The OVATE family proteins (OFPs) are plant-specific proteins that modulate diverse aspects of plant growth and development.  In tomato, OFP20 has been shown to interact with TONNEAU1 Recruiting Motif (TRM) proteins to regulate fruit shape.  In this study, we demonstrated that the mutation of StOFP20 caused a shift from round to oval shaped tubers in a diploid accession C151, supporting the role of StOFP20 in controlling tuber shape.  Its expression reached a maximum in the tuber initiation stage and then decreased as the tuber develops.  To help elucidate the mechanism of tuber shape regulation by StOFP20, 27 TONNEAU1 Recruiting Motif (TRM) proteins were identified and 23 of them were successfully amplified in C151.  A yeast two-hybrid assay identified three TRM proteins that interacted with StOFP20, which was confirmed by firefly luciferase complementation in tobacco leaves.  The OVATE domain was indispensable for the interactions, while the necessity of the M10 motif in TRM proteins varied among the interactions between StOFP20 and the three TRMs.  In summary, both StOFP20 and SlOFP20 directed interactions with TRM proteins, but the corresponding interactants were not completely consistent, implying that they exert regulatory roles through mechanisms that are only partially overlapping.  

Monoclonal antibodies (mAbs) are widely used in virus research and disease diagnosis.  The nucleoprotein (NP) of influenza A virus (IAV) plays important roles in multiple stages of the virus life cycle.  Therefore, generating conserved mAbs against NP and characterizing their properties will provide useful tools for IAV research.  In this study, two mAbs against the NP protein, 10E9 and 3F3, were generated with recombinant truncated NP proteins (NP-1 and NP-2) as immunogens.  The heavy-chain subclass of both 10E9 and 3F3 was determined to be IgG2α, and the light-chain type was κ.  Truncation and site-specific mutation analyses showed that the epitopes of mAbs 10E9 and 3F3 were located in the N terminal 84–89 amino acids and the C terminal 320–324 amino acids of the NP protein, respectively.  We found that mAbs 10E9 and 3F3 reacted well with the NP protein of H1–H15 subtypes of IAV.  Both 10E9 and 3F3 can be used in immunoprecipitation assay, and 10E9 was also successfully applied in confocal microscopy.  Furthermore, we found that the 10E9-recognized ₈₄SAGKDP₈₉ epitope and 3F3-recognized ₃₂₀ENPAH₃₂₄ epitope were highly conserved in NP among all avian and human IAVs.  Thus, the two mAbs we developed could be used as powerful tools in the development of diagnostic methods of IAV, and also surely promote the basic research in understanding the replication mechanisms of IAV.

Nitrate reductase (NR) is a key enzyme for nitrogen assimilation in plants, and its activity is regulated by posttranslational phosphorylation.  To investigate the effects of dephosphorylation of the NIA1 protein on the growth and the physiological and biochemical characteristics of rice under different forms of nitrogen supplies, the phenotypes, nitrogen metabolism and reactive oxygen metabolism were measured in NIA1 phosphorylation site-directed mutant lines (S532D and S532A), an OsNia1 over-expression line (OE) and Kitaake (wild type, WT).  Compared with WT and OE, S532D and S532A have stronger nitrogen assimilation capacities.  When ammonium nitrate served as the nitrogen source, the plant heights, dry weights of shoots and chlorophyll (Chl) contents of S532D and S532A were lower than those of the WT and OE, whereas hydrogen peroxide (H₂O₂), malondialdehyde (MDA) and nitrite contents were higher.  When potassium nitrate served as the nitrogen source, the plant heights, dry weights of shoots and Chl contents of S532D and S532A were higher than those of the WT and OE, there were no significant differences in the contents of H₂O₂ and MDA in the leaves of the test materials, and the difference in nitrite contents among different lines decreased.  When ammonium sulfate served as the nitrogen source, there were no significant differences in the physiological indexes of the test materials, except NR activity.  Compared with ammonium nitrate and ammonium sulfate, the content of NH₄⁺-N in the leaves of each plant was lower when potassium nitrate was used as the nitrogen source.  The qPCR results showed that OsGS and OsNGS1 were negatively regulated by downstream metabolites, and OsNrt2.2 was induced by nitrate.  In summary, when ammonium nitrate served as the nitrogen source, the weak growth of NIA1 phosphorylation site-directed mutant lines was due to the toxicity caused by the excessive accumulation of nitrite.  When potassium nitrate served as the nitrogen source, the assimilation rates of nitrate, nitrite and ammonium salt were accelerated in NIA1 phosphorylation site-directed mutant lines, which could provide more nitrogen nutrition and improve the tolerance of rice to ammonium nitrogen deficiency.  These results could provide a possible method to improve the efficiency of nitrogen utilization in rice under low-nitrogen conditions.  

