Greenhouse gas (GHG) production during ensiling not only causes the nutrient losses of silage but also promotes climate warming. However, there is little information on the production of GHG and strategies for mitigating GHG emissions during ensiling. This work aimed to study the gas production characteristics and techniques for reducing gas emissions during ensiling. Oats and triticale, with Lactiplantibacillus plantarum (LP) or corn meal (CM) addition, were ensiled. The cumulative gas volume rapidly increased and reached to the peak within the first 9 days of ensiling for both forage crops. The highest cumulative gas volume of triticale silage was twice as much as that of oats silage. Triticale silage produced lower carbon dioxide (CO₂) concentration, higher methane (CH₄) and nitrous oxide (N₂O) concentrations than oats silage within the 28 days of ensiling. Adding LP or CM significantly improved the fermentation quality and decreased the gas volume and GHG concentrations of two silages on d56 (except CH₄ of triticale). At the early stage of ensiling, more Enterobacter, Lactococcus and Leuconostoc related to gas production were observed, and adding LP increased the abundance of Lactobacillus and decreased the abundance of bacteria like Kosakonia, Pantoea, Enterobacter and Lactococcus positively correlated with gas volume, CO₂ and N₂O concentrations. These results suggest that gas formation during ensiling mainly occurs in the first 9 days. Adding LP or CM can significantly improve the fermentation quality and decrease the gas volume. This would benefit to reducing GHG emissions in silage production.

Tick-borne encephalitis (TBE) is an important zoonotic viral disease transmitted by ticks. In recent decades, global climate change has increased human exposure to ticks, and mortality rates have gradually risen. Effective vaccines are essential for controlling TBE as specific antiviral treatment is unavailable. Vaccine candidates based on virus-like particles (VLPs) have previously been demonstrated to be efficient in eliciting excellent immune responses against influenza virus and SARS-CoV-2. Here, we constructed TBE virus (TBEV) VLPs containing the envelope and membrane proteins derived from the Far Eastern TBEV strain (WH2012) using an insect cell-baculovirus expression system. Induction of immune responses was investigated in mice following intramuscular injection with the TBEV VLPs vaccine candidates formulated of Poly(I:C) & Montanide ISA 201VG combination adjuvants. Mice produced memory T-cells and serum-specific IgG antibodies that averaged up to 1:10^4.6 and remained at 1:10⁴ (mean) for 24 weeks after three immunizations. TBEV VLPs vaccine was able to provide long-term antibody protection against TBEV, making it a promising subunit vaccine candidate for this disease.

Colistin serves as a crucial treatment for multidrug-resistant (MDR) Gram-negative bacterial infections. However, its excessive use has contributed to an increase in colistin-resistant strains within livestock production (Binsker et al. 2022). The mobile colistin resistance gene, mcr, which reduces colistin affinity by incorporating phosphoethanolamine (pEtN) into the bacterial lipopolysaccharide, significantly compromises the effectiveness of colistin treatments (Liu et al. 2016). To date, ten slightly different variants of the mcr-1 gene, ranging from mcr-1 to mcr-10, have been identified in various bacteria isolated from animals, agricultural settings, humans, and environmental samples (Hussein et al. 2021). Klebsiella pneumoniae（K. pneumoniae）is a major pathogen causing a variety of infections in humans, which is known to harbor mobile genetic elements that help it acquire and spread resistance genes, leading to the emergence of MDR strains (Faccone et al. 2020; Pu et al. 2022). A recent study reported a high prevalence of colistin-resistant K. pneumoniae, accounting for 56% of the isolates in chicken flocks (Mourão et al. 203). Chicken production has the potential to serve as a natural reservoir of colistin resistance K. pneumoniae, posing a public health threat (Talat et al. 2023).

Herein, as part of our routine resistance surveillance, we characterized a colistin-resistant strain of K. pn NXQY01 producing extended-spectrum beta-lactamases (ESBLs) from deceased chicks at a breeder farm in Ningxia, China. Antimicrobial susceptibility testing was performed using the VITEK^® 2 COMPACT System (bioMérieux Inc. France) with the AST-GN 96 card (Lot No. 6862595203), following the manufacturer's recommendations. The K. pn NXQY01 isolates were resistant to multiple antibiotics including ampicillin, ticarcillin/clavulanic acid, cefalexin, cephalothin, cefoperazone, ceftiofur, cefquinome, gentamicin, neomycin, flumequine, enrofloxacin, marbofloxacin, tetracycline, polymyxin B, and sulfamethoxazole/trimethoprim (Table 1). To elucidate the genetic basis of antibiotic resistance in K. pn NXQY01, whole genome sequencing was performed using the PacBio RSII platform (Pacific Biosciences, Menlo Park, California, USA) and de novo assembly. The genome annotation was performed using Diamond software and the NCBI Prokaryotic Genome Annotation Pipeline. The genome of K. pn NXQY01 comprised a circular chromosome, which encompassed 5,279,178 base pairs and exhibited a GC content of 57.28%. Additionally, it harbored six plasmids, namely: pNXQY01-1 (227,409 bp), pNXQY01-2 (110,028 bp), pNXQY01-3 (71,848 bp), pNXQY01-4 (71,087 bp), pNXQY01-5 (66,252 bp), and pNXQY01-7 (8,159 bp). K. pn NXQY01 was determined to be sequence type 15 (ST15) by MLST. Resfinder analysis indicated that the K. pn NXQY01 isolate harbored 28 resistance genes including mcr-1.1, mcr-8.1, blaCTX-M-55, blaCTX-M-65 and others (Supplementary Table 1). Notably, pNXQY01-2 carried mcr-8.1, whereas pNXQY01-5 had both mcr-1.1 and blaCTX-M-55. To our knowledge, this constitutes the first report of an ST15 K. pneumoniae isolate co-harboring mcr-1.1, mcr-8.1 and ESBLs.

The pNXQY01-2 plasmid was 110,028 bp in length and comprised two replicons, characterized as an atypical IncFIB(pQil)/IncFII(K) type (Supplementary Table 2). Homology analysis suggested that the pNXQY01-2 is likely a novel recombinant plasmid resulting from recombination between plasmids pKP4 (Accession No. OL804388.1) and pPK45_NDM1 (accession no. LC521854.1). Fig. 1-A demonstrated that the presence of insertion sequences IS903B flanking the mcr-8 gene indicated its association with a composite transposon formed by IS903B elements. Additionally, pNXQY01-2 might contain ampC1 and ampC2 genes encoding AmpC β-lactamase. As shown in Fig. 1-B, pNXQY01-2-mcr-8.1 was nearly identical to the flanking structures of pKP4 (accession no. OL804388) and p5589 (accession no. CP102079), exhibiting only minor base sequence variations. Notably, the upstream region of the mcr-8 gene on pKP46 shows significant dissimilarities to the corresponding region in pNXQY01-2, primarily due to an incomplete ΔIS903B gene in the upstream mcr-8 of pNQXY01-2. Wu et al. (2018) documented the genetic diversity associated with the mcr-8 gene, noting that the upstream IS903B may be substituted by other insertion sequences, including IS1X2 and ISKpn21, and that the downstream IS903B region is prone to deletions and mutations. Farzana et al. (2020) also observed that in ST15 K. pneumoniae, the IS903B element upstream of mcr-8 is replaced by ISKpn21. It is therefore reasonable to hypothesize that the genetic context surrounding mcr-8, particularly the upstream IS903B element, exhibits instability and may be prone to replacement. Further research should prioritize investigation into this phenomenon.

The plasmid pNXQY01-5 was 66,252 bp in length, and represented a characteristic IncI2-type plasmid harboring the mcr-1 gene (Supplementary Table 2). IncI2 plasmids are mostly found in E. coli, indicating that pNXQY01-5 may facilitate gene transfer from E. coli to K. pneumoniae. The pNXQY01-5 harbored mcr-1.1 and blaCTX-M-55, and mcr-1.1 is situated between positions 23,875 and 25,500 bp (Fig. 1-C). As shown in Figure 1D, both the pNXQY01-5 and pM-199-232 harbored blaCTX-M-55 and mcr-1; however, the genetic contexts of the mcr-1 gene differ between the two plasmids. The pNXQY01-5 carry the insertion sequence ISApl1 upstream of the mcr-1 gene, which is absent in pM-199-232. The mcr-1 gene is frequently associated with plasmids containing the Tn6330 transposon. However, deletion of the ISApl1 elements within Tn6330 can lead to various configurations, such as ISApl1-mcr-1-pap2 or mcr-1-pap2 (Snesrud et al. 2016). Additionally, the genetic context of blaCTX-M-55, termed "ISEc9-blaCTX-M-55-orf-dnaJ", encompassed two hypothetical proteins, indicating a potential insertion pathway for blaCTX-M-55 through this sequence (Figure 1D). The pNXQY01-5 shared a high degree of similarity with the pHNSHP45, which was the first identified carrier of the mcr-1 gene. The primary distinction between them is the presence of the insertion sequence ISEc9, which harbors the blaCTX-M-55 gene, confirming its integration. Present results suggested that the resistance genes mcr-1.1 and blaCTX-M-55 in pNXQY01-5 may have been acquired through the insertion of foreign DNA sequences and are potentially linked to the insertion sequences ISApl1 and ISEc9, respectively.

