In mammals, microRNAs (miRNAs) play key roles in multiple biological processes by regulating the expression of target genes.  Studies have found that the levels of miR-370-5p expression differ significantly in the skins of sheep with different hair colors; however, its function remains unclear.  In this study, we investigated the roles of miR-370-5p in sheep melanocytes and found that the overexpression of miR-370-5p significantly inhibited cell proliferation (P<0.01), tyrosinase activity (P=0.001) and significantly reduced (P<0.001) melanin production.  Functional prediction revealed that the 3´-untranslated region (UTR) of MAP3K8 has a putative miR-370-5p binding site, and the interaction between these two molecules was confirmed using luciferase reporter assays.  In situ hybridization assays revealed that MAP3K8 is expressed in the cytoplasm of melanocytes.  The results of quantitative RT-PCR and Western blotting analyses revealed that overexpression of miR-370-5p in melanocytes significantly inhibits (P<0.01) MAP3K8 expression via direct targeting of its 3´ UTR.  Inhibition of MAP3K8 expression by siRNA-MAP3K8 transfection induced a significant inhibition (P<0.01) of melanocyte proliferation and significant reduction (P<0.001) in melanin production, which is consistent with our observations for miR-370-5p.  Target gene rescue experiments indicated that the expression of MAP3K8 in melanocytes co-transfected with miR-370-5p and MAP3K8-cDNA (containing sites for the targeted binding to miR-370-5p) was significantly rescued (P≤0.001), which subsequently promoted significant increases in cell proliferation (P<0.001) and melanin production (P<0.01).  Collectively, these findings indicate that miR-370-5p plays a functional role in inhibiting sheep melanocyte proliferation and melanogenesis by downregulating the expression of MAP3K8.  

Several viroids in the genus Pospiviroid can infect tomato (Solanum lycopersicum) and cause severe diseases, posing a serious threat to tomato production.  For simultaneous detection of six tomato-infecting pospiviroids - columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), potato spindle tuber viroid (PSTVd), tomato apical stunt viroid (TASVd), tomato chlorotic dwarf viroid (TCDVd), and tomato planta macho viroid (TPMVd), we developed a universal probe based on a highly conserved 61 nt long sequence shared among them.  Compared with their specific probes, the universal probe has a similar, though slightly reduced, detection sensitivity and has the advantages of simple and cost-effective preparation and simultaneous detection of the six pospiviroids.  In addition, the universal probe was used in dot-blot hybridization assays for a large-scale survey of viroid(s) in tomato plantings in China.  Only PSTVd was detected in a few greenhouse-planted tomato plants.  Sequence analysis revealed that these tomato PSTVd isolates may have been introduced from tomato seeds imported from abroad. 

The MADS-box (DAM) gene PpDAM6, which is related to dormancy, plays a key role in bud endodormancy release, and the expression of PpDAM6 decreases during endodormancy release.  However, the interaction network that governs its regulation of the endodormancy release of flower buds in peach remains unclear.  In this study, we used yeast two-hybrid (Y2H) assays to identify a mitogen-activated protein kinase, PpMAPK6, that interacts with PpDAM6 in a peach dormancy-associated SSHcDNA library.  PpMAPK6 is primarily located in the nucleus, and Y2H and bimolecular fluorescence complementation (BiFC) assays verified that PpMAPK6 interacts with PpDAM6 by binding to the MADS-box domain of PpDAM6.  Quantitative real-time PCR (qRT-PCR) analysis showed that the expression of PpMAPK6 was opposite that of PpDAM6 in the endodormancy release of three cultivars with different chilling requirements (Prunus persica ‘Chunjie’, Prunus persica var. nectarina ‘Zhongyou 5’, Prunus persica ‘Qingzhou peach’).  In addition, abscisic acid (ABA) inhibited the expression of PpMAPK6 and promoted the expression of PpDAM6 in flower buds.  The results indicated that PpMAPK6 might phosphorylate PpDAM6 to accelerate its degradation by interacting with PpDAM6.  The expression of PpMAPK6 increased with decreasing ABA content during endodormancy release in peach flower buds, which in turn decreased the expression of PpDAM6 and promoted endodormancy release.

