Flax (Linum usitatissimum L.) is a versatile crop and its seeds are a major source of unsaturated fatty acids.  Stearoyl-acyl carrier protein desaturase (SAD) is a dehydrogenase enzyme that plays a key role in oleic acid biosynthesis as well as responses to biotic and abiotic stresses.  However, the function of SAD orthologs from L. usitatissimum has not been assessed.  Here, we found that two LuSAD genes, LuSAD1 and LuSAD2, are present in the genome of L. usitatissimum cultivar ‘Longya 10’.  Heterogeneous expression of either LuSAD1 or LuSAD2 in Arabidopsis thaliana resulted in higher contents of total fatty acids and oleic acid in the seeds.  Interestingly, ectopic expression of LuSAD2 in A. thaliana caused altered plant architecture.  Similarly, the overexpression of either LuSAD1 or LuSAD2 in Brassica napus also resulted in increased contents of total fatty acids and oleic acid in the seeds.  Furthermore, we demonstrated that either LuSAD1 or LuSAD2 enhances seedling resistance to cold and drought stresses by improving antioxidant enzyme activity and nonenzymatic antioxidant levels, as well as reducing membrane damage.  These findings not only broaden our knowledge of the LuSAD functions in plants, but also offer promising targets for improving the quantity and quality of oil, and the abiotic stress tolerance of oil-producing crops, through molecular manipulation.

The milk fat globule membrane (MFGM) is a complex structure with numerous functions, and its composition is affected by many factors.  There have been few systematic investigations on goat MFGM proteome profiling during lactation.  Individual milk samples from 15 healthy dairy goats were obtained at six lactation time points for investigation of the MFGM proteome using both data-independent acquisition (DIA) and data-dependent acquisition (DDA) proteomics techniques combined with multivariate statistical analysis.  Using the DIA method, 890 variably abundant MFGM proteins were discovered throughout the lactation cycle.  From 1 to 240 d, butyrophilin subfamily 1 member A1, lipoprotein lipase, perilipin-2, and adipose triglyceride lipase were upregulated, while APOE, complement C3, clusterin, and IgG were downregulated.  Furthermore, from 1 to 90 d, annexin A1, annexin A2, and antithrombin-III were downregulated, then upregulated by d 240.  Albumin had a high degree of connectedness, indicating that it was a key protein, according to protein–protein interaction research.  Overall, our findings gave new insights into the biological features of MFGM protein in goat milk throughout lactation, which may aid in the creation of specialized MFGM products and infant formula.

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway play a pivotal role in innate immunity.  Among invertebrates, Domeless receptors serve as the key upstream regulators of this pathway.  In our study on Bactrocera dorsalis, we identified three cytokine receptors: BdDomeless1, BdDomeless2, and BdDomeless3.  Each receptor encompasses five fibronectin-type-III-like (FN III) extracellular domains and a transmembrane domain.  Furthermore, these receptors exhibit the increased responsiveness to diverse pathogenic challenges.  Notably, only BdDomeless3 is upregulated during symbiont-like viral infections.  Moreover, silencing BdDomeless3 enhanced the infectivity of Bactrocera dorsalis cripavirus (BdCV) and B. dorsalis picorna-like virus (BdPLV), underscoring BdDomeless3’s crucial role in antiviral defense of B. dorsalis.  Following the suppression of Domeless3 expression, six antimicrobial peptide genes displayed decreased expression, potentially correlating with the rise in viral infectivity.  To our knowledge, this is the first study identifying cytokine receptors associated with the JAK/STAT pathway in tephritid flies, shedding light on the immune mechanisms of B. dorsalis.

Conservation programs require rigorous evaluation to ensure the preservation of genetic diversity and viability of conservation populations.  In this study, we conducted a comparative analysis of two indigenous Chinese chicken breeds, Gushi and Xichuan black-bone, using whole-genome SNPs to understand their genetic diversity, track changes over time and population structure.  The breeds were divided into five conservation populations (GS1, 2010, ex-situ; GS2, 2019, ex-situ; GS3, 2019, in-situ; XB1, 2010, in-situ; and XB2, 2019, in-situ) based on conservation methods and generations.  The genetic diversity indices of three conservation populations of Gushi chicken showed consistent trends, with the GS3 population under in-situ strategy having the highest diversity and GS2 under ex-situ strategy having the lowest.  The degree of inbreeding of GS2 was higher than that of GS1 and GS3.  Conserved populations of Xichuan black-bone chicken showed no obvious changes in genetic diversity between XB1 and XB2.  In terms of population structure, the GS3 population were stratified relative to GS1 and GS2.  According to the conservation priority, GS3 had the highest contribution to the total gene and allelic diversity in GS breed, whereas the contribution of XB1 and XB2 were similar.   We also observed that the genetic diversity of GS2 was lower than GS3, which were from the same generation but under different conservation programs (in-situ and ex-situ).  While XB1 and XB2 had similar levels of genetic diversity.  Overall, our findings suggested that the conservation programs performed in ex-situ could slow down the occurrence of inbreeding events, but could not entirely prevent the loss of genetic diversity when the conserved population size was small, while in-situ conservation populations with large population size could maintain a relative high level of genetic diversity.

