Cold stress is an important factor that limits apple production.  In this study, we examined the tissue-cultured plantlets of apple rootstocks ‘M9T337’ and ‘60-160’, which are resistant and sensitive to cold stress, respectively.  The enriched pathways of differentially expressed genes (DEGs) and physiological changes in ‘M9T337’ and ‘60-160’ plantlets were clearly different after cold stress (1°C) treatment for 48 h, suggesting that they have differential responses to cold stress.  The differential expression of WRKY transcription factors in the two plantlets showed that MdWRKY40is and MdWRKY48 are potential regulators of cold tolerance.  When we overexpressed MdWRKY40is and MdWRKY48 in apple calli, the overexpression of MdWRKY48 had no significant effect on the callus, while MdWRKY40is overexpression promoted anthocyanin accumulation, increased callus cold tolerance, and promoted the expression of anthocyanin structural gene MdDFR and cold-signaling core gene MdCBF2.  Yeast one-hybrid screening and electrophoretic mobility shift assays showed that MdWRKY40is could only bind to the MdDFR promoter.  Yeast two-hybrid screening and bimolecular fluorescence complementation showed that MdWRKY40is interacts with the CBF2 inhibitor MdMYB15L through the leucine zipper (LZ).  When the LZ of MdWRMY40is was knocked out, MdWRKY40is overexpression in the callus did not affect MdCBF2 expression or callus cold tolerance, indicating that MdWRKY40is acts in the cold signaling pathway by interacting with MdMYB15L.  In summary, MdWRKY40is can directly bind to the MdDFR promoter in order to promote anthocyanin accumulation, and it can also interact with MdMYB15L to interfere with its inhibitory effect on MdCBF2, indirectly promoting MdCBF2 expression, and thereby improving cold tolerance.  These results provide a new perspective for the cold-resistance mechanism of apple rootstocks and a molecular basis for the screening of cold-resistant rootstocks.

Pakchoi (Brassica campestris L. ssp. chinensis) is an important leafy vegetable.  Various light spectra, especially red and blue light, play vital roles in the regulation of nitrate metabolism.  Information on the effects of red and blue light on nitrate metabolism at the transcriptome level in pakchoi is still limited, so this study used RNA sequencing technology to explore this molecular mechanism.  Through pairwise comparisons with white LED light, 3 939 and 5 534 differentially expressed genes (DEGs) were identified under red and blue light, respectively.  By Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses, these unigenes were found to be involved in nitrate assimilation, plant–pathogen interaction, biosynthesis of secondary metabolites, and phenylpropanoid biosynthesis.  The differential effects of light spectra on the nitrate concentration and metabolism-related enzyme activities were also confirmed at the physiological level.  Several signal transduction modules, including Crys/Phys-COP1-HY5/HY5-like, were found to be involved in red and blue light-induced nitrate metabolism, and the transcript levels for this complex were consistent with the observed degree of nitrate assimilation.  The expression patterns of 15 randomly selected DEGs were further validated using qPCR.  Taken together, the results of this study could help improve our understanding of light spectrum-regulated nitrate metabolism in pakchoi at the transcriptome level.

