stage under cold stress, we identified two haplotypes of SiTCD1 (SiTCD1^Hap1 and SiTCD1^Hap2) in 195 accessions, and found that accessions carrying the SiTCD1^Hap2 allele were more tolerant to cold stress than those with the SiTCD1^Hap1 allele at the bud bursting stage.  In summary, our results suggest that SiTCD1 is essential for early chloroplast development under low temperature in foxtail millet.

Fusarium head blight (FHB) is one of the most important and destructive wheat diseases worldwide, threatening both food security and safety.  In this study, a recombinant inbred line (RIL) population with 269 F₆ lines developed from a cross between ‘Nanjing 8611’ and ‘Ocoroni’ was used to map quantitative trait loci (QTLs) for FHB resistance.  Field FHB trials were conducted for three years in Nanjing, China, using point inoculation, and two years in Mexico with spray inoculation.  A high-density genetic map was constructed for the RIL population using the wheat 55 K single nucleotide polymorphism (SNP) array.  A total of 13 QTLs were detected on chromosomes 1B, 2D, 3B, 5D, 6D, and 7A, among which two major QTL, QFhb.CIM-2D.1 and QDon.CIM-3B.1, were stably expressed in this study.  Conditional QTL analysis suggested that QFhb.CIM-2D.1 contributes to reduced deoxynivalenol (DON) content via decreasing FHB severity, whereas QDon.CIM-3B.1 contributed to FHB resistance by directly controlling the accumulation of DON.  Stacking of QFhb.CIM-2D.1 and QDon.CIM-3B.1 exhibited markedly increase in resistance against both FHB and DON.  Furthermore, two Kompetitive Allele-Specific PCR (KASP) markers, KASP-1369 and KASP-8394, tightly linked to QFhb.CIM-2D.1 and QDon.CIM-3B.1, respectively, were developed and successfully validated in their respective genetic populations.  Altogether, our results broaden our understanding of the genetic basis of resistance to FHB and the developed markers will be useful for marker-assisted breeding of wheat. 

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. 

Excessive cadmium (GrCdc) and deficiencies of copper (GrCuc) and magnesium (GrMgc) in grains pose serious human health risks.  Common wheat breeding has reduced genetic diversity within elite germplasm resources, negatively impacting future wheat production.  Thus, identifying loci controlling GrCdc, GrCuc, and GrMgc from tetraploid wheat and introducing them into common wheat is essential for genetic improvement.  In this study, we identified quantitative trait loci (QTL) for GrCdc, GrCuc, and GrMgc using the Wheat 55K single nucleotide polymorphism (SNP) array-based linkage map and phenotypic data across multiple environments in recombinant inbred lines derived from a cross between a wild emmer accession (LM001) and an endemic tetraploid wheat in Sichuan (Ailanmai).  Four major, stably expressed QTL were identified.  Three of these, including QGrCdc.sau-AM-5A for GrCdc, QGrCuc.sau-AM-4A for GrCuc, and QGrMgc.sau-AM-4A for GrMgc, were novel. These loci were validated using tightly linked Kompetitive Allele Specific PCR (KASP) markers in various genetic backgrounds. Several candidate genes (TRIDC5AG052690, TRIDC5BG060070, and TRIDC4AG008520) with sequence variations were predicted to influence Cd, Cu, or Mg absorption and transport within these QTL intervals.  Correlation analysis revealed that GrCdc was not correlated with GrCuc or GrMgc, although GrCuc was significantly correlated with GrMgc.  Furthermore, no significant effects of GrCdc, GrCuc, or GrMgc on agronomic traits were detected, as no correlation between them and any of the eleven agronomic traits investigated was observed.  Additionally, QGrCuc.sau-AM-4A colocalized with QGrMgc.sau-AM-4A, suggesting potential shared physiological and/or genetic control.  Altogether, these stably expressed QTL across environments provide theoretical guidance for further germplasm improvement and fine mapping.