In China, the traditional early and late season double rice (DR) system is declining accompanied by the fast increase of two newly developed cropping systems: ratoon rice (RR) and rice–crawfish (RC).  Three methodologies: economic analysis, emergy evaluation and life cycle assessment (LCA) were employed to evaluate the economics and sustainability of this paddy cropping system change.  Economic analysis indicated that the income and profit of the RC system were far larger than those of RR and DR.  The income to costs ratio of RR and RC increased by 25.5 and 122.7% compared with that of DR, respectively.  RC had the highest emergy input thanks to increasing irrigation water, electricity, juvenile crawfish and forage input while RR showed a lower total emergy and nonrenewable emergy input, such as irrigation water, electricity, fertilizers and pesticides than DR.  The environmental loading ratios decreased by 16.7–50.4% when cropping system changed from DR to RR or from DR to RC while the emergy sustainability indexes increased by 22.6–112.9%.  The life cycle assessment indicated lower potential environmental impacts of RR and RC, whose total environmental impact indexes were 35.0–61.0% lower than that of DR.  Grain yield of RR was comparable with that of DR in spite of less financial and emergy input of RR, but RC had a much lower grain yield (a 53.6% reduction compared to DR).  These results suggested that RR is a suitable cropping system to achieve the food security, economic and environmental goals.

Rice grain yield and quality declines are due to unsuitable temperatures from wide regions and various sowing dates.  This study aimed to evaluate the effects of temperature on rice yield and quality at different phenological periods and obtain suitable temperatures for phenological periods in the Yangtze River Basin, China.  This study conducted experiments on different sowing dates under different areas in the Yangtze River Basin to observe and compare the differences in rice growth, yield, and quality, controlling for regional varieties.  The results showed significant differences in rice growth, yield, and quality among sowing dates and areas, which were related to the average daily temperature during the vegetative period (VT) and the first 20 days of the grain-filling period (GT20).  In addition, there was a smaller variation in the average daily temperature in the reproductive period (RT) than in the two phenological periods.  Therefore, according to the VT and GT20 thresholds of different yields and qualities, the experimental results were divided into four scenarios (I, II, III, and IV) in this study.  In Scenario I, high head rice production (rice grain yield multiplied by head rice rate) and rice quality could be obtained.  The head rice production of Scenarios III and IV was lower than that of Scenario I, by 30.1 and 27.6%, respectively.  In Scenario II, the head rice production increased insignificantly while the chalky grain rate and chalkiness were 50.6 and 56.3% higher than those of Scenario I.  In conclusion, the Scenario I combination with VT ranges of 22.8–23.9°C and GT20 ranges of 24.2–27.0°C or the combination with VT ranges of 23.9–25.3°C and GT20 ranges of 24.2–24.9°C, which can be obtained by adjusting sowing date and selecting rice varieties with suitable growth periods, is recommended to achieve high levels of rice grain yield and quality in the Yangtze River Basin. 

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. 

H7 avian influenza viruses (AIVs) normally circulated among birds before.  From 1996 to 2012, human infections with H7 AIVs (H7N2, H7N3, and H7N7) were reported in Canada, Italy, Mexico, the Netherlands, the United Kingdom and the USA.  Until March 2013, human infections with H7N9 AIVs were reported in China.  Since then, H7N9 AIVs have continued to circulate in both humans and birds.  Therefore, the detection of antibodies against the H7 subtype of AIVs has become an important topic.  In this study, a competitive enzyme-linked immunosorbent assay (cELISA) method for the detection of antibody against H7 AIVs was established.  The optimal concentration of antigen coating was 5 μg mL^–1, serum dilution was 1/10, and enzyme-labeled antibody was 1/3 000.  To determine the cut-off value of cELISA, percent inhibition (PI) was determined by using receiver operating characteristic (ROC) curve analysis in 178 AIVs negative samples and 368 AIVs positive serum samples (n=546).  When PI was set at 40%, the specificity and sensitivity of cELISA were 99.4 and 98.9%, respectively.  This method could detect the antibodies against H7Nx (N1–N4, N7–N9) AIVs, and showed no reaction with AIVs of H1–H6 and H8–H15 subtypes or common avian viruses such as Newcastle disease virus (NDV), Infectious bronchitis virus (IBV) and Infectious bursal disease virus (IBDV), exhibiting good specificity.  This method showed a coincidence rate of 98.56% with hemagglutinin inhibition (HI) test.  And the repeatability experiment revealed that the coefficients of variation (CV) of intra- and inter-batch repetition were all less than 12%.  The data indicated that the cELISA antibody-detection method established in this study provided a simple and accurate technical support for the detection of a large number of antibody samples of H7-AIV.