The oriTfinder analysis revealed that plasmids pNXQY01-2, pNXQY01-3 and pNXQY01-5 possessed a complete conjugative transfer module (Supplementary Figures 1 and 2), enabling autonomous genetic material transfer via conjugation. To evaluate the transferability of mcr gene, conjugal transfer assays were carried out to identify colistin-resistant transconjugants with rifampicin (1000.0 mg L^-1) and colistin (2.0 mg L^-1) as previously described (Wang et al. 2023). PCR analysis of transconjugant NXQY01-600 showed the presence of mcr-1.1, mcr-8.1, blaCTX-M-55, and blaCTX-M-65 genes, indicating horizontal gene transfer. The transconjugant exhibited higher MICs for beta-lactam antibiotics, polymyxin B, and gentamicin (Table 1). The findings indicated that resistance in the transconjugant is likely caused by plasmid-encoded genes, including mcr-1.1 and blaCTX-M-55 on pNXQY01-5, blaCTX-M-65 on pNXQY01-3, and mcr-8.1 on pNXQY01-2. This could lead to the spread of antibiotic resistance, highlighting the importance of increased monitoring and control measures.

To the best of our knowledge, this is the first report of an ST15 K. pneumoniae isolate co-harboring mcr-1.1, mcr-8.1 and ESBLs, signifying a new plasmid-mediated antimicrobial resistance profile. Our research also focused on the genetic characterization of plasmids carrying mcr-1.1 and mcr-8.1, shedding light on their transmission mechanisms. The emergence of isolates from breeder flock harboring both ESBLs and colistin resistance genes narrows therapeutic options and highlights the escalating crisis of antimicrobial resistance in poultry production chain. 

Crop straw incorporation is widely recommended to maintain crop yields and improve soil organic carbon (SOC) stocks as well as soil quality. However, the long-term effects of different straw incorporation practices on the SOC stock remain uncertain. In this study, a long-term experiment (2007 to 2018) with four treatments (MW₀: maize–wheat rotation with no straw incorporation, MW₅₀: maize–wheat rotation with 50% chopped straw incorporation, MW_b50: maize–wheat rotation with 50% burned straw incorporation, and MF₅₀: maize–fallow rotation with 50% chopped straw incorporation) was setup to evaluate the response of the SOC stock to different straw incorporation methods. The results showed that the SOC stock significantly increased by 32.4, 12.2 and 17.4% under the MW₅₀, MW_b50 and MF₅₀ treatments, respectively, after continuous straw incorporation for over a decade, while the SOC stock under MW₀ was significantly reduced by 22.9% after the 11 year long–term experiment. Compared to MW₀, straw incorporation significantly increased organic carbon input, and improved the soil aggregate structure and the ratio of dissolved organic carbon (DOC) to particulate organic carbon (POC), but it did not significantly stimulate soil heterotrophic respiration, resulting in the increased SOC accumulation rate and SOC stocks of bulk soil. The increased ratio of DOC to microbial biomass carbon (MBC) enhanced the relative abundances of Acidobacteria and Proteobacteria but inhibited Bacteroidetes and Chloroflexi, and the bacterial relative abundances were the main reasons for the non-significant increase or even decrease in soil heterotrophic respiration with straw incorporation. The SOC stock would reach an equilibrium based on the results of Rothamsted Carbon (RothC) model simulations, with a long-term equilibrium value of 18.85 Mg ha^-1 under MW₅₀. Overall, the results of the long-term field experiment (2007-2018) and RothC model simulation suggested that maize-wheat rotation with 50% chopped straw incorporation delivered the largest benefits for the SOC stock in calcareous soils of subtropical mountain landscapes over the long term.

Natural rubber is an indispensable material of strategic importance that has critical applications in industry and the military. However, the development of the natural rubber industry is impeded by the red root rot disease of rubber trees caused by Ganoderma pseudoferreum, which is one of the most devastating diseases in the rubber tree growing regions in China. To combat this disease, we screened the antifungal activity of 223 candidate bacterial strains against G. pseudoferreum, and found that Bacillus velezensis strain SF305 exhibited significant antifungal activity against G. pseudoferreum. B. velezensis SF305 had a nearly 70% efficacy against the red root rot disease of rubber trees with the therapeutic treatment (Tre), while it exhibited over 90% protection effectiveness with the preventive treatment (Pre). The underlying biocontrol mechanism revealed that B. velezensis SF305 could reduce the disease severity of red root rot by degrading the mycelia of G. pseudoferreum. An antiSMASH analysis revealed that B. velezensis SF305 contains 15 gene clusters related to secondary metabolite synthesis, 13 of which are conserved in species of B. velezensis, but surprisingly, B. velezensis SF305 possesses 2 unique secondary metabolite gene clusters. One is predicted to synthesize locillomycin, and the other is a novel nonribosomal peptides synthetase (NRPS) gene cluster. Genomic analysis showed that B. velezensis SF305 harbors genes involved in motility, chemotaxis, biofilm formation, stress resistance, volatile organic compounds (VOCs) and synthesis of the auxin indole-3-acetic acid (IAA), suggesting its plant growth-promoting rhizobacteria (PGPR) properties. B. velezensis SF305 can promote plant growth and efficiently antagonize some important phytopathogenic fungi and bacteria. This study indicates that B. velezensis SF305 is a versatile plant probiotic bacterium. To the best of our knowledge, this is the first time a B. velezensis strain has been reported as a promising biocontrol agent against the red root rot disease of rubber trees. 

Kiwifruit bacterial canker, caused by Pseudomonas syringae pv. actinidiae (Psa), is a significant threat to the kiwifruit industry. The two-component signaling systems (TCSs) play a crucial role in regulating the virulence of Pseudomonas syringae (P. syringae), yet their specific function in Psa remains largely unclear. In this study, we found that disrupting the TCS RegAB (encoded by Psa_802/Psa_803) resulted in a notable increase in the pathogenicity of Pseudomonas syringae pv. actinidiae M228 (Psa M228) in host plant and hypersensitive reaction (HR) in nonhost plant. Through comparative transcriptome analysis of the Psa M228 wild-type strain and the regA mutant, we identified the pivotal role of RegA/B in controlling various physiological pathways, including the Type III secretion system (T3SS), a key determinant of Psa virulence. Additionally, we discovered that the RegA does have binding sites in the promoter region of the hrpR/S, and the transcriptional level of the hrpR and other T3SS-related genes increased in the regA deletion strain relative to the Psa M228 wild-type. The DNA-binding affinity of RegA, and therefore the repressor function, is enhanced by its phosphorylation. Our findings unveil the function of TCS RegAB and the regulatory mechanism of T3SS by RegAB in Psa, highlighting the diverse functions of the RegAB system.

Ovulation is paramount for female animal fertility, necessitating a thorough understanding of its process and molecular underpinnings. This study aimed to delineate the goat ovary temporal dynamics of ovulation. Utilizing single-cell sequencing, we analyzed follicular fluid samples obtained at 0, 6, 12, 18, and 24 h post-hCG administration, identifying 4 cell types and 6 myeloid cell subtypes. We elucidated gene expression and functional changes in granulosa cells over the time course of ovulation. Notably, our study detected and confirmed immune cell infiltration at 6 h post-LH peak. Additionally, we observed significant cellular interactions between granulosa cells and macrophages, with granulosa cells expressing IL1RAP, COL4A1, COL4A2 implicated in immune cell regulation, while macrophages expressed EREG to facilitate oocyte maturation. Collectively, our investigation has established a comprehensive single-cell transcriptome atlas of the ovulating goat ovary for advancing exploration into ovulation mechanisms and developing therapies for ovulatory disorders.

The flavonoids produced by legume roots are signal molecules that induce nod genes for symbiotic rhizobium.  Nevertheless, the promoting effects of flavonoids in root exudates in intercropping system on soybean nodulation are still unknown.  A two years of field experiments was carried with maize soybean strip intercropping, i.e., the interspecific row spacing of 30 cm (MS30), 45 cm (MS45), 60 cm (MS60), and sole soybean/maize:SS/MM, and root interaction, i.e., root no barrier (NB) and root polythene-plastic barrier (PB), to evaluate relationships between flavonoids in root exudates and nodulation.  We found that root-root interaction between soybean and maize enhances the nodules number and fresh weight in intercropped soybean.  This enhancement increase gradually with expansion of interspecific distance.  Proportion of nodules with diameter greater than 0.4cm was higher in intercropped soybean with NB than with PB.  The expressions of nodules-related genes (GmENOD40, GmNIN2b and GmEXPB2) were up-regulated.  Furthermore, compared with monocropping, isoflavones secretion of soybean roots reduced, flavonoids and flavonols secretion of maize and soybean roots increased under intercropping.  The secretion of differential metabolites of flavonoids in the rhizosphere of maize and soybean declined with root barrier.  The expressions of GmCHS8 and GmIFS1 in soybean roots were up-regulated and GmICHG was down-regulated under root interaction.  The most of the flavonoids and flavonol compounds were positively correlated with nodule diameter.  The nodules number, the nodules fresh weight and the proportion of nodules with a diameter greater than 0.2 cm increased in different genotypes of soybean treated with maize root exudate, which promoted the improvement of nitrogen fixation capacity.  Therefore, maize-soybean strip intercropping combined with reasonable spacing to enhance the positive effect of underground root interaction, and improve the nodulation and nitrogen fixation capacity of intercropping soybean.