African horse sickness (AHS) is an acute and fatal vector-borne infectious disease of equids, caused by the African horse sickness virus (AHSV).  The World Organization for Animal Health (WOAH) has classified AHS as a notifiable animal disease, and AHS has also been classified as a Class I animal infectious disease in China.  AHS is mainly found in Africa, the Middle East and the Arabian Peninsula.  China is currently recognized by the WOAH as an AHS-free zone.  However, in 2020, there were outbreaks of AHS in 2 countries neighboring China, Thailand and Malaysia (Bunpapong et al. 2021), which increases the risk of the introduction of AHS into China.  Therefore, in order to prevent the occurrence of AHS in China and to further monitor the spread of the disease, the development of rapid, accurate and cost-effective diagnostic methods for the detection of AHSV is essential.  

AHSV is a segmented double-stranded RNA virus belonging to the genus Orbivirus in the family Reoviridae.  It is mainly transmitted by midges (Maurer et al. 2022), and is able to infect all members of the Equidae, including horses, mules, donkeys, and zebras.  AHSV infection causes severe morbidity and mortality (up to 90%) in horses, while mules, donkeys and zebras are less susceptible than horses to the disease (Barnard 1998).  

The AHSV genome contains 10 double-stranded RNA segments, encoding 7 structural proteins (VP1–7) and 4 non-structural proteins (NS1–4).  AHSV is a complex non-enveloped virus with an icosahedral capsid comprising 3 distinct concentric protein layers.  VP2 and VP5 are the components of the outer capsid of the virion.  VP2 is the major determinant of AHSV serotype, and 9 serotypes (AHSV-1 to AHSV-9) have been identified according to the VP2 antigenicity; VP3 and VP7 are the components of the major inner capsid of the virion; VP1, VP4 and VP6 constitute the minor inner capsid of AHSV.  

AHSV RNA segment 7 (vp7) is highly conserved among all AHSV serotypes and is the primary molecular diagnostic target of AHSV.  The VP7 protein encoded by this segment is the major antigen of AHSV and is commonly used as a serological diagnostic for AHSV.  Real-time RT-PCR targeting vp7 is capable of detecting all known types of AHSV and is recommended by the WOAH for the detection of this virus (Aguero et al. 2008; Guthrie et al. 2013; WOAH 2019).  

Recently, molecular diagnostics for infectious diseases have been developed based on clustered regularly interspaced short palindromic repeats-associated Cas endonucleases (CRISPR/Cas) systems combined with isothermal amplification techniques (Chen J S et al. 2018; Gootenberg et al. 2018; Myhrvold et al. 2018).  Some Cas proteins with non-specific endonuclease activity, such as Cas12a, activate auxiliary (non-specific) cleavage of nearby single-stranded non-target nucleic acids upon recognition of the target.  By modifying a single-stranded nucleic acid with a fluorophore quencher, which fluoresces upon cleavage of the Cas12a and crRNA complex, this activity can be used to detect the presence of specific cleavage.  The CRISPR/Cas12-based detection system has certain advantages over traditional nucleic acid diagnostic methods (qPCR), including rapidity, simplicity, low cost, and low equipment requirements.  In this study, we developed a sensitive detection method for AHSV using the CRISPR/Cas12a system combined with reverse transcription-recombinase-assisted amplification (RT-RAA) (CRISPR/Cas12a-RT-RAA), which specifically targets the vp7 RNA of AHSV.  