Maize/soybean intercropping systems are commonly used in developing countries, but few studies have been performed to elucidate the differences in nutrient efficiency and rhizosphere microbiome, especially when maize is intercropped with different soybean varieties. In this study, field experiments were conducted to compare the growth and yield of two soybean (Glycine max) varieties, BD2 and YC03-3, and one maize (Zea mays) variety, Huazhen, in mono-cropped and intercropped cultures. The plant biomass and N content of both crops in BD2/maize intercropping were significantly improved compared to their monoculture, but no such effects were observed in the plants of YC03-3/maize intercropping. The yield of BD2 intercropped with maize exhibited a 37.5% increment above that of BD2 in monoculture. Moreover, 19.2-29.1% longer root length of maize and 19.0-39.4% larger root volume of BD2 were observed in BD2/maize intercropping than in monoculture, but no growth advantage was observed in YC03-3/maize intercropping. Maize showed root avoidance when intercropped with BD2, but space competition when intercropped with YC03-3. 16S rRNA amplicon sequencing showed that compared with the monoculture system, rhizobacteria community composition in BD2/maize intercropping changed more significantly than that of the YC03-3/maize intercropping system. In BD2/maize intercropping, most of the rhizobacteria community biomarker bacteria of BD2 were positively correlated with plant biomass, as well as plant P and N content. Maize tended to recruit Rhizobiales and Proteobacteria, which showed positive correlation with plant biomass and N content, respectively, as well as soil available N. In conclusion, soybean varieties determined the advantages of maize/soybean intercropping through root-root interactions and modification of rhizobacteria communities. Our insight emphasizes a linkage between root traits and the rhizobacteria community, which shows the importance of optimizing intercropping systems by selection of appropriate crop varieties.

Deep learning-based intelligent recognition algorithms are increasingly recognized for their potential to address the labor-intensive challenge of manual pest detection. However, their deployment on mobile devices has been constrained by high computational demands. Here, we developed GBiDC-PEST, a mobile application that incorporates an improved, lightweight detection algorithm based on the you only look once (YOLO) series single-stage architecture, for real-time detection of four tiny pests (wheat mites, sugarcane aphids, wheat aphids, and rice planthoppers). GBiDC-PEST incorporates several innovative modules, including GhostNet for lightweight feature extraction and architecture optimization by reconstructing the backbone, the Bi-directional Feature Pyramid Network (BiFPN) for enhanced multiscale feature fusion, Depthwise convolution (DWConv) layers to reduce computational load, and the Convolutional Block Attention Module (CBAM) to enable precise feature focus. The newly developed GBiDC-PEST was trained and validated using a multitarget agricultural tiny pest dataset (Tpest-3960) that covered various field environments. GBiDC-PEST (2.8 MB) significantly reduced the model size to only 20% of the original model size, offering a smaller size than the YOLO series (v5 ~ v10), higher detection accuracy than YOLOv10n and v10s, and faster detection speed than v8s, v9c, v10m and v10b. In Android deployment experiments, GBiDC-PEST demonstrated enhanced performance in detecting pests against complex backgrounds, and the accuracy for wheat mites and rice planthoppers was improved by 4.5-7.5% compared with the original model. The GBiDC-PEST optimization algorithm and its mobile deployment proposed in this study offer a robust technical framework for the rapid, onsite identification and localization of tiny pests. This advancement provides valuable insights for effective pest monitoring, counting, and control in various agricultural settings.

Rapeseed is globally important as an oil crop to provide essential edible plant oils for various agricultural products. Currently, manipulating plant height with branching effectively balances biomass and yields. However, the genetic mechanisms to control plant height largely remains unknown in rapeseed. To address this gap, we isolated an extremely dwarf mutant (dm1) from ethyl-methanesulfonate (EMS) mutagenesis, and revealed the dwarfism is result from a significant reduction of cell length. Bulk segregant analysis (BSA) identified BnaA10.CYP90A1 and BnaC09.CYP90A1 as the causative genes of dm1. Both genes encoded the proteins homologous to the Arabidopsis cytochrome P450 AtCPD/AtCYP90A1, which is cruicial for brassinosteroid (BR) biosynthesis. In concern with that, we demonstrated the reduced levels of bioactive BRs, castasterone (CS), and its precursor 6-deoxoCS in dm1, resulting in the down-regulation of various genes for cell expansion. The reduced BR levels also caused negative feedback to promote the expression of BR biosynthetic genes in dm1. Furthermore, we proved that the single mutation of BnaA10.CYP90A1 gene conferred semi-dwarfism, potentially beneficial for producing ideal type of plant to improve cultivars with a balance on yield and machinery harvest through genetic modifications. Collectively, these findings highlighted the critical role of BnaCYP90A1s in BR biosynthesis and validated their influence on the regulation of plant height in rapeseed.

To ensure the reliability of learned information, most insects require multiple intervals of experience before storing the information as Long-term memory (LTM), and this requirement has been validated in insects from the behavioral to the molecular level. Recent studies have shown that some insects can form LTM after a single experience, although the mechanisms underlying one-trial LTM formation are not well understood. Therefore, understanding the mechanisms underlying rapid learning and subsequent preference formation in insects is crucial. Here we show that the agricultural pest Bactrocera dorsalis can rapidly form LTM, which is dependent on protein synthesis, and that the formation of LTM requires high energy support at the cost of reduced survival. Furthermore, based on a liquid chromatography-mass spectrometry (LC-MS) metabolomics approach, we found that LTM-related processes are sequentially coupled to two processes for energy generation, the TCA cycle and oxidative phosphorylation. This was further confirmed by blocking these energy generation processes. Our results provide a theoretical basis for the development of behavioral modulators in oriental fruit flies that target energy generation intermediate metabolites, as well as a new perspective on the rapid formation of LTM in insects.