High-moisture extrusion technology should be considered one of the best choices for producing plant-based meat substitutes with the rich fibrous structure offered by real animal meat products.  Unfortunately, the extrusion process has been seen as a “black box” with limited information about what occurs inside, causing serious obstacles in developing meat substitutes.  This study designed a high-moisture extrusion process and developed 10 new plant-based meat substitutes comparable to the fibrous structure of real animal meat.  The study used the Feature-Augmented Principal Component Analysis (FA-PCA) method to visualize and understand the whole extrusion process in three ways systematically and accurately.  It established six sets of mathematical models of the high-moisture extrusion process based on 8 000 pieces of data, including five types of parameters.  The FA-PCA method improved the R² values significantly compared with the PCA method.  The Way 3 was the best to predict product quality (Z), demonstrating that the gradually molecular conformational changes (Yⁿ´) were critical in controlling the final quality of the plant-based meat substitutes.  Moreover, the first visualization platform software for the high-moisture extrusion process has been established to clearly show the “black box” by combining the virtual simulation technology.  Through the software, some practice work such as equipment installation, parameter adjustment, equipment disassembly, and data prediction can be easily achieved.

Salmonella Enteritidis (SE) is a zoonotic and vertically transmitted pathogen, often colonized in the reproductive tract of adult poultry, which can result in direct contamination of eggs and threaten human health.  Previous studies have revealed that some pattern recognition receptors and resistance genes were involved in regulating immune responses to SE invasion in birds.  However, the role of these immune response genes was not independent, and the interactions among the genes remained to be further investigated.  In this study, SE burden and colonization were determined in reproductive tissue after the ducks were SE-infected, and RNA-sequencing was performed to construct co-expression networks by weighted gene co-expression network analysis (WGCNA).  The result showed that SE could be isolated from 22% of infected-birds in any segment of the reproductive tract and the SE was readily colonized in the stroma, small follicle, isthmus, and vagina of the reproductive tracts in morbid ducks.  The top central, highly connected genes were subsequently identified three specific modules in the above four tissues at the defined cut-offs (P<0.01), including 60 new candidate regulators and 125 transcription factors.  Moreover, those 185 differentially expressed genes (DEGs) in these modules were co-expressed.  Moreover, the hub genes (TRAF3, CXCR4 and IL13RA1) were identified to act with many other genes through immune response pathways including NF-kappaB, Toll-like receptor, steroid biosynthesis, and p53 signaling pathways.  These data provide references that will understand the immune regulatory relationships during SE infection, but also assist in the breeding of SE-resistant lines through potential biomarkers.

The sugar beet cyst nematode, Heterodera schachtii, is a major parasite of sugar beet which has been recognized and listed as a quarantine nematode in China and more than 20 countries and regions worldwide.  A survey for important nematodes was undertaken in the sugar beet planting area of China during 2015–2018, and numerous cysts were collected from sugar beet fields in Xinyuan County, Xinjiang Uygur Autonomous Region of China.  The observations of morphological and morphometric characteristics revealed that cysts, vulval cones and second-stage juveniles of the Xinjiang population were in the same range of each other and within those of other reported H. schachtii populations.  Molecular analysis of rDNA-ITS, 28S-D2/D3 and mtDNA cytochrome c oxidase subunit 1 (COI) gene sequences suggested that the Xinjiang population clustered in a branch with those foreign populations, and the sequence similarity was as high as 99.81–100%.  Moreover, this result was confirmed by PCR assay with species-specific primer SHF6 and rDNA2 of H. schachtii, and the pathogenicity test confirmed successful Xinjiang population reproduction in both plant hosts.  In conclusion, based on morphological and molecular characterization, this study confirmed that the cyst nematode population collected from sugar beet fields in Xinjiang is H. schachtii.  As far as we know, this is the first report of H. schachtii on sugar beets in Xinjiang, China.