To generate a CRISPR/Cas12-based AHSV detection system, a Cas12a from the Lachnospiraceae bacterium, LbCas12a protein, was first expressed in an Escherichia coli system and purified with Strep-Tactin Sepharose resin (Appendix A).  A single-stranded DNA (ssDNA) reporter labeled with a fluorophore and a quencher at the 2 termini (5´-6-FAM-TTATT-BHQ-3´) was synthesized by Sangon Biotech (Shanghai, China).  Ten crRNAs were designed to target the conserved region of the vp7 sequence of all AHSV strains (Appendices B and C).  These crRNAs with a repeat sequence were prepared using in vitro transcription following a previously described method (Wang et al. 2023).  In order to screen for an optimal crRNA for the sensitive detection of AHSV, 10 crRNAs were individually tested using a 25 μL CRISPR/Cas12-based reaction volume containing 0.4 μmol L^–1 LbCas12a, 0.4 μmol L^–1 ssDNA reporter, 1.2 μmol L^–1 crRNA, 10⁹ copies μL^–1 vp7 plasmid DNA (pMD18-T-vp7, containing the entire vp7 sequence) and 2.5 μL NEBuffer 2.1 (10×).  The reaction was performed at 37°C for 50 min on a qPCR thermal cycler (Applied Biosystems QuantStusio 5 Real-Time PCR System, USA) with fluorescence measurements taken every 30 s.  Fluorescence detection suggested that crRNA10 showed the highest efficiency in this reaction system (Fig. 1-A).  Therefore, crRNA10 was identified as the best option for the AHSV CRISPR/Cas12a detection platform and was used in the subsequent experiments.

Recombinase-assisted amplification (RAA) is an isothermal amplification technique that has been widely used to detect microbial pathogens (Chen C et al. 2018; Wang et al. 2020; Xue et al. 2020).  Recent studies have increasingly integrated the RAA assay with the CRISPR-Cas system, which provides a second detection step for amplification products, increasing detection sensitivity and specificity, and enabling more convenient and intuitive determination of detection results (Li et al. 2023).  Six specific RAA primers specifically targeting vp7 were designed based on the flanking sequence of the crRNA10 region (Appendix D).  To screen for the optimal RAA primer pair for the sensitive detection of AHSV, a standard RAA reaction was performed with pMD18-T-vp7 (at a concentration of 10⁹ copies μL^–1) as a template, using the RAA Nucleic Acid Amplification Kit (Qitian, China) and following the manufacturer’s instructions.  Following RAA amplification at 37°C for 30 min, the products of the RAA amplification were used as substrates for the CRISPR/Cas12a system detection.  As shown in Fig. 1-B, the strongest fluorescence signals for the CRISPR/Cas12a-RAA assay were detected when the F3/R3 primer pair was used.  The results showed that the F3/R3 primer pair had the best amplification efficiency and was therefore selected for the establishment of the CRISPR/Cas12a-RAA detection platform and used for subsequent experiments.

To evaluate the sensitivity of the CRISPR/Cas12a-RAA detection platform, we prepared vp7 RNA in vitro using the HiScribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs, USA), using vp7 PCR products and with T7 promoter sequences as templates.  A total of 1 μL of vp7 RNA at different concentrations was used as a template for the RT-RAA reaction using RT-RAA Nucleic Acid Amplification Kit (Qitian, Wuxi, China) for 30 min, and then 1 μL of RAA amplification product was extracted and used as a substrate for the CRISPR/Cas12a detection system and reacted for 30 min.  The CRISPR/Cas12a-RT-RAA assay was developed in this way.  As shown in Fig. 1-C, the detection limit of the CRISPR/Cas12a-RT-RAA assay was 10 copies of the vp7 mRNA molecule per reaction.  However, the detection limit of the real-time RT-PCR assay established by Aguero in 2008 was 100 copies of the vp7 RNA molecule per reaction (Aguero et al. 2008) (Fig. 1-D).  In 2015, WOAH organized the AHS reference laboratory to conduct a comparison of trials to evaluate different conventional detection methods, and confirmed that the real-time RT-PCR established by Aguero in 2008 was one of the best detection methods for diagnosing AHSV (WOAH 2019).  Our results suggest that the CRISPR/Cas12a-RT-RAA assay has higher sensitivity compared to the real-time RT-PCR assay.