Exploring the suitability of biochar for improving soil quality under different water and salt conditions is important for maintaining soil health and productivity in the arid regions of Northwestern China. We compared the effects of biochar application practices on soil physical, chemical and biological properties under different irrigation and water salinity levels in a two-year field experiment in a mulched and drip-irrigated maize field in Gansu province, China. Eight treatments in total included the combination of two biochar addition rates of 0 t ha^-1 (B0) and 60 t ha^-1 (B1), two irrigation levels of full (W1) and deficit irrigation (W2; W2=1/2 W1) and two water salinity levels of fresh water (S0, 0.71 g L^-1) and brackish water (S1, 4.00 g L^-1). The minimum dataset method was used to calculate the soil quality index (SQI) under different treatments. Deficit and brackish water irrigation significantly reduced SQI by 3.80-9.80% through reducing some soil physical, chemical and biological properties. Biochar application significantly increased the SQI by 6.13 and 10.40% under full irrigation with fresh and brackish water, respectively. Biochar addition enhanced the relative abundance of beneficial bacteria (e.g., Proteobacteria, Patescibacteria) in the soil in all water-salt treatments. The partial least squares path model showed that biochar application significantly enhanced the SQI mainly by improving soil aggregation and pore structure under particular water-salt conditions. This research provides an important basis for utilizing biochar to improve soil quality in arid regions of Northwest China under various water-salt conditions.

The combined application of organic manure and chemical fertilizers is an effective way to enhance soil organic carbon (SOC) sequestration through its influences on organic carbon (OC) input and the stability of SOC fractions. However, there is limited information on the carbon sequestration efficiency (CSE) of chemically separated SOC fractions and its response to OC input under long-term fertilization regimes, especially at different sites. This study used three long-term fertilization experiments in Gongzhuling, Zhengzhou and Qiyang spanning 20 years to compare the stocks and CSE in four different OC fractions (very labile OC, labile OC, less labile OC, and non-labile OC) and their relationships with annual OC input. Three treatments of no fertilization (CK), chemical nitrogen, phosphorous, and potassium fertilizers (NPK), and chemical NPK combined with manure (NPKM) were employed. The results showed that compared with CK, NPKM resulted in enhanced SOC stocks and sequestration rates as well as CSE levels of all fractions irrespective of experimental site. Specifically for the very labile and non-labile OC fractions, NPKM significantly increased the SOC stocks by 43 and 83%, 77 and 86%, and 73 and 82% in Gongzhuling, Qiyang, and Zhengzhou relative to CK, respectively. However, the greatest changes in SOC stock relative to the initial value were associated with non-labile OC fractions in Gongzhuling, Zhengzhou, and Qiyang, which reached 6.65, 7.16, and 7.35 Mg ha^-1 under NPKM. Similarly, the highest CSE was noted for non-labile OC fractions under NPKM followed sequentially by the very labile OC, labile OC, and less-labile OC fractions, however a CSE of 8.56% in the non-labile OC fraction for Gongzhuling was higher than the values of 6.10 and 4.61% in Zhengzhou and Qiyang, respectively. In addition, the CSE for the passive pool (very labile+labile OC fractions) was higher than the active pool (less-labile+non-labile OC fractions), with the highest value in Gongzhuling. The redundancy analysis revealed that the CSEs of fractions and pools were negatively influenced by annual OC input, mean annual precipitation and temperature, but positively influenced by the initial SOC and total nitrogen contents. This suggests that differential stability of sequestered OC is further governed by indigenous site characteristics and variable amounts of annual OC input.