The efficient colonization of plant-beneficial Pseudomonas spp. is a prerequisite for their biocontrol capacity. Prior work revealed that the PcoI/PcoR quorum-sensing (QS) system plays a pivotal role in the root colonization of P. fluorescens 2P24. During the colonization, strain 2P24 has faced diverse impacts from plant-derived reactive oxygen species and other environmental stress. However, the molecular mechanism by which the PcoI/PcoR QS system is regulated under unfavored conditions remains unclear. Thus, in this study, the role of the (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthase/hydrolase SpoT in the PcoI/PcoR QS system of P. fluorescens was investigated. Our data indicated that the deficiency of relA and spoT genes remarkably improved the expression of the pcoI gene, whereas the mutation of the spoT gene significantly repressed the expression of the pcoI gene. We further demonstrated that the regulation of the PcoI/PcoR QS system by (p)ppGpp was dependent on the function of the trmE gene, which encodes a tRNA modification GTPase. Furthermore, the mutation of relA, spoT, or both significantly influenced the motility, biofilm formation, oxidative stress, osmotic tolerance, and rhizosphere colonization. Collectively, our data indicated that the (p)ppGpp signaling pathway mediated by the relA gene and spoT gene was important to the function of the PcoI/PcoR QS system and had important implications for the understanding of the molecular mechanism of (p)ppGpp in epiphytic fitness via TrmE of P. fluorescens.

Farmers in China often use nitrogen (N) fertilizers to ensure adequate crop growth. However, injudicious applications have increased the risk of environmental pollution, lower maize yields, and reduced profits for farmers.  Appropriate N fertilizer management is crucial for improving yield and nitrogen use efficiency (NUE). This study conducted a three-year experiment involving nine N treatments (0, 45, 90, 135, 180, 225, 270, 315, and 360 kg ha^-1) on a field under nitrogen fertilizer precision management (NFPM) in Northeast China.  These results were compared with studies published within the past decade that analyzed yield and dry matter (DM) content under two management practices in Northeast China: conventional nitrogen fertilization management (CNFM) and water-saving fertilization management (WSFM).  The findings reveal that maize yield increases with rising N application rates up to 270 kg ha^-1, after which yield decreases.  The kernel number (KN) and kernel weights (KW) of maize grown under NFPM were 13.7 and 14.7% higher than those grown under WSFM, respectively.  Furthermore, they surpassed crops grown under CNFM by 38.4 and 21.2%, respectively.  The maximum total yield of the NFPM treatment was 41.8 and 78.8% higher than WSFM and CNFM, respectively.  Additionally, compared with CNFM and WSFM, NFPM significantly increased nitrogen use efficiency (NUE) across various N-level treatments. Optimizing nitrogen management could help farmers achieve higher yields and promote sustainable agricultural development.

Glycosylphosphatidylinositol (GPI) anchoring is one of the common post-translational modifications in eukaryotic cells. In fungi, it exerts a wide range of biological functions by targeting proteins to the cell wall, but only few studies focus on the roles of GPI anchoring in plant pathogenic fungi. Here, we reveal a role of GPI anchoring in the maize fungal pathogen Cochlibolus heterostrophus. We found that GPI-anchored proteins were widely accumulated in hyphae, appressorium and infection hyphae of C. heterostrophus. Deletion of ChGPI7, which encodes a key enzyme involved in the biosynthesis of GPI anchors, resulted in significant reduction of vegetative growth and conidiation, as well as virulence due to impairment of appressorium formation and invasive growth. The ∆Chgpi7 mutants also showed severe defects in cell wall integrity, resulting in a significant reduction of stress resistance. Deletion of ChGPI7 and hydrofluoric acid (HF) pyridine treatment both led to removal of cell wall GPI-anchored proteins and exposure of chitin, the results suggested that GPI anchored proteins could protect chitin from host immune recognition. A total of 124 proteins were predicted to be GPI anchored proteins in C. heterostrophus, including a putative cell wall glycoprotein ChFEM1. Deletion of ChFEM1 also resulted in significant reduction in virulence and defects in infection structures, as well as cell wall integrity. We further found that cell wall localization and protein abundance of ChFEM1 were affected by ChGPI7. Our results showed that GPI anchoring regulates cell wall integrity and immune evasion for infection of C. heterostrophus.

With the progress of urbanization, rural tourism has emerged as a popular leisure activity in China.  The crop field with pattern art has been growing in popularity over the years, using strains with colorful leaves to create a variety of impressive designs (Xu 2024).  A lot of successful cases in rice field has not only attracted tourists but also increased the income of farmers (Song et al. 2020).  Plants with colorful leaves also have gained significant popularity in ornamental agriculture.  Therefore, the development of plants with colorful leaves have gained significant popularity in ornamental agriculture.

Betalains are natural tyrosine-derived pigments in red-violet and yellow hues, which are exclusively found in plants of the Caryophyllales order (Azeredo 2009).  In addition to their attractive colors, betalains have demonstrated robust antioxidant activity, thereby demonstrating potential health-promoting properties, including anticancer, hypolipidemic, hepatoprotective, anti-inflammatory and antidiabetic activities (Polturak and Aharoni 2018).  Biosynthesis of betalains has been extensively investigated and only needs three enzymatic reactions to convert tyrosine into betalain.  Tyrosine is initially hydroxylated on the benzene ring, resulting in the formation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by the P450 oxygenase CYP76AD1.  Then, L-DOPA could be further oxidized into cyclo-DOPA by CYP76AD1, or undergoes catalysis by L-DOPA 4,5-dioxygenase (DODA) to yield betalamic acid.  Subsequently, betalamic acid is condensed with cyclo-DOPA into betanidin in the absence of any enzymatic activity.  Finally, a sugar moiety is added to betanidin by a glucosyltransferase to generate the betalain (Khan and Giridhar 2015).  An artificial open reading frame named RUBY containing the three key genes (CYP76AD1, DODA and glucosylstransferase) has been engineered to enable betalain production in Arabidopsis, tomato, carrot and cotton (He et al. 2020; Grützner et al. 2021; Deng et al. 2023; Ge et al. 2023; Li et al. 2023).  Therefore, there is an impetus to engineer colorful-leaved peanuts through expression of the RUBY gene.

Peanut (Arachis hypogaea L.) is an important oilseed crop worldwide, which grown on 30 million ha between latitudes 40°N and 40°S (Hariharan et al. 2023).  In China, the area of peanut planting has exceeded 4.46 million ha in 2022 (FAO 2022), indicating great potential for ornamental agriculture.  However, peanut leaves are predominantly green, which limits the development of sightseeing agriculture in peanut.  Hence, the RUBY gene was employed to genetically modify peanuts with colorful leaves in this study.

To create peanut plants with colorful leaves, a transgenic construct named pBinBarRuby was developed, harboring the RUBY gene driven by 35S-promoter (Fig. 1-A).  Then this construct was introduced into the peanut cultivar Zhonghua 12 (ZH12) through Agrobacterium tumefaciens-mediated transformation (Huai et al. 2023).  The transformed callus turned red-violet, indicative of betalain biosynthesis, whereas the wild-type (WT) callus displayed a green coloration.  Subsequently, the transgenic shoots also exhibited a red-purple color, while the WT shoots retained their green coloration.  Upon the emergence of roots, the entire transgenic plantlets showed a vibrant purple hue in leaves, stems and roots (Fig. 1-B).  Finally, a total of 18 independent positive T₀ transgenic plants were successfully obtained.  Each independent transgenic peanut plant accumulated betalain, but the accumulation patterns were different depending on the expression level of the RUBY gene.  The transgenic plants exhibited purple leaves, orange flowers, purple pod shell, purple testa and purple embryo, whereas the WT control showed green leaves, yellow flowers, white pod shell, red testa and white embryo (Fig. 1-C).  The T₁ progeny of RUBY peanut exhibited the same morphological characteristics as T₀ generation, indicating that the betalain can be inherited by subsequent generations.

Five transgenic lines displaying red-purple color were selected and were confirmed by PCR. The expression levels of RUBY were detected in leaves of the five transgenic lines by qRT-PCR.  As expected, the expression of RUBY was not detectable in the WT, while was detected in transgenic lines (Fig. 1-D).  The betalain contents in leaves of WT and the five transgenic lines were determined.  In WT leaves, the betalain content was 0.57 mg g^-1, while a significant increase ranging from 0.95 to 2.03 mg g^-1 was observed in transgenic purple leaves.   Compared to WT, the transgenic lines exhibited a substantial enhancement in betalain contents ranging from 0.66 to 2.55-fold (Fig. 1-E).