MicroRNA (miRNA) has vital regulatory effects on the proliferation, differentiation and secretion of ovarian granulosa cells, but the role of miR-99a-5p in goat ovarian granulosa cells (GCs) is unclear.  Both miR-99a-5p and Frizzled-5 (FZD5) were found to be expressed in GCs in goat ovaries via fluorescence in situ hybridization and immunohistochemistry, respectively, and FZD5 was verified (P<0.001) as a target gene of miR-99a-5p by double luciferase reporter gene experiments.  Furthermore, FZD5 mRNA and protein expression were both found to be regulated (P<0.05) by miR-99a-5p in GCs.  Moreover, the overexpression of miR-99a-5p or knockdown of FZD5 suppressed (P<0.05) estradiol and progesterone secretion from the GCs, as determined by ELISA.  In summary, miR-99a-5p inhibits target gene FZD5 expression and estradiol and progesterone synthesis in GCs.  Our study thus provides seminal data and new insights into the regulatory mechanisms of follicular development in the goat and other animals.

Ascorbate peroxidase (APX) plays a key role in scavenging reactive oxygen species (ROS) in higher plants.  However, there is very little information available on the APXs in kiwifruit (Actinidia), which is an economically and nutritionally important horticultural crop with exceptionally high ascorbic acid (AsA) accumulation.  This study aims to identify and characterize two cytosolic APX genes (AcAPX1 and AcAPX2) derived from A. chinensis ‘Hongyang’.  The constitutive expression pattern was determined for both AcAPX1 and AcAPX2, and showed relatively higher expression abundances of AcAPX1 in leaf and AcAPX2 in root.  Transcript levels of AcAPX1 and AcAPX2 were increased in kiwifruit roots treated with NaCl.  Subcellular localization assays using GFP-fusion proteins in Arabidopsis protoplasts showed that both AcAPX1 and AcAPX2 are targeted to the cytosol.  Recombinant AcAPX1 or AcAPX2 proteins were successfully expressed in the prokaryotic expression system and their individual ascorbate peroxidase activities were determined.  Finally, constitutive over-expression of AcAPX1 or AcAPX2 could dramatically increase total AsA, glutathione level and salinity tolerance under NaCl stress in Arabidopsis thaliana.  Our findings revealed that cytosolic AcAPX1/2 may play an important protective role in the responses to unfavorable environmental stimuli in kiwifruit.

Light heterogeneity leads to anatomically and physiologically heterogeneous features in leaves.  However, little attention has been paid to the effects of nonuniform illumination on the anatomical and photosynthetic performance on both sides along the leaf main vein.  This study explored such effects by combining in situ determination in the field with shading simulation in the phytotron, on pima cotton that has cupping leaves.  Photosynthetic characteristics and morphological structures were measured in the field on both sides along the main vein of eastward, westward, southward, and northward leaves.  The results showed that the difference in photosynthetic capacity between the two sides along the main vein in different directions was closely related to the daily photo irridiance (DPI).  This result indicates that the photosynthetic heterogeneity between the two sides is related to their intercepted light energy.  The conclusion was further verified by the shading simulation experiments.  Photosynthetic capacity and leaf thickness of the unshaded sides of leaves in the half-shaded treatment decreased, compared to those in the unshaded treatment.  Therefore, it is conjectured that the development of  photosynthetic characteristics on one side is systematically regulated by that on the other side.  The study provides theoretical guidance on accessing the feasibility of sampling and directional planting.   