Global climate warming is characterized by diurnal and seasonal asymmetry, with greater increases at nighttime and in winter and spring, and growing evidence has recognized that night-warming in winter and spring significantly impacts winter wheat production.  Pre-crop straw returning is the principal method for straw utilization currently and in the future, but the interactions between straw returning and night-warming on wheat yield and NUE (N use efficiency) still remain elusive.  Here, a consecutive three-year field experiment with two straw treatments (S0, straw removal; S1, straw returning) and two warming treatments (W0, no warming control; W1, night-warming) found that both S1 and W1 improved wheat grain yield and NUE, with W1 exhibiting more pronounced improvements.  Notably, the interaction between S1 and W1 (S1W1) further enhanced yield and NUE by 13.0 and 16.5% compared to S0W0 through increasing grain number and 1,000-grain weight, respectively (three-year average).  Additionally, root growth and topsoil inorganic N content exhibited reductions in S1 before jointing, thus reducing plant dry matter and N accumulation.  However, W1 exhibited an opposite trend, thereby mitigating these negative effects.  Simultaneously, under S1W1, increased N translocation to grain and post-anthesis dry matter accumulation, driven by greater N distribution to leaves and higher N metabolism enzyme activity, enhanced both yield and NUE.  This improvement was supported by better root morphology and biomass, particularly in the 0−40 cm soil layer, boosting plant N absorption.  Additionally, elevated soil N-acquiring enzyme activity after jointing increased the net N mineralization rate and microbial biomass N, enhancing soil N-supply capacity.  As a result, post-jointing inorganic N content rose in the 0−20 cm layer while decreasing at 20−60 cm, thus reducing the apparent N surplus.  Collectively, straw returning, night-warming, and their interactions enhanced more root distribution and N-supply capacity after jointing in the topsoil layer to increase plant N uptake and its translocation to grains, along with post-anthesis dry matter accumulation, ultimately improving grain yield and NUE.

Re-domestication of diploid potato (Solanum tuberosum) into a seed crop is an innovative breeding method to accelerate genetic improvement. Seed propagation would allow hybrid production and mix superior alleles. However, almost all diploid potatoes in nature are self-incompatible (SI). Gametophytic self-incompatible (GSI) is a widespread SI in Solanaceae and is controlled by the S locus that contains a ribonuclease (S-RNase) and multiple F-box (SLFs); however, the genetic diversity of the S locus in potato is unclear. In this study, we identified 21 S-RNase alleles involved in SI from 194 diploid potato accessions by large-scale transcriptome sequencing. The levels of amino acid similarity among different S-RNase proteins varied from 31.3 to 95.8%. S2 alleles is the most widespread in 194 diploid potato and is mainly distributed in the S. tuberosum Group Phureja. Based on genomic annotation and expression analysis, we identified 12 potential functional SI male-determinant genes, S-locus F-box (SLFs), encoding F-box proteins in the S2 locus on a genomic region of approximately 13 Mb. Comparative genomics analysis showed that eight SLF genes are relatively conserved among four homozygous S locus. The Ka and Ks analysis suggested that S-RNase and intra-haplotypic SLF genes have undergone co-evolved. These findings can not only help to select suitable pollinators but also to combine more hybrid combinations and make full use of heterosis to accelerate diploid potato breeding.

Phytopathogenic fungi can weaken the effectiveness of anti-fungal chemicals from plants and artificial synthesis through xenobiotic detoxification system. Nevertheless, the transcription factors responsible for transcriptional activation of xenobiotic detoxification genes in phytopathogenic fungi are rarely reported. Here, we show that a GATA transcription factor SsGATA1 is regulating the transcription of drug efflux pump genes, thus contributing to the tolerance of various types of chemical fungicides, including propiconazole, caspofungin and azoxystrobin in Sclerotinia sclerotiorum. Similarly, SsGATA1 also plays the role of tolerance to isothiocyanate and flavonols, two reported as broad-spectrum anti-fungal chemicals, by mediating the transcription of isothiocyanates hydrolase SsSaxA. Importantly, SsGATA1 positively regulates pathogenicity, which is attributed to the up-regulation of hydrolases and SsSaxA during infection. Furthermore, SsGATA1 is responsible for tolerance to several stresses. Our findings demonstrated that SsGATA1 plays roles in multidrug resistance and pathogenicity by activating the transcription of hydrolases and xenobiotic detoxification genes.

Genotype imputation is essential for increasing marker density and maximizing the utility of existing SNP array data in animal breeding. Although a wide range of software is available for genotype imputation, a comprehensive benchmark in pigs is still lacking. In this study, we benchmarked 24 combinations of genotype imputation software for SNP arrays in pigs, comprising six independent pre-phasing software (fastPHASE, MaCH, BIMBAM, Eagle, SHAPEIT, Beagle) and four distinct imputation software (pbwt, Minimac, IMPUTE, Beagle), using 1,602 whole-genome sequencing (WGS) pigs from a multibreed pig genomics reference panel (PGRP) in PigGTEx. Our results indicated that the combination of Beagle for pre-phasing and Minimac for imputation achieves the highest imputation accuracy with a concordance of 0.983, especially for low-frequency SNPs (MAF<0.05). Finally, we proposed three recommended strategies: i) the combination of Beagle and Minimac is optimal for achieving the highest accuracy; ii) the combination of Beagle and Beagle is recognized for its convenience and relatively high accuracy despite it being memory-intensive; iii) the combination of Eagle and pbwt is feasible for its minimal computational cost with relatively high accuracy. This study provides valuable insights for implementing genotype imputation for pig SNP arrays toward sequence data and offers a basis for applications in livestock and poultry breeding.