The betalains exhibit remarkable attractiveness as natural pigments due to their vibrant color and relatively simple biosynthesis pathway.  The RUBY gene is a synthetic cassette consisting of the three key genes for involved in the betalain biosynthesis pathway (He et al. 2020), which has been used in cotton to produce pink fibers (Ge et al. 2023; Li et al. 2023).  Due to the powerful antioxidant properties of betalain, the RUBY gene has been expressed in vegetable and fruits including tomatoes, cucumbers and carrots (Grützner et al. 2021; Deng et al. 2023; Liao et al. 2023).  For ornamental purposes, the RUBY gene was applied used in Torenia fournieri, Gentiana scabra and Portulaca grandiflora to change the flower colors (Sakuta et al. 2021; Nishihara et al. 2023, 2024).  In this study, the RUBY gene was used to modify the colors of both of leaves and flowers, and generated novel peanut germplasms which are not present in current varieties (Fig. 1-C).  The distinctiveness of purple leaves compared to green leaves highlights the potential application of purple-leaved peanuts in field pattern artistry.  This is an eco-friendly and health-conscious alternative to synthetic colorants for the cultivation of ornamental plants.

The RUBY gene has also been widely employed as a prominent reporter to visualize transgenic events in Arabidopsis, rice and soybean (He et al. 2020; Wang et al. 2023; Chen et al. 2024).  In this study, the efficiency of identifying transgenic peanut plants using the RUBY reporter was investigated.  The red-violet pigment could be observed at very early stage of callus formation (Fig. 1-B), indicating that the screening could be started very early as well.  Introduction of RUBY facilitated the discrimination between transformed and untransformed callus.  Compared to the DsRed2 reporter, RUBY is clearer and much more convenient to operate during tissue culture condition.  Therefore, the utilization of RUBY as a visible reporter proved to be highly advantageous in the monitoring of transgenes within peanut plants.

In summary, we successfully created novel peanut germplasms with purple leaves by heterologous expression of the RUBY gene, thereby showcasing their potential application in field pattern artistry.  Meanwhile, we also demonstrated that RUBY can serve as a visible reporter in peanut transformation without the need for any additional instrumentation.  Additionally, we provided an eco-friendly and health-conscious alternative to synthetic colorants for the cultivation of ornamental plants.

The auxin response factor (ARF) is the key component of phytohormone auxin signal pathway and is crucial in regulating a plurality of functions throughout plant life cycle.  Although ARFs’ structure and function have been well studied in Arabidopsis, such knowledge is far from being sufficient for cereal crops, especially wheat, rice and maize.  This review is based on a comprehensive retrospection into the studies on ARFs in the three cereal crops, consisting of four parts: (1) characterization of the domains of 23, 25 and 33 ARF family members in wheat, rice and maize, respectively; (2) revision of nomenclatures for previously reported ARFs to the family numbers based on sequence alignment, and summary of ARFs’ functions including the regulation of agronomic traits and response to biotic/abiotic stresses; (3) highlight of general regulatory models for fundamental physiological and reproductive traits from miRNA-ARFs, IAA-ARF-LBD, IAA-ARF-Auxin response gene, and IAA-ARF-ERF (4) prospects to promising future ARF research for anticipated agronomic traits.  Hopefully, the review will facilitate the understanding of ARF functions in the three cereal crops and promote its application in molecular breeding for achieving optimal plant architecture, higher yield, and wide adaptability.

Cutting farming-related methane emissions from ruminants is critical in the battle against climate change. Since scientists initially investigated the potential of marine macroalgae to reduce methane emissions, using seaweeds as an anti-methanogenic feed additive has become prevailing in recent years. Asparagopsis taxiformis is the preferred species because it contains a relatively higher concentration of bromoform. As a type of halogenated methane analogue, bromoform contained in A. taxiformis can specifically inhibit the activity of coenzyme M methyltransferase, thereby blocking the ruminal methanogenesis. However, bromoform is a potential toxin and ozone-depleting substance. In response, current research focuses on the effects of bromoform-enriched seaweed supplementation on ruminant productivity and safety, as well as the impact of large-scale cultivation of seaweeds on the atmospheric environment. The current research on seaweed still needs to be improved, especially in developing more species with low bromoform content, such as Bonnemaisonia hamifera, Dictyota bartayresii, and Cystoseira trinodis. Otherwise, seaweed is rich in bioactive substances and exhibits antibacterial, anti-inflammatory, and other physiological properties, but research on the role of these bioactive compounds in methane emissions is lacking. It is worthy of deeper investigation to identify more potential bioactive compounds. As a new focus of attention, seaweed has attracted the interest of many scientists. Nevertheless, seaweed still faces some challenges as a feed additive to ruminants, such as the residues of heavy metals (iodine and bromine) and bromoform in milk or meat, as well as the establishment of a supply chain for seaweed cultivation, preservation, and processing. We have concluded that the methane-reducing efficacy of seaweed is indisputable. However, its application as a commercial feed additive is still influenced by factors such as safety, costs, policy incentives, and regulations.

Crops produced using the practice of continuous cropping can become seriously damaged by plant-parasitic nematodes, which are an important continuous cropping obstacle. As a typical and important perennial economic crop, dragon fruit is prone to serious plant-parasitic nematode infestation, but whether it encounters this problem due to continuous cropping remains unclear. Here, we studied plant-parasitic nematodes (Meloidogyne spp. and Tylenchorhynchus sp.) in the soil and roots, soil nematode communities, the metabolic footprint, soil integrated fertility, and the yield of intensively planted dragon fruit under non-continuous cropping (Y1) and under 3 (Y3) and 5 (Y5) years of continuous cropping, to determine the potential roles of continuous cropping obstacles and other factors that affect the yield of this fruit. The greatest numbers of plant-parasitic nematodes in the soil and roots were observed in Y5, in which the associated yield was reduced and the dragon fruit was severely stressed. Further analysis of the composition, diversity, and ecological function indices of soil nematodes showed that the soil ecological environment had deteriorated after 3 years of continuous cropping, with Y5 showing the worst results. Similarly, the soil at Y5 had a significant inhibitory effect on the growth and reproduction of Caenorhabditis elegans. Mantel test analysis and a random forest model showed that soil available phosphorus, soil exchangeable calcium, and soil nematode abundance and diversity were significantly related to yield. Partial least squares path modeling revealed that soil fertility and soil nematode diversity directly impact the yield of continuously cropped dragon fruit. In summary, continuous cropping obstacles occurred in year 5 of intensive dragon fruit cultivation, with soil nematode diversity and soil fertility determining the crop yield.

There is growing interest in introducing ecological risks (ERs) and ecosystem services (ESs) into environmental policies and practices. However, the integration of ESs and ERs into actual decision-making remains insufficient. We simulated the spatiotemporal dynamics of ESs (e.g., carbon storage, water yield, habitat quality, and soil conservation) and ERs in the upper reach of the Yellow River (URYR) from 2000 to 2100. Additionally, we explored their relationships by combining the InVEST model and a landscape ecological risk model with CMIP6 data. Our main findings showed that regional ERs change in response to land use and environmental dynamics. Specifically, the ER area decreased by 27,673 m² during 2000-2020, but it is projected to increase by 13,273, 438, and 68 m² under the SSP1-2.6, SSP2-4.5, and SSP5-8.5 scenarios, respectively. We also observed remarkable spatial differences in ESs and ERs between past and future scenarios. For instance, the source area of the URYR exhibited high ESs and low ERs (P<0.001), while the ESs and ERs are declining and increasing, respectively, in the northeastern URYR (P<0.05). Finally, we proposed a spatial optimization framework to improve ESs and reduce ERs, which will support regional sustainable development.

The response of plant functional diversity to external disturbances not only effectively predicts changes in the ecosystem but it also reflects how plant communities use external environmental resources. However, research on how different herbivore assemblages affect plant functional diversity is limited. Therefore, this study systematically explored the effects of three typical herbivore assemblages (yak grazing, Tibetan sheep grazing, and mixed grazing by yaks and Tibetan sheep) on species richness, plant functional diversity, and soil physicochemical properties in alpine grasslands on the Qinghai-Tibet Plateau. This study further investigated the primary mechanisms driving the changes in plant functional diversity. The results indicate four key aspects of this system: (1) Grazing significantly enhanced plant functional diversity, particularly when the mixed grazing by yaks and Tibetan sheep was applied at a ratio of 1:2. This ratio showed the most substantial improvement in the functional dispersion index and Rao's quadratic entropy index. (2) Compared to enclosed treatments, grazing increased species richness and β-diversity, contributing to higher plant functional diversity. (3) Grazing treatments affected various plant traits, such as reducing plant community height and leaf thickness while increasing specific leaf area. However, the impact on plant functional diversity was most pronounced under the mixed grazing by yaks and Tibetan sheep at a ratio of 1:2. (4) Species α-diversity was positively correlated with plant functional diversity. Changes in plant functional diversity were primarily regulated by variations in soil physicochemical properties. Specifically, increases in soil available nitrogen significantly promoted changes in plant functional diversity, while increases in soil available potassium and bulk density had a significant inhibitory effect on these changes. Long-term grazing significantly reduced the height of plant communities in alpine meadows, while a balanced mixture of yak and Tibetan sheep grazing, especially at a ratio of 1:2, enhanced plant functional diversity the most. This suggests that, under these conditions, the use of external environmental resources by the plant community is optimized.