Genes involved in chemosensation are essential for odorant-mediated insect behaviors.  Odorant receptors (ORs) bind and respond to pheromones and plant volatiles, regulating insect behaviors such as mating and host-plant selection, while ionotropic receptors (IRs), which are present at lower levels in insects than ORs, influence ion channels, especially in agricultural pests.  Asian corn borer, Ostrinia furnacalis, is the main pest of maize that causes huge economic losses in Asia.  Twenty-one OfurIRs have been identified, but none has been characterized.  In this study, tissue-specific expression profiling, phylogenetic analysis, and electroantennography (EAG) analysis were applied to characterize the evolution, expression, and the potential function of OfurIRs.  It was found that 20 OfurIRs were highly expressed in the antennae, except for OfurIR75p3, whereas 10 and nine OfurIRs were highly expressed in the proboscis and genitalia, respectively, indicating that these OfurIRs were functionally associated with feeding and oviposition.  EAG results showed that seven acids elicited responses in the antennae of O. furnacalis and that 2-oxopentanoic acid displayed a significant female-biased response.  Combined with the phylogenetic analysis, 10 OfurIRs in clade 4 were roughly predicted to be candidate receptors for 2-oxopentanoic acid and other tested acids.  These results provide basic information about OfurIRs and may help advance the knolwedge on the olfactory system of O. furnacalis. 

Crop straw return after harvest is considered an important way to achieve both agronomic and environmental benefits.  However, the appropriate amount of straw to substitute for fertilizer remains unclear.  A field experiment was performed from 2016 to 2018 to explore the effect of different amounts of straw to substitute for fertilizer on soil properties, soil organic carbon (SOC) storage, grain yield, yield components, nitrogen (N) use efficiency, phosphorus (P) use efficiency, N surplus, and P surplus after rice harvesting.  Relative to mineral fertilization alone, straw substitution at 5 t ha^–1 improved the number of spikelets per panicle, effective panicle, seed setting rate, 1 000-grain weight, and grain yield, and also increased the aboveground N and P uptake in rice.  Straw substitution exceeding 2.5 t ha^–1 increased the soil available N, P, and K concentrations as compared with mineral fertilization, and different amounts of straw substitution improved SOC storage compared with mineral fertilization.  Furthermore, straw substitution at 5 t ha^–1 decreased the N surplus and P surplus by up to 68.3 and 28.9%, respectively, compared to mineral fertilization.  Rice aboveground N and P uptake and soil properties together contributed 19.3% to the variation in rice grain yield and yield components.  Straw substitution at 5 t ha^–1, an optimal fertilization regime, improved soil properties, SOC storage, grain yield, yield components, N use efficiency (NUE), and P use efficiency (PUE) while simultaneously decreasing the risk of environmental contamination.

The rice cultivars carrying dep1 (dense and erect panicle 1) have the potential to achieve both high grain yield and high nitrogen use efficiency (NUE).  However, few studies have focused on the agronomic and physiological performance of those cultivars associated with high yield and high NUE under field conditions.  Therefore, we evaluated the yield performance and NUE of two near-isogenic lines (NILs) carrying DEP1 (NIL-DEP1) and dep1-1 (NIL-dep1) genes under the Nanjing 6 background at 0 and 120 kg N ha^–1.  Grain yield and NUE for grain production (NUEg) were 25.5 and 21.9% higher in NIL-dep1 compared to NIL-DEP1 averaged across N treatments and planting years, respectively.  The yield advantage of NIL-dep1 over NIL-DEP1 was mainly due to larger sink size (i.e., higher total spikelet number), grain-filling percentage, total dry matter production, and harvest index.  N utilization rather than N uptake contributed to the high yield of NIL-dep1.  Significantly higher NUEg in NIL-dep1 was associated with higher N and dry matter translocation efficiency, lower leaf and stem N concentration at maturity, and higher glutamine synthetase (GS) activity in leaves.  In conclusion, dep1 improved grain yield and NUE by increasing N and dry matter transport due to higher leaf GS activity under field conditions during the grain-filling period.