Root-knot nematodes (RKNs) are the most economically damaging plant-parasitic nematodes globally. Xinjiang, encompassing one-sixth of China's landmass, currently lacks comprehensive data regarding the occurrence, distribution, and genetic variation of RKNs infecting vegetables within its borders. Hence, identifying RKNs species and genetic diversity is crucial for devising comprehensive management strategies. Between 2021 and 2023, We present a survey of 130 samples, collected from 86 counties across 14 cities in Xinjiang, aiming to comprehensively understand the occurrence, distribution, damage, and species of vegetable RKNs. The results indicated that 57 out of 130 samples collected from the cities of Hami, Tulufan, Ili, Bayingol, Hotan, Aksu, Kashgar, and Kizilsu in Xinjiang were infected by RKNs, suggesting an expansion of RKN disease in the vegetable-producing regions of Xinjiang. The infected vegetable roots were found to harbor Meloidogyne incognita and M. hapla, with M. incognita being the most prevalent species. A phylogenetic analysis targeting the COI regions of M. incognita revealed significant evolutionary and genetic disparities between Xinjiang and Southeastern China RKN populations. Haplotype analysis of the COI gene revealed that M. incognita populations are categorized into three major lineages: Asia, Europe, and a combined lineage encompassing both America and Africa. Notable gene flow patterns were observed among M. incognita populations, with significant migrations from Europe and America to Asia, specifically from Southeastern China towards Xinjiang. This study's findings indicate a consistent increase in the detrimental effects of vegetables production caused by RKNs in Xinjiang. Implementing effective prevention and control measures is crucial to mitigate the spread of RKNs.

Infectious Bursal Disease (IBD) is an acute, highly contagious disease that affects chicks (MüLler et al. 2003). IBD mainly damages the immune organs of chicks, especially the central immune organ, causing immune suppression in diseased chicks (MULLER et al, 2012). The pathogenic Infectious Bursal Disease virus (IBDV) is a member of the Avira virus genus in the Birnaviridae family. (Dobos et al, 1979; Müller et al, 1979; Harkness et al, 1975). IBDV is prevalent worldwide, causing serious economic losses to the global poultry industry. Currently, vaccination remains the most cost-effective way to prevent IBDV.

Subunit vaccines are based on specific components of pathogens and are typically located on their surface. Therefore, subunit vaccines are considered safer than completely pathogen based inactivated or attenuated live vaccines. VP2 protein is the main host protective antigen that contains most neutralizing sites and is the target protein for developing subunit vaccines using various expression systems. Nowadays, IBDV is widely prevalent, but commercial vaccines are no longer effective in preventing it. It is suspected that it is related to genetic changes in the VP2 gene, causing immune escape. Therefore, in this experiment, IBDV variant strain isolated in the laboratory was used to prepare IBDV subunit vaccine to protect sensitive chicks from IBDV infection.

Adjuvants can enhance the immunogenicity of antigens and enhance their immune efficacy. With the development of biotechnology and the upgrading of vaccines, adjuvants have become an important part of improving vaccine quality. Montanide™ ISA 78 VG is an oil in water adjuvant enhanced by immunostimulatory compounds. Its ingredients are based on fortified oil, which can induce antigen-specific cell-mediated immune responses, thereby enhancing protection against viral diseases. MONTANIDE™ GEL is a semi transparent flowable gel with good and stable fluidity. It is easy to mix the antigen phase and adjuvant, and easy to prepare with low viscosity. Aluminum hydroxide adjuvant is the oldest used vaccine adjuvant and remains the primary adjuvant in animal vaccines to this day. This study selected IBDV VP2 protein as the immunogen and combined it with three adjuvants, while comparing it with the widely used white oil adjuvant. By immunizing chicks and comparing the effects of different adjuvants on immune protection, this provides a reference for the development of universal IBDV vaccines.

The IBDV VP2 protein was successfully expressed in E. coli through a prokaryotic expression system, and the protein exists in the form of inclusion bodies. The BCA method was used to detect the protein concentration, which was determined to be 1.5 mg mL^-1 by the BCA assay kit. The IBDV VP2 subunit was emulsified with Montanide^TM ISA 78 VG, Montanide^TM Gel P, Aluminium hydroxide (Seppic Shanghai Chemical Specialities Co., China), and white oil (ExxonMobil, China) adjuvantat a ratio of 3:7, 9:1, 9:1 and 1:1 (w/v) , while ensuring the same antigen content to produce the IBDV VP2 subunit vaccines. IBDV VP2 subunit vaccines without adjuvant also were compared and PBS was served as control. The preparation methods of each vaccine group were shown in Appendix C.