Nsa CMS, a type of cytoplasmic male sterility (CMS) in rapeseed, originates from the cross between Xinjiang wild rapeseed (Sinapis arvensis) and Xiangyou 15 (Brassica napus L.).  Although this CMS variant shows promising applications, the factors contributing to its sterility and their underlying mechanisms remain unclear.  To the best of our knowledge, we successfully assembled and analyzed the mitochondrial genome of 1258A (Nsa CMS) for the first time.  This mitochondrial genome, spanning 263,010 bp, contains 91 genes, including 33 protein-coding and 36 orf genes.  Our analysis identified a novel mitochondrial gene, orf113b, and a mutated, truncated gene, orf146, both likely linked to the sterility observed in 1258A.  ORF113b and ORF146 were found to impede cell growth, disrupt gene expression associated with complexes I, III, and V of the mitochondrial oxidative phosphorylation pathway, and trigger reactive oxygen species (ROS) production.  Additionally, transcriptome data analysis revealed key nuclear genes co-expressed with orf113b and orf146, suggesting that their aberrant expression may be influenced by retrograde signaling from the mitochondria.  This signaling could lead to atypical programmed cell death (PCD) in the tapetum layer, resulting in pollen sterility.  In conclusion, our study not only provides the first characterization of the Nsa CMS mitochondrial genome but also identifies orf113b and orf146 as crucial to pollen sterility.  Furthermore, it suggests that ROS induced by these mitochondrial genes may play a central role in the abnormal regulation of nuclear genes essential for pollen development, offering new insights into the molecular mechanisms underlying Nsa CMS. 

Plant roots interact with diverse fungi that are essential for maintaining the productivity and sustainability of pasture ecosystems, but how these root-associated fungi (RAF) differ between forage species and how they respond to nutrient enrichment and fungicide application are not well understood. Here, we constructed an 11-year experiment involving fungicide application (with or without) nested within four levels of experimental nitrogen (N) addition treatments in an alpine pasture, and the RAF communities, root traits, tissue nutrients, and shoot biomass of two dominant forage species (Carex capillifolia and Elymus nutans) were analyzed. The RAF community composition showed striking differences between the plant species and was strongly affected by both N addition level and fungicide applications. Fungicide, but not N application, dramatically reduced the RAF richness of all functional guilds in both plant species, and fungicide also simplified the co-occurrence network of the RAF for C. capillifolia. The RAF community correlated strongly with root traits, whereas their relationships became weakened or even vanished at the level of the individual plant species. The importance of RAF to plant nutrients and productivity varied between plant species, with significant contributions in C. capillifolia but not in E. nutans. This is the first report elucidating the long-term effect of fungicides on RAF in alpine pastures, and our findings emphasize the host-specific responses of RAF community structure and function to anthropogenic disturbances.

Cover cropping is a diversifying agricultural practice that can improve soil structure and function by altering the underground litter diversity and soil microbial communities. Here, we tested how a wheat cover crop alters the decomposition of cucumber root litter. A three-year greenhouse litterbag decomposition experiment showed that a wheat cover crop accelerates the decomposition of cucumber root litter. A microcosm litterbag experiment further showed that wheat litter and the soil microbial community could improve cucumber root litter decomposition. Moreover, the wheat cover crop altered the abundances and diversities of soil bacterial and fungal communities, and enriched several putative keystone OTUs, such as Bacillus spp. OTU1837 and Mortierella spp. OTU1236, that were positively related to the mass loss of cucumber root litter. The representative bacterial and fungal strains B186 and M3 were isolated and cultured. In vitro decomposition tests demonstrated that both B186 and M3 had cucumber root litter decomposition activity and a stronger effect was found when they were co-incubated. Overall, a wheat cover crop accelerated cucumber root litter decomposition by altering the soil microbial communities, particularly by stimulating certain putative keystone taxa, which provides a theoretical basis for using cover crops to promote sustainable agricultural development. 

With the development of international trade and frequent personnel exchanges, biological invasion is showing a rapidly growing trend worldwide. Insects are ectothermic animals, so their geographical distribution is due largely to their high and low temperature tolerances. To study the temperature response mechanisms of Bemisia tabaci MED cryptic species, miRNA-seq technology was used to unravel the miRNA library of B. tabaci MED in three field populations (TP, HB, and HK) from cities with different environmental temperatures. We identified 12 differentially expressed miRNAs in response to temperature stress, and Bta-miR-998 and Bta-miR-129 were shown to be associated with temperature tolerance. In addition, we predicted and verified the target genes associated with the temperature tolerance imparted by Bta-miR-998 and Bta-miR-129. The results showed that the down-regulated target gene of Bta-miR-129, BtMGAT3, significantly reduced the heat tolerance and another down-regulated target gene, BtRGS7, affected the cold tolerance of B. tabaci MED. These results indicate that gene expression regulated by miRNAs is an important temperature response mechanism in B. tabaci MED. This study reveals the important regulatory role of miRNA in insect temperature adaptation and provides a new avenue for studying the regulation of insect gene expression by miRNA.

Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have proven to be highly effective in managing some key pests. However, the evolution of resistance by the target pests threatens the sustainability of Bt crops. The L31S mutation in a tetraspanin encoded by HarmTspC5 (previously known as HaTSPAN1) has been shown to confer dominant resistance to the Bt protein Cry1Ac in Helicoverpa armigera, a globally damaging lepidopteran pest. However, the broader implications of the L31S mutation in the tetraspanins of other lepidopteran species remain unclear. The evolutionary analyses in this study indicate that TspC5s have evolved in a species-specific manner among the lepidopteran insects. To investigate the role of TspC5s in conferring dominant resistance to Cry1Ac, we used the piggyBac-based transformation system to generate four transgenic H. armigera strains that express exogenous TspC5 variants from three phylogenetically close species (Helicoverpa zea, Helicoverpa assulta and Heliothis virescens) and one phylogenetically distant species (Plutella xylostella). In comparison with the background SCD strain of H. armigera, the transgenic strains expressing HzeaTspC5-L31S, HassTspC5-L31S, or HvirTspC5-L31S exhibited significant resistance to Cry1Ac (10.0-, 21.4-, and 81.1-fold, respectively), whereas the strain expressing PxylTspC5-L27S remained susceptible. Furthermore, the Cry1Ac resistant phenotypes followed an autosomal dominant inheritance pattern and were closely linked to the introduced mutant TspC5s. These findings reveal the conserved role of TspC5s from Helicoverpa and Heliothis species in mediating the dominant resistance to Cry1Ac, and they provide crucial insights for assessing resistance risks related to mutant tetraspanins and devising adaptive resistance management strategies for these major lepidopteran pests.

The efficient genome editing tool (the CRISPR/Cas12a system) has been used in research on plant functionional genomics and improvement of agronomic traits.  In this study, CRISPR/Cas12a system was optimized by using the endogenous pGhαGloA promoter in cotton.  Using this system, crRNAs was driven by the Pol II pGhaGloA promoter to construct the pGhRBE3-pGhαGloA-GhPGF vector and carry out genetic transformation.  The vector could work efficiently in all positive transgenic plants and the editing efficiency at the crRNA1 target site was up to 93.37%, and the editing efficiency of the crRNA2 target was up to 88.24%, which is significantly higher in editing efficiency of the pGhRBE3 system with Pol III promoter-Ubi 6.7 promoter, this result indicates that the Pol II promoter is more suitable for expressing multiple sgRNA or crRNA than the pol III promoter in cotton.  The vector mainly generated the editing type of fragment deletion and the deletion size was in the range of 3-12 bp with the editing sites spanning at the 14th to 29th bases downstream of the protospacer adjacent motif (PAM).  All the targeted mutation loci were stably inherited from T₀ to T₂ generation and three transgene-free lines with target site mutations of GhPGF gene were obtained and these glandless and gossypol-free/(low contents) cotton germplasm will play key role for healthy cottonseeds oil/cake production.  Therefore, the CRISPR/Cas12a system driven by the pGhαGloA promoter can efficiently edit target genes in cotton, which provides a powerful tool for cotton functionional genomics and genetic improvement.

The commercialization of genetically modified crops has increased food production, improved crop quality, reduced pesticide use, promoted changes in agricultural production methods, and become an important new productivity to deal with insect pests and weeds while reducing cultivated land area.  This article provides a comprehensive examination of the global distribution of genetically modified crops in 2023.  It discusses the internal factors that are driving this, such as the increasing number of genetically modified crops and the increased variety of commodities.  It also provides information support and application direction guidance for the new productivity of global agricultural science and technology.