To test the specificity of the CRISPR/Cas12a-RT-RAA assay to AHSV, other equine viral and bacterial pathogens were tested, including equine infectious anemia virus (EIAV), equine influenza virus (EIV), equine arthritis virus (EAV), equine herpesvirus-1 (EHV-1), equine herpesvirus-4 (EHV-4), Streptococcus equi subspecies equi (S. equi), and Salmonella enterica subsp. enterica serovar Abortusequi (S. Abortusequi).  All of these pathogens were stored in our laboratory and RNA/DNA from these pathogens was prepared as previously described (Chen et al. 2022).  As shown in Fig. 1-E, no fluorescence was observed when these equine pathogens were tested using the CRISPR/Cas12a-RT-RAA assay, whereas significant fluorescence was observed when the vp7 mRNA was tested, indicating that this CRISPR/Cas12a-RT-RAA method is highly specific for the detection of AHSV.

Due to the lack of AHSV-positive samples, we evaluated the performance of the CRISPR/Cas12a-RT-RAA assay in clinical practice by adding vp7 mRNAs to mRNAs extracted from equine blood or tissue samples as positive controls, and equine blood or equine tissue mRNAs without vp7 mRNAs as negative controls.  A total of 20 equine mRNA samples, including 5 equine blood cell mRNA samples (S1–5), 5 equine blood cell mRNA samples with vp7 mRNA (S6–10), 5 equine tissue (heart, liver, spleen, lung and kidney) mRNA (S11–15), and 5 equine tissue mRNA with vp7 mRNA (S16–20), were prepared and assessed using the CRISPR/Cas12a-RT-RAA assay.  As shown in Fig. 1-F, strong fluorescence signals were detected in all equine mRNA samples with vp7 mRNA, but not in any equine mRNA samples without vp7 mRNA.  This result demonstrates that the CRISPR/Cas12a-RT-RAA assay is able to detect vp7 mRNA efficiently in samples collected under complex conditions and can be used as a back-up technology for the early field detection of AHSV.

In conclusion, we reported the development and validation of a CRISPR/Cas12a-combined RT-RAA-based detection assay for AHSV with high specificity, sensitivity and convenience.  This assay targets the vp7 mRNA, a highly conserved segment of the AHSV genome that has not been observed to cross-react with the nucleic acids of 7 common equine pathogens, including EIAV, EIV, EAV, EHV-1, EHV-4, S. equi, and S. Abortusequi.  Notably, this assay was 10 times more sensitive than real-time RT-PCR.  In addition, the signal generated by the assay would be directly visible to the naked eye under UV light without the need for special instrumentation.  Therefore, the CRISPR/Cas12a combination RT-RAA assay developed here has the potential to be used as an alternative to traditional real-time RT-PCR assays for the rapid diagnosis of AHSV infection.  We will continue to optimize and improve the assay and expect that it will allow the detection of AHSV in the field and improve the early warning and diagnosis of AHS.

In 2013, peste des petits ruminants (PPR) re-emerged in China and spread to the majority of provinces across the country. The disease was effectively controlled through a vaccination campaign employing live attenuated vaccines, although sporadic cases still occurred. However, limited information is currently available regarding the peste des petits ruminants virus (PPRV) endemic in China. Here, a PPRV strain (HLJ/13) was isolated from a field sample in China by using Vero cells expressing goat signalling lymphocyte activation molecule. Phylogenetic analysis indicated that HLJ/13 belonged to lineage Ⅳ. Subsequent intranasal and subcutaneous inoculation of goats with a dose of 2×10⁶ TCID₅₀ of HLJ/13 resulted in the development of typical clinical symptoms of PPR, including pyrexia, ocular and nasal discharges, stomatitis, and diarrhea. All infected goats succumbed to the disease by day 8. To gain further insight, viral loading, pathological examination and immunohistochemical analyses were conducted, elucidating the main targets of HLJ/13 as the respiratory system, digestive tract and lymphoid organs. Employing the goat infection model established above, the goat poxvirus-vectored PPR vaccine, which was previously developed and could be used as DIVA (differentiating infected from vaccinated animals) vaccine, provided complete protection against the challenge of HLJ/13. It is important to note that this study represents the first comprehensive report delineating the biology and pathogenicity characterization, and infection model of PPRV isolated in China.