Nitrate reductase (NR) is an important enzyme for nitrate assimilation in plants, and post-translational phosphorylation regulates NR activity.  To evaluate the impact of the dephosphorylation of nitrate reductase 1 (NIA1) protein on NR activity, nitrogen metabolism and plant growth, NIA1 phosphorylation site directed mutant lines (S532D and S532A) and an OsNia1 over-expression line (OE) were constructed, and the phenotype, NIA1 protein and its phosphorylation level, NR activity, nitrate metabolism and reactive oxygen metabolism of the transgenic lines were analysed.  Exogenous NIA1 protein was not phosphorylated in S532D and S532A mutant lines, and their NR activities, activity states of NR and assimilation efficiencies of NO3–-N were higher than those in Kitaake (WT) and OE.  The changes in these physiological and biochemical indexes in the OE line were less than in S532D and S532A compared to WT.  These results suggest that the removal of transcriptional level control had little effect on nitrogen metabolism, but the removal of post-translational modification had a profound effect on it.  With the removal of NIA1 phosphorylation and the improvement in the nitrate assimilation efficiency, the plant height and chlorophyll content of S532D and S532A decreased and the hydrogen peroxide and malondialdehyde contents of rice seedlings increased, which may be related to the excessive accumulation of nitrite as an intermediate metabolite.  These results indicated that the phosphorylation of NR may be a self-protection mechanism of rice.  The reduced phosphorylation level of nitrate reductase improved the assimilation of nitrate, and the increased phosphorylation level reduced the accumulation of nitrite and prevented the toxic effects of reactive oxygen species in rice. 

Inoculation of wheat (Triticum aestivum L.) leaves with wheat powdery mildew fungus (Blumeria graminis f. sp. tritici) induces the cell death in adventitious roots.  Reactive oxygen species (ROS) play a key role in respond to biotic stress in plants.  To study the involvement of ROS and the degree of cell death in the wheat roots following inoculation, ROS levels and microstructure of root cells were analyzed in two wheat cultivars that are susceptible (Huamai 8) and resistant
(Shenmai 8) to powdery mildew fungus.  At 18 d after powdery mildew fungus inoculation, only Huamai 8 displayed the leaf lesions, while root cell death occurred in both varieties.  Huamai 8 had a high level of ROS accumulation, which is associated with increased root cell degradation, while in Shenmai 8, there was little ROS accumulation correlating with slight root cell degradation.  The molecular study about the expression levels of ROS scavenging genes (MnSOD and CAT) in wheat roots showed that these genes expression decreased after the leaves of wheat was inoculated.  The difference between Huamai 8
and Shenmai 8 on subcellular localization of H₂O₂ and O₂^–· was corresponded with the different down-regulation of the genes encoding for superoxide dismutase and catalase in two wheat cultivars.  These results suggested that ROS were involved in the process by which powdery mildew fungus induced cell death in wheat roots.

   In 2013, a human influenza outbreak caused by a novel H7N9 virus occurred in China.  Recently, the H7N9 virus acquired multiple basic amino acids at its hemagglutinin (HA) cleavage site, leading to the emergence of a highly pathogenic virus.  The development of an effective diagnostic method is imperative for the prevention and control of highly pathogenic H7N9 influenza.  Here, we designed and synthesized three pairs of primers based on the nucleotide sequence at the HA cleavage site of the newly emerged highly pathogenic H7N9 influenza virus.  One of the primer pairs and the corresponding probe displayed a high level of amplification efficiency on which a real-time RT-PCR method was established.  Amplification using this method resulted in a fluorescent signal for only the highly pathogenic H7N9 virus, and not for any of the H1–H15 subtype reference strains, thus demonstrating high specificity.  The method detected as low as 39.1 copies of HA-positive plasmid and exhibited similar sensitivity to the virus isolation method using embryonated chicken eggs.  Importantly, the real-time RT-PCR method exhibited 100% consistency with the virus isolation method in the diagnosis of field samples.  Collectively, our data demonstrate that this real-time RT-PCR assay is a rapid, sensitive and specific method, and the application will greatly aid the surveillance, prevention, and control of highly pathogenic H7N9 influenza viruses.