Understanding of how combinations of agronomic options can be used to improve the grain yield and nitrogen use efficiency (NUE) of winter wheat is limited.  A three-year experiment involving four integrated management strategies was conducted from 2013 to 2015 in Tai’an, Shandong Province, China, to evaluate changes in grain yield and NUE.  The integrated management treatments were as follows: current practice (T1); improvement of current practice (T2); high-yield management (T3), which aimed to maximize grain yield regardless of the cost of resource inputs; and integrated soil and crop system management (T4) with a higher seeding rate, delayed sowing date, and optimized nutrient management.  Seeding rates increased by 75 seeds m^–2 with each treatment from T1 (225 seeds m^–2) to T4 (450 seeds m^–2).  The sowing dates were delayed from T1 (5th Oct.) to T2 and T3 (8th Oct.), and to T4 treatment (12th Oct.).  T1, T2, T3, and T4 received 315, 210, 315, and 240 kg N ha^–1, 120, 90, 210 and 120 kg P₂O₅ ha^–1, 30, 75, 90, and 45 kg K₂O ha^–1, respectively.  The ratio of basal application to topdressing for T1, T2, T3, and T4 was 6:4, 5:5, 4:6, and 4:6, respectively, with the N topdressing applied at regreening for T1 and at jointing stage for T2, T3, and T4.  The P fertilizers in all treatments were applied as basal fertilizer.  The K fertilizer for T1 and T2 was applied as basal fertilizer while the ratio of basal application to topdressing (at jointing stage) of K fertilizer for both T3 and T4 was 6:4.  T1, T2, T3, and T4 were irrigated five, four, four and three times, respectively.  Treatment T3 produced the highest grain yield among all treatments over three years and the average yield was 9 277.96 kg ha^–1.  Grain yield averaged across three years with the T4 treatment (8 892.93 kg ha^–1) was 95.85% of that with T3 and was 21.72 and 6.10% higher than that with T1 (7 305.95 kg ha^–1) and T2 (8 381.41 kg ha^–1), respectively.  Treatment T2 produced the highest NUE of all the integrated treatments.  The NUE with T4 was 95.36% of that with T2 and was 51.91 and 25.62% higher than that with T1 and T3, respectively.  The N uptake efficiency (UPE) averaged across three years with T4 was 50.75 and 16.62% higher than that with T1 and T3, respectively.  The N utilization efficiency (UTE) averaged across three years with T4 was 7.74% higher than that with T3.  The increased UPE with T4 compared with T3 could be attributed mostly to the lower available N in T4, while the increased UTE with T4 was mainly due to the highest N harvest index and low grain N concentration, which consequently led to improved NUE.  The net profit for T4 was the highest among four treatments and was 174.94, 22.27, and 28.10% higher than that for T1, T2, and T3, respectively.  Therefore, the T4 treatment should be a recommendable management strategy to obtain high grain yield, high NUE, and high economic benefits in the target region, although further improvements of NUE are required.

   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.

japonica rice is mainly distributed in Northeast China and accounts for 44.6% of the total cultivated area of japonica rice in China.  The comprehensive using of inter-subspecies heterosis is the main breeding mode of super japonica rice varieties in this region.  Improving rice quality at relative high yielding level is the current research focus.  Performing crosses between indica and japonica lines allows for the recombination of regulatory genes and genetic backgrounds, leading to complicated genetic rice quality characteristics, which can be used to explore patterns of quality improvement.  In the present study, we utilize recombinant inbred lines (RILs) derived from indica-japonica hybridization to analyze the effect factors of rice quality derived from genetic factors, which contain both regulatory genes concerning rice quality and genetic backgrounds’ random introduction frequency coming from indica (Di value), and the improvement strategy was further discussed.  The regulatory genes involved in amylase content (Wx) and nitrogen utilization efficiency (NRT1.1B) were the major factors affecting the amylose content (AC) and protein content (PC) in RILs, respectively.  Both the Di value and the major grain width gene (GS5) had regulatory effects on milled rice width (MRW) in RILs, and their interaction explained the major variance of MRW in the RILs.  With the mediation of MRW and chalkiness degree (C), Di value had a further impact on head rice rate (HR), which was relatively poor when the Di value was over 40%.  In Northeast China, the Di value should be lowered by backcrossing or multiple crosses during the breeding of indica-japonica hybridization to maintain the whole better HR and further to emphasize the use of favorable genes in individual selection.