The chicks without specific-pathogens-free (SPF) provided by Zhejiang Lihua Agricultural Technology Co., Ltd were subdivided into six immunized groups. The grouping of chicks and the proportion of adjuvants were shown in Table 1. All of the chicks were subcutaneously vaccinated into their necks. Primary vaccination was given on day 7, and revaccination was given on day 14. At 28 days of age, half of the chicks in each group were separated and infected with 0.2 mL of IBDV-LY21/2 (1×10⁶ EID₅₀ 0.1mL^-1) (Huang et al, 2023). The positive control group was not vaccinated but was infected, and six infection groups were established thereafter.

During the immunization process, it was observed that the Gel P, Adjuvant-free and PBS control groups' vaccines were fully absorbed 1 minute after injection, partially absorbed at intermediate dosage in ISA 78 VG, Aluminium hydroxide, not absorbed in White oil. Within 48 h after injection, there was redness, warmth and swelling at the injection site in White oil. Within 5 days after injection into GroupIII-white oil, the reaction at the injection site got better and disappeared on its own. In addition, the weight gain trend of chicks in different injection groups was similar, indicating that vaccination had no significant effect on weight gain of chicks.

The clinical reactions of experimental animals after infection induced by IBDV, including virus shedding, morphometric identification, and gross lesions, were shown in Figure 1-A. There is no gross clinical symptoms or mortality of vaccinated chicks after challenged by IBDV. All the unvaccinated animals succumbed to the disease within 10 days post-challenge (dpc) and caused atrophy of the cloacal bursa. After calculating the group's bursa index for virus attack after immunization (Sharma et al., 1989; Lucio & Hitchner, 1979) , the results showed that the BBIX of the ISA 78 VG, Gel P, and Aluminum hydroxide groups were greater than 0.7. Although the BBIX of the white oil and Adjuvant-free groups were greater than 0.7, the BBIX was significantly smaller than the above three groups, and the BBIX of the positive control group was less than 0.7. After 10 days of post-immunization challenge, there were no significant pathological changes in the bursa in the ISA 78 VG, Gel P, and Aluminum hydroxide groups compared to the Negative control group. In the positive control group, there were significant lesions in the bursa with atrophy of the bursa (Figure 1-B). This proved that the vaccine prepared in this experiment can inhibit the atrophy of the chicken's bursa and had a preventive effect on chickens infected with IBDV.

The immune efficacy of IBDV VP2 subunit vaccines prepared with different adjuvants was evaluated. Serum collected on day 7 after the prime-boost manner and on days 7, 14, and 21 after the second dose were detected using indirect ELISA method. The percentage of antibodies in the serum of the ISA 78, GLP, and Aluminum hydroxide groups can detect high antibody levels on the 7th day after the first immunization, and positive antibodies can still be detected on the 28th day after secondary immune enhancement. The white oil group achieved an antibody-positive rate of 80% on the 14th day after secondary immune enhancement. The adjuvant-free group only detected antibodies with a 50% positive rate at the end of the experiment and continued to decrease antibody levels (Figure 1-C).

According to the statistical results of the cell neutralization experiment of each immune group at 96 h p.i., it can be observed that on the 7th day after the first immunization, only the adjuvant-free group did not achieve a neutralization titer of 1:16. The results showed that the neutralizing antibody production time in this experiment was earlier than the antibody production time previously studied, which was 14 days after immunization (Li et al, 2020). On the 7th day after the second immunization, the neutralizing titers of all groups significantly increased. On the 21st day after immunization, the antibody level reached its highest, with the Aluminum hydroxide group having the highest neutralizing antibody level. On the 28th day after immunization, the neutralizing titer of the adjuvant-free group decreased to 1:16, while the antibody level of the ISA 78 VG, Gel P, and Aluminum hydroxide groups remained at a high level. The level of neutralizing antibodies was shown in Figure 1-C.

The IL-2 ELISA detection kit was used to detect IL-2 levels, and the results showed that ISA 78 VG, Gel P, and Aluminum hydroxide groups could produce higher levels of IL-2 (Figure 1-D).