Wheat is a staple cereal crop that is crucial for food security and human health.  Improving wheat quality has become an essential task for breeders to meet escalating market demand.  In this study, a set of wheat-Aegilops tauschii introgression lines was developed from a cross between the high-yielding wheat variety Jimai 22 and Ae. tauschii Y215.  A high-density genetic map containing 2,727 single nucleotide polymorphisms (SNPs) was constructed using a 55K SNP array to conduct quantitative trait loci (QTL) analysis for grain quality-related traits.  Eight QTL were identified for grain protein content (GPC), starch content, and wet gluten content in the two environments.  Among them, a major and environmentally stable QTL, qGPC4D, for GPC was identified, with favorable alleles contributed by Ae. tauschii Y215.  Subsequently, qGPC4D was narrowed down to a 9.88 Mb physical interval through further fine mapping utilizing the introgression lines.  Additionally, three linked SNP of qGPC4D were converted into high-throughput kompetitive allele-specific PCR (KASP) markers and validated in the introgression population.  These findings offer promising candidate genes, elite introgression lines, and KASP markers for wheat high-quality breeding. 

Oral immunization is an alternative or supplementary approach that can significantly improve dog vaccination coverage, especially for free-roaming dogs. Safe and effective oral rabies vaccines for dogs are still being sought. In our previous studies, we generated a genetically modified rabies virus (RABV) ERA strain, rERAG_333E, containing a mutation from arginine (Arg, R) to glutamic acid (Glu, E) at residue 333 of the G protein (G_333E). Our previous results demonstrated that rERAG_333E was safe for adult mice and dogs, and oral vaccination with rERAG_333E induced a strong and long-lasting protective immune response in dogs. Here, we further investigated the safety and immunogenicity of rERAG_333E in non-target species, including suckling mice, rhesus monkeys, foxes, raccoon dogs, piglets, goats, and sheep. Suckling mice studies demonstrated that the G_333E mutation significantly reduced the virulence of the ERA strain. All of the suckling mice aged 10 days and above survived and showed no apparent signs of disease after intracerebral inoculation with rERAG_333E. Animal studies demonstrated that rERAG_333E was safe in rhesus monkeys, foxes, raccoon dogs, piglets, goats, and sheep. None of those animals inoculated orally with 10 times the intended field dose of rERAG_333E showed abnormal clinical signs before and after the booster immunization with Rabvac 3, an inactivated rabies vaccine. Meanwhile, oral inoculation with rERAG_333E induced strong neutralizing antibody (NA) responses to RABV in rhesus monkeys, foxes, raccoon dogs, and piglets. These results demonstrated that rERAG_333E has the potential to serve as a safe oral rabies vaccine for dogs.

The milk fat globule membrane (MFGM) is a complex structure with numerous functions, and its composition is affected by a number of circumstances. There have been few systematic investigations on goat MFGM proteome profiling during lactation. Individual milk samples from 15 healthy dairy goats were obtained at six lactation time points for investigation of the MFGM proteome using both data-independent acquisition (DIA) and data-dependent acquisition (DDA) proteomics techniques combined with multivariate statistical analysis. Using the DIA method, 890 variably abundant MFGM proteins were discovered throughout the lactation cycle. From 1 to 240 d, butyrophilin subfamily 1 member A1, lipoprotein lipase, perilipin-2, and adipose triglyceride lipase were upregulated, while APOE, complement C3, clusterin, and IgG were downregulated. Furthermore, from 1 to 90 d, annexin A1, annexin A2, and antithrombin-III were downregulated, then upregulated by d 240. Albumin had a high degree of connectedness, indicating that it was a key protein, according to protein-protein interaction research. Overall, our findings gave new insights into the biological features of MFGM protein in goat milk throughout lactation, which may aid in the creation of specialized MFGM products and infant formula.

The host intestinal microbiota has emerged as the third element in the interactions between hosts and enteric viruses, and potentially affects the infection processes of enteric viruses. However, the interaction of porcine enteric coronavirus with intestinal microorganisms during infection remains unclear. In this study, we used 16S-rRNA-based Illumina NovaSeq high-throughput sequencing to identify the changes in the intestinal microbiota of piglets mediated by porcine epidemic diarrhea virus (PEDV) infection and the effects of the alterations in intestinal bacteria on PEDV infection and its molecular mechanisms. The intestinal microbiota of PEDV-infected piglets had significantly less diversity than the healthy group and different bacterial community characteristics. Among the altered intestinal bacteria, the relative abundance of Clostridium perfringens was significantly increased in the PEDV-infected group. A strain of C. perfringens type A, named DQ21, was successfully isolated from the intestines of healthy piglets. The metabolites of swine C. perfringens type A strain DQ21 significantly enhanced PEDV replication in porcine intestinal epithelial cell clone J2 (IPEC-J2) cells, and PEDV infection and pathogenicity in suckling piglets. Palmitic acid (PA) was identified as one of those metabolites with metabolomic technology, and significantly enhanced PEDV replication in IPEC-J2 cells and PEDV infection and pathogenicity in suckling piglets. PA also increased the neutralizing antibody titer in the immune sera of mice. Furthermore, PA mediated the palmitoylation of the PEDV S protein, which improved virion stability and membrane fusion, thereby enhancing viral infection. Overall, our study demonstrates a novel mechanism of PEDV infection, with implications for PEDV pathogenicity.