As a critical food crop, sweetpotato (Ipomoea batatas (L.) Lam.) is widely planted all over the world, but it is deeply affected by Sweetpotato Virus Disease (SPVD).  The present study utilized short tandem target mimic (STTM) technology to effectively up-regulate the expression of laccase (IbLACs) by successfully inhibiting the expression of miR397.  The upstream genes in the lignin synthesis pathway were widely up-regulated by feedback regulation, including phenylalanine ammonialyase (PAL), 4-coumarate-CoAligase (4CL), hydroxycinnamoyl CoA:shikimatetransferase (HTC), caffeicacid O-methyltransferase (COMT), and cinnamyl alcohol dehydrogenase (CAD).  Meanwhile, the activities of PAL and LAC increased significantly, finally leading to increased lignin content.  Lignin deposition in the cell wall increased the physical defence ability of transgenic sweetpotato plants, reduced the accumulation of SPVD transmitted by Bemisia tabaci (Gennadius), and promoted healthy sweetpotato growth.  The results provide new insights for disease resistance breeding and green production of sweetpotato. 

Environmental conditions greatly affect the growth of rice. To investigate the geographic differences in yield formation of single-season high-yielding hybrid rice in southern China, experiments were conducted in 2017 and 2018 in the upper and middle–lower reaches of the Yangtze River with 10–30 main locally planted high-yielding hybrid cultivars used as materials. Compared with rice planted in the middle–lower reaches of the Yangtze River, rice planted in the upper reaches has a longer tillering duration, higher accumulated temperature (≥10°C) during tillering period, but lower accumulated temperature and solar radiation from initial booting to maturity. Yield traits comparison between the upper and the middle–lower reaches of Yangtze River showed that the former had 48.1% more panicles per unit area while the latter had 46.4% more grains per panicle; the rice yield in the former was positively correlated with the seed setting rate and the dry matter accumulation before heading, while the latter was positively correlated with grains per panicle and dry matter accumulation from booting to maturity. Comparison of the same variety Tianyouhuazhan planted in different regions showed there was a significant positive correlation between panicle number and the duration of and accumulated temperature during the tillering period (r=0.982**, r=0.993**, respectively), and between grains per panicle and accumulated solar radiation during booting period (r=0.952*). In the upper reaches of the Yangtze River, more than 90% of cultivars with an yield of greater than 11 t ha^–1 had an effective panicle number of 250–340 m^–2, and there was a significant negative correlation between seed setting rate and grains per panicle; therefore, the high-yielding rice production in these regions with a long effective tillering period (>40 d) should choose varieties with moderate grains per panicle, adopt crop managements such as good fertilizer and water measures during vegetative growth period to ensure a certain number of effective panicles, and to increase the dry matter accumulation before heading. While in regions with a short effective tillering period (<20 d) but good sunshine conditions during the reproductive growth period, such as the middle–lower reaches of the Yangtze River, high-yielding rice production should choose cultivars with large panicles, adopt good water and fertilizer managements during the reproductive growth period to ensure the formation of large panicles and the increase of dry matter accumulation after heading.

Somatic embryogenesis (SE) is one of the most important steps during regeneration of cotton, but the molecular mechanism of SE remains unclear.  SOMATIC EMBRYOGENSIS RECEPTOR KINASE (SERK) gene is known to function in SE.  A homolog GhSERK2 (accession number: JF430801) was cloned from Upland cotton and characterized for its functions in SE.  GhSERK2 expressed in different tissues and showed higher expression level in floral organs than vegetative ones with the highest levels in ovule and anther.  GhSERK2 expressed during SE with a high level at globular embryos stage.  Upon treatment with indole-3-butytic acid (IBA), the transcription level of GhSERK2 was induced and promoted SE subsequently.  A 2-day treatment of 2,4-dichlorophenoxyacetic acid (2,4-D) induced the expression of GhSERK2, but treatments of 2,4-D for longer periods sharply inhibited the GhSERK2 transcription level of embryogenic callus (EC).  The levels of hormones, including 3-indoleacetic acid (IAA), abscisic acid (ABA), and brassinosteroid (BR), were increased in the initial calli induced from the over-expression of GhSERK2 cotton.  Our results indicated that GhSERK2 expression was associated with induction of SE and closely related to hormone levels during tissue culture in Upland cotton, and the gene might play an important role in regeneration of cotton.