IBDV replication following challenge exposure of the experimental animals was summarized in Figure 7. The total viral RNA of IBDV from bursa tissues of chicks at day 10 post infected with the LY21/2 strain of IBDV, and viral loads were determined by qRT-PCR technique. The vaccine's efficacy in preventing virus isolation from swabs was consistent with tissue samples. Compared to the control group, the number of viral RNA copies is the lowest in the ISA 78 VG group among the immunization groups. The collected pharyngeal and anal swabs of chicks were used to detect detoxification, and the results showed that ISA 78 VG group had the lowest detoxification level, with the GLP group being the second lowest. ISA 78 VG, Gel P, white oil, and Aluminum hydroxide could reduce virus infection in chicks (XIONG et al, 2013) (Fig. 1-E).

In order to evaluate the pathological changes caused by IBDV infection, paraffin sections were prepared for H&E staining and tunel detection of all bursa tissues. The  VP2 subunit vaccine prepared with three adjuvants had normal bursa structure, while some follicles in the white oil and Adjuvant-free groups showed atrophy and vacuolization. The positive control group showed a decrease in lymphocytes, infiltration of macrophages, atrophy of follicles, and proliferation of connective tissue. Tunel detection showed that the ISA 78 VG, Aluminium hydroxide, and Gel P groups showed less apoptosis in the bursa, while the white oil and Adjuvant-free groups showed significant apoptosis with green fluorescence . Indicating that the prepared vaccine had a protective effect on the bursa and had not caused any damage to the bursa (Figure 1-F).

This experiment provides preliminary information on the duration of protection that vaccination can provide for chicks. The safety and efficacy of subunit vaccines prepared with different adjuvants and without adjuvant were evaluated on chicks. Under the same dose of antigen (IBDV), the protective effects of ISA 78 VG, Gel P, and Aluminum hydroxide adjuvants evaluated were enhanced to prevent infection with this pathogen. To confirm whether ISA 78 VG and Aluminum hydroxide can effectively reduce antigen load, long-term evaluation is necessary. A previous study showed that classical strain vaccines can completely prevent classical strain infections, but can only provide partial protection against nVarIBDV (Eterradossi et al, 2020). nVarIBDV strain vaccines can not only provide complete immune protection against homologous nVarIBDV, but also against heterologous vvIBDV variant strains (Wang et al, 2021). The immune protection level of the VP2 protein subunit vaccine prepared in this experiment against other strains have not been studied and further research is needed.

In this study, we report the discovery of a novel bocaparvovirus, identified through viral metagenomic analysis of fecal samples from goats presenting with diarrhea. The complete genomic sequence of this virus shows the highest identity with the ECBOV-tdf70 strain, which was found in the wild animal Elaphodus cephalophus. Its NS1 protein shares 91.2% amino acid identity with the ECBOV-tdf70 strain. According to ICTV criteria, this strain should be classified as same species along with the ECBOV-tdf70 strain. The positive rate of diarrheal fecal samples versus non-diarrheal samples indicates the potential role of viruses in goat diarrhea. The complete VP1 genes of the five strains obtained in this study shared 74.4 - 99.2% nucleotide identity, and 63.7 - 99.1% amino acid identity. This study represents the first report of Bocaparvovirus infection in goats, providing valuable insights into the epidemiology and genetic diversity of the virus.

Streptococcus suis has garnered increasing attention due to its implication in severe infections in both swine and humans, as well as its development of multidrug resistance. The phenomenon of collateral sensitivity, whereby resistance to one antibiotic leads to increased sensitivity to another, provides new opportunities for mitigating the evolution of resistance. In this study, we evolved resistance in S. suis to 11 clinically used antibiotics and characterized the resulting collateral sensitivity profiles, revealing a complex network of interactions. Based on our findings, we identified dozens of such drug pairs and demonstrated collateral sensitivity to gamithromycin in ciprofloxacin-resistant S. suis both in vitro and in vivo. Gamithromycin effectively limits the evolution of resistance and reduces the mutant selection window for ciprofloxacin-resistant S. suis strains. Mechanistic studies indicated that the heightened sensitivity of ciprofloxacin-resistant S. suis to gamithromycin was associated with increased intracellular gamithromycin accumulation due to membrane potential alterations and reduced functions of proton motive force (PMF)-dependent efflux pumps. Furthermore, collateral sensitivity-based treatments significantly resensitized ciprofloxacin-resistant S. suis strains to gamithromycin, resulting in superior efficacy, lower pharmacodynamic targets, and higher treatment success rates in a murine thigh infection model. Our results indicate that gamithromycin sensitivity in S. suis is a collateral consequence of resistance to ciprofloxacin, providing valuable insight for the strategic design of collateral sensitivity-based antibiotic therapies for S. suis infections.