Peanut (Arachis hypogaea L.) is an important oil and edible protein crop. The fatty acid composition not only influences the quality of peanut oil but also impacts flavor, shelf life, and consumer health. Peanut oil comprises approximately 80% oleic acid (C18:1) and linoleic acid (C18:2), 10% palmitic acid (C16:0), and the remaining 10% includes stearic acid (C18:0), arachidic acid (C20:0), gadoleic acid (C20:1), behenic acid (C22:0), and lignoceric acid (C24:0). To unravel the genetic foundation of fatty acid content and delve into QTL localization, we utilized high-density SNP microarrays for genotyping the RIL population of 'SunOleic 97R' × 'NC94022'. A genetic linkage map was constructed with 3141 SNP markers, covering a total genetic distance of 3051.81 cM. We identified 60 quantitative trait loci (QTLs) associated with fatty acids which distributed in 11 linkage group, with phenotypic variance explained (PVE) ranging from 1.37% to 44.92%. Notably, the QTL qFAT_A05.1 and qFAT_A08.1 are multiple-effect loci contributing to various fatty acid compositions. Moreover, we identified 15 haplotypes for the QTL qFAT_A05.1 and qFAT_A08.1 through genotyping 178 peanut germplasms. Haplotypes analysis in natural population confirmed the closely relationship of the QTLs with the content of oil, oleic acid, lignoceric acid, palmitic acid and behenic acid. This study serves as a valuable reference for selecting improved peanut genotypes with superior oil quality and desirable fatty acid composition.

Tea's popularity and flavor are influenced by factors like cultivation and processing methods, shaping techniques also has an impact on tea flavor. Targeted metabolomics and chemometrics were employed to study how shaping techniques affect the flavor of milk-flavored white tea (MFWT) in this study. The results showed that the tea cake sample with the shortest pressing time (Y90) has the highest amino acid content and milky aroma intensity. There were variations in amino acids, catechins, and soluble sugars among MFWT samples with different shaping techniques. The total contents of amino acids and catechins in tea cake sample (Y90) were significantly lower than those in loose tea sample (SC) and bundle-like tea sample (SG), while the total sugar content was significantly higher than that in SC (P<0.05). Additionally, the content of volatiles presenting milky aroma (VIP&OAV>1) in Y90 remained lower relative to SC and SG (P<0.05), but the proportion was not different from that in SC and SG, minimally affecting the overall flavor. The short-time pressing method could be suitable for mass production of MFWT. These findings provide insights into improving the tightness of the appearance of MFWT with minimal impact on tea flavor.

The lodging issue is a significant factor that hinders the enhancement of oil flax production efficiency in northern China.  Crop lodging or not, and lignin content in the stems are closely related, and how nitrogen fertilizer and potassium fertilization interaction regulate lignin biosynthesis in the stems of oil flax requires further in-depth study.  Therefore, the research aims to enhance the lodging resistance and facilitate an increased yield of oil flax.  We are examined the interaction of different nitrogen fertilizers (75, 150, and 225 kg N ha^-1) and potassium fertilizers (60 and 90 kg K₂O ha^-1) on oil flax lignin metabolism, lodging resistance, and grain yield in 2022 and 2023 growing seasons.  The results indicated that nitrogen and potassium fertilizer levels and their interaction treatments are facilitated lignin accumulation, achievement of lodging resistance and high yield.  Compared to CK, the 75-150 kg N ha^-1 in combination with 60 kg K₂O ha^-1 treatments significantly enhanced the enzyme activities (TAL, PAL, POD, and CAD) and the gene expressions (4CL1 and F5H3) for lignin syntheses, lignin content dramatically increased 29.63-43.30%, improved stem bending strength and lodging resistance index, and grain yield increased 23.27-32.34%.  Correlation analysis indicated that nitrogen and potassium fertilizer positively regulated the relative enzyme activities and genes expression for lignin to facilitated lignin biosynthesis and accumulation, and enhanced stem bending strength and lodging resistance index.  Positive correlations were found among the relative enzyme activities and gene expressions for lignin, lodging resistance and grain yield.  To summarize, 75-150 kg N ha^-1 in conjunction with 60 kg K₂O ha^-1 treatment was promoted to lignin biosynthesis and accumulation, enhanced the lodging resistance and grain yield of oil flax in the dryland farming region of central Gansu.  Furthermore, it served as a technical guide for cultivating stress tolerance and high-yield oil flax in the dryland farming region.

Due to the breeding of dense-resistant and lodging-resistant varieties in maize production, dense planting has become an effective means for achieving high and stable yields, while excellent hybrids are a prerequisite for reasonable dense planting in maize production.  Nonetheless, the photosynthetic mechanism of improving plant density tolerance of maize hybrids released at different era in China remains unclear.  This study aims to investigate the 40-year breeding effort for enhanced photosynthetic trait at different densities, and elucidate the physiological and ecological mechanisms of improving the density tolerance of maize hybrids.  We conducted a 3-year study in 2019, 2020, and 2021.  From 1970 to 2009, a comparison was made between the eight major hybrids promoted in China, divided into four decades, under three planting densities (45,000 (D1), 67,500 (D2), and 90,000 (D3) plants ha⁻¹).  At high density, modern hybrids had more rational canopy structure and leaf photosynthetic performance compared with old hybrids and specific leaf nitrogen has decreased slightly.  Among all treatments, the modern hybrids (2000s) were able to maintain higher net photosynthetic rate and photosynthetic nitrogen utilization efficiency (PNUE) at D3 density, and therefore possessed the highest grain yield (GY), which was 118.47% higher than that of the old hybrids (1970s).  Leaf area duration after anthesis, total chlorophyll content, photosynthesis key enzyme activities, and maximum efficiency of PSII photochemistry were all positively correlated with GY, with PNUE was more significantly correlated with GY indeed and is a key indicator for maize hybrids optimization.  Based on these results, breeders should continue to conduct hybrid selections under adverse and high-density conditions, focusing on the optimization of population structure and the continuous improvement of photosynthetic capacity, searching for the optimal leaf nitrogen-content status, so as to select and breed high-yielding and density-tolerance hybrids, which resulted in a sustained increase in maize GY.

Drought (D) caused by global warming is severely affecting crop growth and agricultural production (Gupta et al. 2020).  Stomata is an important window for photosynthesis and water transpiration, and stomatal density is closely related to drought response (Herrmann and Torii 2021).  Foxtail millet (Setaria italica), an important drought tolerant crop, plays an important role in ensuring food and nutrition security (Muthamilarasan and Prasad 2017).  Compared to with other crops, the drought-resistance mechanism of foxtail millet is not fully understood with only SiDPY1, SiYTH1 and SiMYBS3 currently identified and characterised (Liu et al. 2023; Luo et al. 2023; Zhao et al. 2023).  Therefore, their drought resistance mechanism is not fully understood.

In this study, a drought resistant cultivar named MT3 was identified through drought screening. MT3 exhibited significantly higher drought resistance compared to Jingu21 (JG21) (Fig. 1-A; Appendix A).  Statistical analysis of physiological data for normal and drought-treated 15-day seedlings showed that the shoot of MT3 was shorter than that of JG21, with no significant difference in root length. However, the root system of JG21 was denser than that of MT3.  In addition, the dry and fresh weight of MT3 was higher than that of JG21, and MT3 also had a larger leaf area (Appendix A).  Further testing after 15 days of drought showed that MT3 had higher chlorophyll content, lower intercellular carbon dioxide concentration, lower stomatal conductance, and lower transpiration rate compared to JG21 (AppendixB).  Importantly, the leaf water loss rate of MT3 was significantly lower than that of JG21 (Fig. 1-B).  The degree of shrinkage in detached leaves of JG21 was significantly higher than that of MT3, further confirming the lower water loss rate of MT3 (Appendix C). Previous studies have shown a strong correlation between leaf water loss, water use efficiency, and stomatal density (Hughes et al. 2017).  In this study, we performed a comparison of water use efficiency and stomatal density between different treatments.  Our results showed that MT3 had a higher water use efficiency compared to JG21 (Fig. 1-C).  In addition, MT3 had a significantly lower stomatal density than JG21 (Fig. 1-D; Appendix D).  The analysis also revealed no significant differences in individual stomatal size (Appendix D).  These results suggest that the reduced stomatal density in MT3 may contribute to reduced water loss and improved drought tolerance.

Based on the above physiological data analysis and relevant literature reading (Qi et al. 2013).  To further explore the underlying molecular mechanism transcriptomic analysis was performed (Fig. 1-E; Appendix E).  Following the observation and analysis of stomatal density, it was found that MT3 exhibited significantly lower stomatal density than JG21 (Appendix D). T ranscriptomics was then employed to analyse the genes related to the regulate stomatal development.  The analysis of the entire regulatory network revealed that EPFs was expressed in high amounts (Fig. 1-F), and the detection of other key genes that regulate stomatal development did not reveal clear regulatory trends (Appendix F).  Subsequently, the results were validated through qRT-PCR, demonstrating consistency with the transcriptome data (Fig. 1-G).  Furthermore, we conducted a comparative analysis of drought-related homologous genes in cereals (Appendix G) (Yang and Qin 2023).  This revealed that there was no significant difference in their expression levels, which also demonstrated that the enhanced drought resistance of MT3 was attributable to the high expression of EPFs.  Furthermore, the research examined the expression levels of EPFs in various parts of JG21, with the highest levels observed in the leaves (Appendix H). 