The quality or structure of a wheat population is significantly affected by the compositions of tillers. Little has been known about the physiological basis for the differences of productive capacity among tillers. Two winter wheat cultivars, Shannong 15 (SN15) and Shannong 8355 (SN8355), were used to investigate the differences of productive capacity among tillers and analyze the physiological mechanisms that determine the superior tiller group. Low-position tillers (early initiated tillers) had a higher yield per spike than high-position tillers (late initiated tillers) in both cultivars, which was due to their more grain number per spike, more fertile spikelet per spike, less sterile spikelet per spike and higher grain weight. According to cluster analysis, tillers of SN15 were classified into 2 groups: superior tiller group including main stem (0), the first primary tiller (I) and the second primary tiller (II); and inferior tiller group including the third primary tiller (III) and the first secondary tiller (I-p). Tillers of SN8355 were classified into 3 groups: superior tiller group (0 and I), intermediate tiller group (II and III) and inferior tiller group (I-p). In comparison with other tiller groups, the superior tiller group had higher photosynthetic rate of flag leaves, higher antioxidant enzyme (SOD, POD and CAT) activities and lower levels of lipid peroxidation in leaves, higher grain filling rate in both superior and inferior grains during grain filling, higher single-stem biological yield and larger single-stem economic coefficient. Correlation analysis showed that yield per spike was positively and significantly correlated with the flag leaf photosynthetic rate, grain filling rate, the antioxidant enzyme activities and soluble protein content (except for SN15 at 5 days post-anthesis (DPA)) of flag leaf, the single-stem biological yield, and the single-stem economic coefficient. Remarkable negative correlation was also found between yield per spike and MDA content of flag leaf. These results suggested that superior tiller group had stronger leaf photosynthetic capacity, more predominance in terms of grain filling, slower senescence rate, higher biological yield and larger economic coefficient, and therefore, showed greater productive capacity than other tiller groups.

indica and japonica are the two most important subspecies of Asian cultivated rice. Identifying mechanisms responsible for population differentiation in these subspecies is important for indica-japonica hybridization breeding. In this study, subspecies and economic trait differentiation patterns were analyzed using morphological and molecular (InDel and Intron Length Polymorphism) data in F2 and F3 populations derived from indica-japonica hybridization. Populations were grown in Liaoning and Guangdong provinces, China, with F3 populations generated from F2 populations using bulk harvesting (BM) and single-seed descent methods (SSD). Segregation distortion was detected in F3-BM populations, but not in F3- SSD or in F2 populations. Superior performance was observed with respect to economic traits in Liaoning compared with that in Guangdong and 1 000-grain weight (KW), seed setting rate (SSR) and grain yield per plant (GYP) were significantly correlated with indica and japonica subspecies types. Analysis of molecular and morphological data demonstrated that the environment is the main factor giving rise to population differentiation in indica-japonica hybridization. In addition, we also found that KW, SSR and GYP are related to subspecies characteristics and kinship, which is possibly a significant factor resulting in economic trait differentiation and determining environmental adaptability. Our study has provided new insights into the process of population differentiation in these subspecies to inform indica-japonica hybridization breeding.

Wuschel-related homeobox (WOX) genes play essential, specific, and sometimes redundant roles in plant embryo development, shoot and root meristem maintenance, and plant development. Though much information was quickly gained with members of the WOX gene family of Arabidopsis, monocotyledonous crops, and gymnospermous conifers, little is known about perennial woody plants. In this study, we isolated the first WOX gene family member from grape (Vitis vinifera L. cv. Cabernet Sauvignon), and named it VvWOX4 based on its characteristic domains and phylogenetic analysis. The identity of VvWOX4 was validated by MALDI-TOF MS and Western blot with polyclonal antibody against Arabidopsis thaliana Wuschel. Functional analysis showed that VvWOX4 markedly increased shoot primordia structures when overexpressed under CaMV 35S promoter in tobacco. A different expression pattern was found for VvVOX4 compared with AtWUCHEL and its expression was detected in unique organs of grapevines. Besides the expression in the vegetative shoot apical meristem (SAM) of grape shoot tips, VvWOX4 is expressed in dormant winter buds, inflorescence, young leaves, and tendril tips, but not in root tips. In young leaves, the expression of VvWOX4 is strongly upregulated by wounding, and also by plant growth regulators such as 2 mg L-1 2,4-D, 1 mg L-1 NAA and 1 mg L-1 BAP treatments, while downregulation was monitored by 1 mg L-1 IBA treatment, and there was no response to 0.5 mg L-1 GA3 treatment. Together, our results revealed the first member of grape WOX gene family and indicated different roles and regulation of VvWOX4 in the perennial woody crop grapevine.