Ketosis, a common metabolic disease during early lactation, is associated with high circulating levels of β-hydroxybutyrate (BHB). A portion of BHB that reaches the mammary gland is utilized as precursor for synthesis of fatty acids. Recent findings from nonruminant studies revealed that long chain fatty acyl-CoA ligase 4 (ACSL4) could play a role in the regulation of cellular fatty acid metabolism, but the mechanisms by which ACSL4 mediates cellular lipid metabolism in response to BHB remains unclear. To achieve the aims, we conducted in vivo or in vitro analyses using bovine mammary gland biopsies and the immortalized mammary epithelial cell line (MAC-T). The in vivo study (n = 6 cows group^-1) involved healthy cows (plasma BHB < 0.60 mmol L^-1) or ketotic cows (plasma BHB > 2.0 mmol L^-1) from which mammary gland tissue was biopsied. In vitro, MAC-T cells were challenged with 0, 0.3, 0.6, 1.2, or 2.4 mmol L^-1 BHB for 24 h to determine an optimal dose. Subsequently, MAC-T were incubated with 1.2 mmol L^-1 BHB for 0, 3, 6, 12, 24, or 48 h. Furthermore, MAC-T cells were treated with small interfering ACSL4 (siACSL4) for 24 h or ACSL4 overexpression plasmid (pcACSL4) for 36 h followed by a challenge with 1.2 mmol L^-1 BHB for 24 h. Results showed that increased mRNA and protein abundance of lipogenic genes were linked to both mammary gland and in vitro challenge with BHB. BHB increased fatty acid content by activating ACSL4 expression, whereas inhibition of ACSL4 reduced BHB-induced reactive oxygen species (ROS) overproduction, enhancement of mitochondrial membrane potential, increase in fatty acid content, and lipid droplet accumulation. Furthermore, we also elevated ACSL4 expression with an overexpression plasmid to clarify its molecular role in response to BHB challenge. ACSL4 overexpression enhances BHB-induced lipid droplet accumulation by increased fatty acid content. Overall, the information showed that ACSL4 is crucial for the process of producing fatty acids from exogenous BHB. Reduced ACSL4 decreased fatty acid content and lipid droplet accumulation, improved mitochondrial function, directed more fatty acids towards oxidation. Thus, ACSL4 plays an important role in determining the fate of intracellular fatty acids and BHB in BMECs.

The Huang-Huai-Hai wheat region (HHHR) is characterized by the largest cultivation area and yield among all the major wheat-producing regions in China.  Over the past 70 years, significant advances in wheat breeding have been achieved in this region, resulting in high and stable yields as well as improved disease resistance.  However, there is a notable deficiency in the systematic molecular-level analyses of wheat breeding advantages in HHHR.  To bridge this gap, we used a Wheat 55K SNP array to evaluate 384 accessions from a core collection of wheat germplasms across China to systematically analyze the distribution patterns of beneficial haplotypes associated with traits related to yield and powdery mildew resistance specific to HHHR.  Our findings indicate that varieties from HHHR demonstrate significantly superior performance in terms of yield-related traits and powdery mildew resistance compared to those from other wheat regions.  Using genome-wide association studies (GWAS) analysis, we identified the QTNs associated with both grain yield and powdery mildew resistance.  Importantly, beneficial haplotypes were found at significantly higher frequencies in the HHHR than in other wheat-growing regions.  Based on these haplotypes, the MFP-a gene was identified as potentially regulating jasmonic acid synthesis while also playing a role in grain development and conferring powdery mildew resistance.  Furthermore, identity by descent (IBD) analysis revealed specific conserved genomic segments that have become fixed through selective breeding practices in HHHR, which may serve as invaluable resources for the targeted enhancement of yield and disease resistance traits in other wheat-growing areas.  Finally, using the Aimengniu breeding lineage as a case study, we elucidated the genetic basis underlying the key founder parental formations utilized in breeding programs.  This study not only provides essential references and guidance for future molecular breeding initiatives in China but also has implications for enhancing wheat production worldwide.

赤霉素在植物生长发育中起着重要作用。作物矮化主要与赤霉素合成或信号转导途径的突变有关。然而，对作物中与赤霉素合成和调控有关的基因知之甚少。本研究分离到一株极端矮化的自然矮杆突变体dwarf4。基于BSA-seq，证实了由SiDWARF4编码的古巴焦磷酸合成酶（CPS）在谷子GA生物合成过程中催化了牻牛儿基牻牛儿基焦磷酸（GGPP）向古巴焦磷酸（CPP）的转化。在dwarf4中，CPS酶底物GGPP水平升高，导致GAs含量降低。外源GA₃处理可以恢复dwarf4的异常表型。SiDWARF4敲除系表型与dwarf4一致。与对照Ci846相比，GGPP含量升高，GAs含量降低，与dwarf4的结果一致。研究结果对改良谷子株型和提高谷子种植密度具有一定的指导意义。