Phylogenetic trees were constructed using EPF families from Arabidopsis, rice, barley, and millet, thereby revealing close relationships between their relatives and those of rice (Appendix I).  In this study, we synthesized small peptides known as EPFs, which are capable of exerting their function through exogenous application.  The synthesized EPF peptide was applied to plants in order to observe phenotypic changes and conduct physiological analyses.  The results demonstrated that EPF enhanced the plant’s response to drought, reducing the rate of leaf water loss and the density of stomata (Appendix J).  Based on the results of transcriptome analysis and qRT-PCR, we selected the two highest expressed EPF genes for transgenic analysis in millet (Appendix K).  The analysis of EPF gene expression levels in the overexpressed lines using qRT-PCR revealed significantly higher expression levels compared to the wild-type Ci846 (Appendix K).  A subsequent drought treatment indicated that the wild-type Ci846 exhibited more severe wilting compared to the overexpressed lines (OE2-11 and OE1-2) (Fig. 1-H).  Upon rewatering, the Ci846 plants, exhibited complete mortality whereas the overexpressed lines resumed normal growth (Fig. 1-H).  To ascertain whether EPFs regulate drought resistance by adjusting stomatal density, a comparison of physiological data revealed a comparable trend in the overexpressed lines and MT3 (Appendices L and M), with significantly lower water loss rate in the overexpressed lines compared to Ci846 (Fig. 1-I).  In comparison the wild type, the overexpression lines exhibited higher water use efficiency and lower stomatal density (Fig. 1-J and K; Appendix N).  These findings provide further evidence that EPFs enhance water use efficiency and drought tolerance by regulating stomatal density in foxtail millet.

By regulating stomatal density, EPFs reduce the rate of water loss from leaves enhance water use efficiency, and prolonging the survival of millet plants under drought conditions.  In conclusion this study describes the function and physiological effects of overexpression of a natural epidermal model factor in grasses.  The regulation of SiEPF2 expression levels has enhanced our comprehension of the mechanisms underlying stomatal development in grasses, resulting in a notable reduction in stomatal density in a range of millet plants.  This has led to a significant improvement in drought resistance and water use efficiency.

In future climates with high atmospheric CO₂ concentrations and areas of reduced water availability (Franks et al. 2015), targeted genetic modification of stomatal conductances, such as EPFs, represents a viable approach to enhance crop water use efficiency and drought resistance without compromising food yield.  This indicates that stomatal development represents an attractive target for breeders seeking to develop future crops.

In agriculture, a significant challenge revolves around low phosphate (Pi) use efficiency, leading to increased costs and environmental issues while causing a shortage of phosphorus (P) resources.  The TaPHT1;6 gene, which encodes a high-affinity Pi transporter (PHT), plays a crucial role in Pi absorption and transport.  In this study, the promoter and coding regions of three TaPHT1;6 gene copies on chromosomes 5A, 5B, and 5D were individually amplified and sequenced from 167 common wheat (Triticum aestivum L.) cultivars.  Sequence analysis revealed 16 allelic variation sites within the promoters of TaPHT1;6-5B among these cultivars, forming three distinct haplotypes: Hap1, Hap2, and Hap3.  Field trials were conducted over two years to compare wheat genotypes with these haplotypes, with a focus on assessing plant dry weight, grain yield, P content, Pi fertilizer absorption efficiency, and Pi fertilizer utilization efficiency.  Results indicated that Hap3 represented the favored Pi-efficient haplotype.  The dual-luciferase reporter assay demonstrated that the Hap3 promoter, carrying the identified allelic variation sites, exhibited higher gene-driven capability, leading to increased expression levels of the TaPHT1;6-5B gene.  Based on these allelic variation sites, a distributed cleaved amplified polymorphic site marker (dCAPS-571) was developed to distinguish Hap3 from the other two haplotypes, presenting an opportunity for breeding Pi-efficient wheat cultivars.  This study successfully identified polymorphic sites on TaPHT1;6-5B associated with Pi efficiency and developed a functional molecular marker to facilitate future breeding endeavors.

Population stress has compelled the whole world to think about increasing, protecting, and finding ways to develop the best plant species for assured productivity in spite of all environmental odds.  In addition to climate change, fluctuations in nature and human activities pose serious environmental degradation, threatening global food security through physical stress in the environment.  Significant environmental constraints on agricultural yield worldwide include salt stress, water deficiency stress, nutritional imbalances (including mineral toxicity and deficiencies), and temperature extremes.  Abiotic factors, such as agronomic factors, climatic factors, and nutrient availability in the soil, influence crop yield.  Plants initiate and develop various possible stress mechanisms for their survival, which can be molecular, cellular and physiological.  Abiotic stress has a high impact on crop growth and productivity, either in single forms or in combined forms.  For example, drought stress causes a decrease in leaf area, plant height, and crop development.  Cold stress reduces plant development and crop efficiency, resulting in productivity loss.  Salinity stress not only contributes to water stress in plants, but it can also adversely influence cytosolic metabolism, cell development, membrane function, and increase reactive oxygen species (ROS) generation.  Higher concentrations of CO₂ could potentially improve global precipitation, resulting in increased rainfall, which can adversely affect crop development.  Crops under excessive water stress have a lower percentage of amylose but a higher crude protein content.  This in turn, affects the quality and quantity of crop production by hindering seed germination and causing growth damage due to the combined effects of higher osmotic potential and ion toxicity.  In response to abiotic stress, plants evolve a variety of escape-avoidance and tolerance mechanisms, which include physiological adaptation and integrated cellular or molecular responses.  Therefore, the main purpose of the current review paper is to investigate the effect of abiotic stress on morpho-physiological, biochemical and molecular activities in various crops.  Moreover, we concentrate on crop inter-relativity with abiotic stress to react to and adapt to for survival, which can be a basic roadmap for the selection of species or the development of new tolerant species in the future.  

Maize (Zea mays L.) is a globally significant crop that plays a crucial role in feeding the growing global population.  Among its various traits, plant height is particularly important as it affects yield, lodging resistance, ecological adaptability, and other important factors.  Traditional methods for measuring plant height often lack cost-efficiency and accuracy.  In this study, we employed a light detection and ranging (LiDAR) sensor mounted on an unmanned aerial vehicle (UAV) to collect point cloud data from 270 doubled haploid (DH) lines.  This innovative application of UAV-based LiDAR technology was explored for high-throughput phenotyping in maize breeding.  We constructed high-density genetic maps and assessed plant height at both single-plant and row scales across multiple developmental stages and genetic backgrounds.  Our findings revealed that for many varieties and small areas, single-plant-scale estimation accuracy was superior to row-scale estimation, with an R⊃2; of 0.67 versus 0.56 and an RMSE of 0.12 m vs. 0.17 m, respectively.  Two high-density genetic maps were constructed based on SNP markers.  In Sanya and Xinxiang, the F₁DH and F₂DH populations identified 12 and 20 QTLs (quantitative trait loci) for plant height, respectively.  The study successfully identified and validated QTLs associated with plant height, revealing novel genetic loci and candidate genes.  This research highlights the potential of UAV-based remote sensing to advance precision agriculture by enabling efficient, large-scale phenotyping and gene discovery in maize breeding programs.

Dud dormancy is a complex physiological process of perennial woody plants living in temperate regions, and can be affected by various phytohormones. Cytokinin oxidase/dehydrogenases (CKXs) are a group of enzymes essential for maintaining cytokinin homeostasis, but a comprehensive analysis in peach is lacking. Here, a total of 51 CKX members from different species, including six from peach, eleven from apple, nine from poplar, seven from Arabidopsis, eight from strawberry and ten from rice, which were identified using The Simple HMM Search tool of TBtools and a BLASTP program, were classified into four groups using phylogenetic analysis. Conserved motif and gene structure analysis of these 51 CKX members showed that ten conserved motifs were identified, and each CKX gene contained at least two introns. Cis-element analysis of PpCKXs showed that all of the PpCKX genes have light-responsive elements and at least one hormone-responsive element. The obviously changed relative expression levels of six PpCKX genes in peach buds from endodormancy to bud-break were observed by qRT-PCR. Among them, the expression trend of PpCKX6 was almost opposite that of PpEBB1, a positive bud-break regulator in woody plants, around the bud-break stage. Y1H, EMSA, and dual-luciferase assays indicated that PpEBB1 negatively regulated PpCKX6 through direct binding to a GCC box-like element located in the promoter region of PpCKX6. In addition, a transient assay showed that overexpression of PpCKX6 delayed bud-break of peach. These results indicate that the PpCKX genes play an important role in the dormancy-regrowth process and PpCKX6 may act downstream of PpEBB1 directly to regulate bud-break process, which further improves the hormone-regulatory network of dormancy-regrowth of woody plants, and provides new insights for molecular breeding and genetic engineering of peach.