    Plant height (PH) is one of the most important agronomic traits of rice, as it directly affects the lodging resistance and the high yield potential. Meanwhile, PH is often constrained by water supply over the entire growth period. In this study, a recombinant inbred line (RIL) derived from Xiaobaijingzi and Kongyu 131 strains grown under drought stress and with normal irrigation over 2 yr (2013 and 2014), respectively (regarded as four environments), was used to dissect the genetic basis of PH by developmental dynamics QTL analysis combined with QTL×environment interactions. QTLs with net effects excluding the accumulated effects were detected to explore the relationship between gene×gene interactions and gene×environment interactions in specific growth period. A total of 26 additive QTLs (A-QTLs) and 37 epistatic QTLs (E-QTLs) associated with PH were detected by unconditional and conditional mapping over seven growth periods. qPH-2-3, qPH-4-3, qPH-6-1, qPH-7-1, and qPH-12-5 could be detected by both unconditional and conditional analyses. qPH-4-3 and qPH-7-5 were detected in four stages (periods) to be sequentially expressed QTLs controlling PH continuous variation. QTLs with additive effects (A-QTLs) were mostly expressed in the period S3|S2 (the time interval from stages 2 to 3), and QTL×environment interactions performed actively in the first three stages (periods) which could be an important developmental period for rice to undergo external morphogenesis during drought stress. Several QTLs showed high adaptability for drought stress and many QTLs were closely related to the environments such as qPH-3-5, qPH-2-2 and qPH-6-1. 72.5% of the QTLs with a and aa effects detected by conditional analysis were under drought stress, and the PVE of QTLs detected by conditional analysis under drought stress were also much higher than that under normal irrigation. We infer that environments would influence the detection results and sequential expression of genes was highly influenced by environments as well. Many QTLs (qPH-1-2, qPH-3-5, qPH-4-1, qPH-2-3) coincident with previously identified drought resistance genes. The result of this study is helpful to elucidating the genetic mechanism and regulatory network underlying the development of PH in rice and providing references to marker assisted selection.

Segregation analysis of the mixed genetic model of major gene plus polygene was used to identify the major genes for cotton yield-related traits using six generations P1, P2, F1, B1, B2, and F2 generated from the cross of Baimian 1 × TM-1. In addition to boll size and seed index, the major genes for the other five traits were detected: one each for seed yield, lint percentage, boll number, lint index; and two for lint yield. Quantitative trait locus/loci (QTL) mapping was performed in the F2 and F2:3 populations of above cross through molecular marker technology, and a total of 50 QTL (26 suggestive and 24 significant) for yield-related traits were detected. Four common QTL were discovered: qLP-3b(F2)/qLP-3(F2:3) and qLP-19b (F2)/qLP-19(F2:3) for lint percentage, qBN-17(F2)/qBN-17(F2:3) for boll number, and qBS-26b(F2)/qBS-26(F2:3) for boll size. Especially, qLP- 3b(F2)/qLP-3(F2:3), not only had LOD scores >3 but also exceeded the permutation threshold (5.13 and 5.29, respectively), correspondingly explaining 23.47 and 29.55% of phenotypic variation. This QTL should be considered preferentially in marker assisted selection (MAS). Segregation analysis and QTL mapping could mutually complement and verify, which provides a theoretical basis for genetic improvement of cotton yield-related traits by using major genes (QTL).

Water used in agriculture consumes much energy, mainly due to pumping water for irrigation, but the water-energy nexus is always neglected in arid and semi-arid areas. Based on hydrological observation data, irrigation data and socioeconomic data over the past 50 yr, this study has derived a detailed estimate of greenhouse gas (GHG) emissions from agricultural water use in the Minqin Oasis. Results show that the decreasing water supply and increasing demand for agriculture has caused severe water deficits over the past 50 yr in this region. The groundwater energy use rate rose by 76% between 1961 and 2009 because of the serious decline in groundwater levels. An increase in pump lift by an average 1 m would cause GHG emission rates to rise by around 2%. Over the past 10 yr, the GHG emissions from groundwater accounted for 65-88% of the total emissions from agricultural water. GHG emissions for diverted water varied from 0.047 to 0.074 Mt CO2e as the water input increased. Long distance conveyance and high pump lifts need more electricity input than groundwater abstraction does. Government policies have had a favorable effect on total emissions by reducing water abstraction. But groundwater depletion, exacerbated by a growing population and an expansion in arable land, remains the principal energy-water nexus challenge in the region. In response to the increasing water-energy crisis, energy-saving irrigation technology, matching to cost efficiencies, and better coordination between different infrastructural agencies could be feasible ways of rendering the water and energy sectors more sustainable over the long term.