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.

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.

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.

Sustainable increase in maize yield is severely constrained by the continuing reduction in topsoil depth due to irrational farming practices and the effects of climate change. However, the mechanisms by which topsoil depth affects crop physiology and biochemistry remain unclear, particularly with respect to photosynthesis and carbon assimilation.  To investigate the effects of topsoil depth on maize photosynthetic processes, carbon assimilation, and yield in the field, we used a two-factor random block design with five topsoil depths of 10 cm (S1), 20 cm (S2), 30 cm (S3), 40 cm (S4), and 50 cm (S5) at two planting densities of 60,000 plants ha⁻¹ (conventional density, D1) and 90,000 plants ha⁻¹ (high density, D2).  Increasing topsoil depth significantly increased maize grain yield, with maximum increases of 61.7% in D1 and 72.1% in D2.  Increasing topsoil depth also increased chlorophyll content, maximum photochemical efficiency (F_v/F_m), actual photochemical efficiency (ΦPSII), and photosynthetic enzyme activities, including ribulose-1,5-bisphosphate carboxylase (Rubisco), phosphoenolpyruvate carboxylase (PEPC), and pyruvate orthophosphate dikinase (PPDK).  With the increases in those parameters, plants maintained the highest net photosynthetic rate (P_nmax) when reaching the light saturation point, with maximum increases of 68.0% in D1 and 75.7% in D2, thereby increasing dry matter production at physiological maturity.  The accumulation of ¹³C-photosynthates in maize stem, leaf, and grain increased with the increase in topsoil depth, indicating increases in carbon assimilation capacity, distribution efficiency, and photosynthetic capacity.  In summary, increasing topsoil depth is an important factor in ensuring high and stable maize yields, and the increase in yield is closely related to the physiological differences caused by changes in topsoil depth.

Long-term manure application has the potential to alleviate soil acidification, and increase carbon sequestration and nutrient availability, thus improving cropland fertility. However, the mechanisms behind greenhouse gas N₂O emissions from acidic soil mediated by long-term manure application remain poorly understood. Herein, we investigated N₂O emission and its linkage with gross N mineralization and nitrification rates, as well as nitrifying and denitrifying microbes in an acidic upland soil subjected to 36-year fertilization treatments, including an unfertilized control (CK), inorganic fertilizer (F), 2 x rate of inorganic fertilizer (2F), manure (M), and the combination of inorganic fertilizer and manure (FM) treatments. Compared to the CK treatment (1.34 μg N kg^–1 d^–1), fertilization strongly increased N₂O emissions by 34-fold on average, with more pronounced increases in the manure-amendment (10.6–169 μg N kg^–1 d^–1) than those in the inorganic fertilizer treatments (3.26–5.51 μg N kg^–1 d^–1). The manure amendment-stimulated N₂O emissions were highly associated with increased soil pH, mean weight diameter of soil aggregates, substrate availability (e.g., particulate organic carbon, NO₃⁻ and available phosphorus), gross N mineralization rates, denitrifier abundances and the (nirK+nirS)/nosZ ratio. These findings suggest that the increased N₂O emissions primarily resulted from alleviated acidification, increased substrate availability and improved soil structure, thus enhancing microbial N mineralization and favoring N₂O-producing denitrifiers over N₂O consumers. Moreover, AOB rather than AOA positively correlated with soil NO₃⁻ concentration and N₂O emissions, indicating that nitrification indirectly contributed to N₂O production by supplying NO₃⁻ for denitrification. Collectively, manure amendment potentially stimulates N₂O emissions, primarily resulting from alleviated soil acidification and increased substrate availability, thus enhancing N mineralization and denitrifier-mediated N₂O production. Our findings suggest that consideration should be given to the greenhouse gas budgets of agricultural ecosystems when applying manure for managing the pH and fertility of acidic soils.