Dud dormancy is a complex physiological process of perennial woody plants living in temperate regions, and can be affected by various phytohormones. Cytokinin oxidase/dehydrogenases (CKXs) are a group of enzymes essential for maintaining cytokinin homeostasis, but a comprehensive analysis in peach is lacking. Here, a total of 51 CKX members from different species, including six from peach, eleven from apple, nine from poplar, seven from Arabidopsis, eight from strawberry and ten from rice, which were identified using The Simple HMM Search tool of TBtools and a BLASTP program, were classified into four groups using phylogenetic analysis. Conserved motif and gene structure analysis of these 51 CKX members showed that ten conserved motifs were identified, and each CKX gene contained at least two introns. Cis-element analysis of PpCKXs showed that all of the PpCKX genes have light-responsive elements and at least one hormone-responsive element. The obviously changed relative expression levels of six PpCKX genes in peach buds from endodormancy to bud-break were observed by qRT-PCR. Among them, the expression trend of PpCKX6 was almost opposite that of PpEBB1, a positive bud-break regulator in woody plants, around the bud-break stage. Y1H, EMSA, and dual-luciferase assays indicated that PpEBB1 negatively regulated PpCKX6 through direct binding to a GCC box-like element located in the promoter region of PpCKX6. In addition, a transient assay showed that overexpression of PpCKX6 delayed bud-break of peach. These results indicate that the PpCKX genes play an important role in the dormancy-regrowth process and PpCKX6 may act downstream of PpEBB1 directly to regulate bud-break process, which further improves the hormone-regulatory network of dormancy-regrowth of woody plants, and provides new insights for molecular breeding and genetic engineering of peach.

Nitrogen (N) uptake is regulated by water availability, and a water deficit can limit crop N responses by reducing N uptake and utilization. The complex and multifaceted interplay between water availability and the crop N response makes it difficult to predict and quantify the effect of water deficit on crop N status. The nitrogen nutrition index (NNI) has been widely used to accurately diagnose crop N status and to evaluate the effectiveness of N application. The decline of NNI under water-limiting conditions has been documented, although the underlying mechanism governing this decline is not fully understood. This study aimed to elucidate the reason for the decline of NNI under water-limiting conditions and to provide insights into the accurate utilization of NNI for assessing crop N status under different water-N interaction treatments. Rainout shelter experiments were conducted over three growing seasons from 2018 to 2021 under different N (75 and 225 kg N ha^-1, low N and high N) and water (120 to 510 mm, W0 to W3) co-limitation treatments. Plant N accumulation, shoot biomass (SB), plant N concentration (%N), soil nitrate-N content, actual evapotranspiration (ET_a), and yield were recorded at the stem elongation, booting, anthesis and grain filling stages. Compared to W0, the W1 to W3 treatments exhibited NNI values that were greater by 10.2 to 20.5%, 12.6 to 24.8%, 14 to 24.8%, and 16.8 to 24.8% at stem elongation, booting, anthesis, and grain filling, respectively, across the 2018-2021 seasons. This decline in NNI under water-limiting conditions stemmed from two main factors. First, reduced ET_a and SB led to a greater critical N concentration (%N_c) under water-limiting conditions, which contributed to the decline in NNI primarily under high N conditions. Second, changes in plant %N played a more significant role under low N conditions. Plant N accumulation exhibited a positive allometric relationship with SB and a negative relationship with soil nitrate-N content under water-limiting conditions, indicating co-regulation by SB and the soil nitrate-N content. However, this regulation was influenced by water availability. Plant N accumulation sourced from the soil nitrate-N content reflects soil N availability. Greater soil water availability facilitated greater absorption of soil nitrate-N into the plants, leading to a positive correlation between plant N accumulation and ET_a across the different water-N interaction treatments. Therefore, considering the impact of soil water availability is crucial when assessing soil N availability under water-limiting conditions. The findings of this study provide valuable insights into the factors contributing to the decline in NNI among different water-N interaction treatments and can contribute to the more accurate utilization of NNI for assessing winter wheat N status. 

Increasing oil content is a key objective in peanut breeding programs.  Accurate identification of quantitative trait loci (QTLs) with linked markers for oil content can greatly aid in marker-assisted selection for high-oil breeding.  In this study, a high-density bin map was constructed by resequencing a recombinant inbred line (RIL) population (ZH16×J11) consisting of 295 lines.  The bin map contained 4,212 loci and had a total length of 1,162.3 cM.  Ten QTLs for oil content were identified in six linkage groups.  Notably, two of these QTLs, qOCB03.1 and qOCB06.1, were consistently detected in a minimum of three environments and explained up to 13.62% of phenotypic variation.  They have not been reported in previous studies and thus are novel QTLs.  The combination of favorable alleles from the qOCB03.1 and qOCB06 in the RIL population could increase oil content across multiple environments from 1.50 to 2.46%.  Two InDel markers linked to qOCB03.1 and qOCB06.1 were developed and validated to be associated with oil content in another RIL population (ZH10×ICG12625) with diverse phenotypes.  Additionally, the high-resolution map allowed for the precise positioning of qOCB03.1 and qOCB06.1 within a 1.77 Mb-interval on chromosome B03 and a 1.51 Mb- interval on chromosome B06, respectively.  Annotation of genomic variants, analysis of transcriptome sequencing, and evaluation of the allelic effects in 292 peanut varieties revealed two candidate genes associated with oil content for each of the two QTLs.  The identification of candidate genes in this study can enable the map-based cloning of key genes controlling oil content in peanut.  Furthermore, these novel and stable QTLs and their tightly linked markers are valuable for marker-assisted breeding for increased oil content in peanut.

Filament-like plant proteins are intermediate filament proteins that play a major part in the development and growth of plants.  However, no systematic identification and characterization have been conducted on the FPP family in plants.  Fifty nine FPP candidates were found in this work by analyzing the genomes of two dicots and four monocots.  Phylogenetic analysis and multicollinearity mapping showed the relatively conserved evolution of FPP genes in monocots.  Herein, eight OsFPPs were characterized and found to be induced or repressed by abiotic stresses.  Additional genetic evidence shows that OsFPP7-overexpressing rice exhibited increased sensitivity to abscisic acid during the germination stage, disrupted Na⁺/K⁺ homeostasis, and disrupted the balance of reactive oxygen species during the seedling stage when exposed to salt stress.  Conversely, knockout of osfpp7 alleviated ABA sensitivity, safeguarded the antioxidant system and sodium ion transport system, and thus enhanced rice salt tolerance.  The cytoskeleton, FPPs’ function in controlling salt stress and plant stress tolerance mechanisms are all further elucidated by our findings.

The plant circadian clock temporally drives gene expression through the day and coordinates various physiological process with diurnal environmental changes. It is essential to confer plant fitness and competitive advantage to survive and thrive under natural condition by circadian control of gene transcription. Chinese cabbage (Brassica rapa ssp. pekinensis) is an economically important vegetable crop worldwide; however, there is little information concerning its circadian clock system. Here we uncovered that gene expression patterns were affected by circadian oscillators at both transcriptional and post-transcriptional levels in Chinese cabbage. Time-course RNA-seq analyses were conducted on two short-period lines (SPcc-1 and SPcc-2) and two long-period lines (LPcc-1 and LPcc-2) under constant light. We showed that 32.7-50.5% of the genes were regulated by the circadian oscillator and the expression peak of cycling genes appeared even earlier in short-period lines compared to long-period lines. In addition, approximately 250 splicing events showed circadian regulation, of which intron retention (IR) accounted for a large proportion. Rhythmically spliced genes included the clock genes LATE ELONGATED HYOCOTYL (BrLHY), REVEILLE 2 (BrRVE2) and EARLY FLOWERING 3 (BrELF3). We also found that the circadian oscillator could notably influence the diurnal expression patterns of genes that are associated with glucose metabolism via photosynthesis, Calvin cycle and tricarboxylic acid (TCA) cycle at both transcriptional and post-transcriptional levels. Taken together, our results demonstrate that circadian-regulated physiological processes contribute to Chinese cabbage growth and development.

Sugarcane has recently attracted increased attention for its potential as a source of sugar and bioethanol, and increasing its yields is essential to ensure the sugar security and bioenergy production. Intergeneric hybridization is a highly efficient method to produce new genetic variants of crop plants, particularly those species with high ploidy such as sugarcane (Saccharum spp.). Tripidium arundinaceum exhibits many desirable agronomic traits, and has been widely studied to produce hybrid with improved stress tolerance and other characteristics in sugarcane breeding. However, the genetic relationship between T. arundinaceum and Saccharum species, and the individual T. arundinaceum chromosomal composition in sugarcane hybrids are still elusive. Here we used whole-genome single-nucleotide polymorphisms (SNPs) to ascertain the phylogenetic relationships between these species and found that T. arundinaceum was more closely related to Saccharum than Sorghum, in contrast to the previous narrow genetic analyses utilizing chloroplast DNA. Additionally, oligonucleotide (oligo)-based chromosome-specific painting derived from Saccharum officinarum was found to be able to distinctly identify the chromosomes of T. arundinaceum. We developed the oligo- genomic in situ hybridization (GISH) system, for the first time, to unveil the novel chromosome translocations and the individual chromosomal transmission of T. arundinaceum in sugarcane progeny. Notably, we discovered the chromosomal transmission of T. arundinaceum exhibited several inheritances, including n, 2n, and over 2n in BC₁ progenies. Such inheritances may be resulted from the first division restitution (FDR), FDR + nondisjunction of a chromosome with sister chromatids in the second meiosis division/second division restitution (FDR + NSC/SDR) model during meiosis. These results will be of substantial benefit in further selection of T. arundinaceum chromosome for sugarcane genetic improvement.