Basic vegetative period (BVP) is an important trait for determining flowering time and adaptation to variable environments. A short BVP barley mutant is about 30 d shorter than its wild type. Genetic analysis using 557 F2 individuals revealed that the short BVP is governed by a single recessive gene (BVP-1) and was further validated in 2 090 F3 individuals. The BVP-1 gene was first mapped to barley chromosome 1H using SSR markers. Comparative genomic analysis demonstrated that the chromosome region of BVP-1 is syntenic to rice chromosome 5 and Brachypodium chromosome 2. Barley ESTs/genes were identified after comparison with candidate genes in rice and Brachypodium; seven new gene-specific markers were developed and mapped in the mapping populations. The BVP-1 gene co-segregated with the Mot1 and Ftsh4 genes and was flanked by the gene-specific markers AK252360 (0.2 cM) and CA608558 (0.5 cM). Further analysis demonstrated that barley and wheat share the same short BVP gene controlling early flowering.

The joint analysis of the mixed genetic model of major gene and polygene was conducted to study the inheritance of cryotolerance in cotton during the overwintering period. H077 (G. hirsutum L., weak cryotolerance) and H113 (G. barbadence L., strong cryotolerance) were used as parents. Cryotolerance of six generation populations including P1, P2, F1, B1, B2, and F2, from each of the two reciprocal crosses H077×H113 and H113×H077 were all investigated. The results showed that cryotolerance in cotton during the overwintering period was accorded with two additive major genes and additivedominance polygene genetic model. For cross H077×H113, the heritabilities of major genes in B1, B2, and F2 were 83.62, 76.84, and 90.56%, respectively; and the heritability of polygene could only be detected in B2, which was 7.76%. For cross H113×H077, the heritabilities of major genes in B1, B2, and F2 were 67.42, 68.95, and 83.40%, respectively; and the heritability of polygene was only detected in F2, which was 6.51%. In addition, the whole heritability in F2 was always higher than that in B1 and B2 in each cross. Therefore, for the cryotolerance breeding of perennial cotton, the method of single cross recombination or single backcross should be adopted to transfer major genes, and the selection in F2 would be more efficient than that in other generations.

To develop a modified live vaccine (MLV) against porcine reproductive and respiratory syndrome virus (PRRSV), virulent CH-1a strain was attenuated by serial passages up to 130 passage (P130) in Marc-145 cells. The virulence and immune efficacy of the attenuated CH-1a were evaluated in pigs. The results showed that animals inoculated with P130 did not develop any clinical sign of the disease, but produced rapid and effective humoral immune responses against PRRSV challenge, indicating that attenuated CH-1a P130 is the candidate as the effective vaccine against PRRSV. To define the potential mutations in the attenuated CH-1a genome, we sequenced and analyzed the ORF5 gene of CH-1a strain of different passages (P39, P55, P65, P70, P85, P100, P115, P120, P125, and P130) and found that three mutations (C5Y, H38Q and L146Q) which may be related with the attenuation of CH-1a. In addition, we also found a unique restriction enzyme site (TspEI) in the ORF5 gene of attenuated CH-1a, which can be used as a genetic marker to distinguish original and attenuated CH-1a.

Synchronizing the nitrogen (N) supply of slow- and controlled-release N fertilizers (SCRNFs) with rice N demand is pivotal in substituting multiple urea applications by a single basal application of SCRNFs.  Traditional assessment of N supply characteristics focuses mainly on N release patterns, which are only applicable to coated SCRNFs and ignore N transformation mechanisms, thus raising the need for a more universal and reliable index.  Based on the ability of crop N status to detect N deficiency or excess, we hypothesized that employing leaf N balance index (NBI) as a measure of N status could provide new insights into assessing N supply characteristics of SCRNFs.  We conducted field experiments involving four individual SCRNFs-humic acid urea (HAU), sulfur-coated urea (SCU), urease inhibitor urea (UIU), and polymer-coated urea (PCU)- and their four combined forms, along with the high-yield urea split application as control (CK).  The results showed that NBI dynamics relative to CK could reflect the N supply potential of different SCRNFs while classifying them as short-, medium-, and long-acting fertilizers.  Combinations that incorporated the long-acting SCRNF (PCU) consistently outperformed others in yield (by 5.5%) and N use efficiency (by 42.8%) by providing a more consistent and efficient N supply throughout the rice growth cycle.  Grain yield showed negative correlation with the difference in NBI dynamics between SCRNFs and CK, indicating synchronizing N supply between one-time application of SCRNFs and conventional high-yield fertilization is the key for high yield.  Our findings identify the potential of N status diagnosed by leaf NBI to evaluate N supply characteristics of SCRNFs and emphasize the significant role of synchronized N supply for a one-time SCRNF application.