The yellow seed trait is preferred by breeders for its potential to improve the seed quality and commercial value of Brassica napus.  In the present study, we produced yellow seed mutants using a CRISPR/Cas9 system when the two BnPAP2 homologs were knocked out.  Histochemical staining of the seed coat demonstrated that proanthocyanidin accumulation was significantly reduced in the pap2 double mutants and decreased specifically in the endothelial and palisade layer cells of the seed coat.  Transcriptomic and metabolite profiling analysis suggested that disruption of the BnPAP2 genes could reduce the expression of structural and regulated genes in the phenylpropanoid and flavonoid biosynthetic pathways.  The broad suppression of these genes might hinder proanthocyanidin accumulation during seed development, and thereby causing the yellow seed trait in B. napus.  These results indicate that BnPAP2 might play a vital role in the regulatory network controlling proanthocyanidin accumulation.

Auxin response factors (ARFs) play key roles throughout the whole process of plant growth and development, and mediate auxin response gene transcription by directly binding with auxin response elements (AuxREs).  However, their functions in abiotic stresses are largely limited, especially in apples.  Here, the auxin response factor gene MdARF2 (HF41569) was cloned from apple cultivar ‘Royal Gala’ (Malus×domestica Borkh.).  Phylogenetic analysis showed that ARF2 proteins are highly conserved among different species and MdARF2 is the closest relative to PpARF2 of Prunus persica, but they differ at the DNA level.  MdARF2 contains three typical conserved domains including the B3 DNA-binding domain, Auxin_resp domain and AUX_IAA domain.  The subcellular localization demonstrated that MdARF2 is localized in the nucleus.  The three-dimensional structure prediction of the proteins showed that MdARF2 is highly similar with AtARF2, and they contain helices, folds, and random coils.  The promoter of MdARF2 contains cis-acting elements which respond to various stresses, as well as environmental and hormonal signals.  Expression analysis showed that MdARF2 is widely expressed in all tissues of apple, with the highest expression of MdARF2 in root.  Functional analysis with a series of MdARF2 transgenic apple calli indicated that MdARF2 can reduce the sensitivity to ABA signaling and enhance salt tolerance in apple.  In summary, the results of this research provide a new basis for studying the regulation of abiotic stresses by ARFs.

Recent studies have shown that the R2R3-MYB transcription factor MdMYB73 is involved in salt stress response in apple. However, no research was done whether MdMYB73 mediated cold tolerance in apple or not. In this study, we found that the expression of MdMYB73 was obviously induced by cold stress. Functional analysis showed that MdMYB73 significantly increased cold tolerance in transgenic apple calli and Arabidopsis. Quantitative real-time PCR (qRT-PCR) assay indicated that the expression levels of cold-responsive genes including MdCBF2, MdCBF3, MdCBF4, and MdCBF5 were obviously enhanced in MdMYB73 transgenic calli, suggesting that MdMYB73 increased cold tolerance via C-repeat binding factor (CBF) cold response pathway. Finally, we found that soluble sugar, which provides an osmoticum for cells, was increased in MdMYB73 transgenic calli compared to that in the wild type control. These findings provide a new insights into how MdMYB73 is involved in cold stress response.

    The community structure and diversity of root-associated bacteria have been tentatively investigated using polymerase chain reaction (PCR) amplification methods in several studies. However, the homology between small submit ribosomal (SSU) rRNA genes of plant plastids and mitochondria and that of bacteria have hindered in these studies. To address this issue, in this paper, we adopted the methods of locked nucleic acid (LNA) oligonucleotide-PCR clamping with 454 pyrosequencing to analysis the root-associated bacterial community compositions in soybean and corn. Results showed that plant chloroplast and mitochondria genes were effectively inhibited from PCR amplification in the root samples with LNA oligonucleotides (LNA (+)), and PCR amplicons with LNA (+) had higher bacterial operational taxonomic unit (OTU) numbers and ACE, Chao1, and Shannon indices, as well as a lower Simpson index than the corresponding samples without LNA oligonucleotides (LNA (–)). Those findings suggested that the methods of this study provide a much more detail description of root-associated bacterial communities. In the soybean LNA (+) sample, Pseudomonas, Bradyrhizobium and Flavobacterium were the three most abundant genera, whereas the top two predominant genera in corn LNA (+) samples were Streptomyces and Niastella. The presence and absence of major genera varied between soybean and corn, suggesting the root-associated bacterial communities differed between two crops. The rare phylotypes and uncultured root-associated bacterial members detected in this study inferred that the root-associated bacterial communities are highly complex and information on their taxonomic affiliates potentially gives the clues for selecting the optimal medium and method to isolate the novel bacteria for further functional analysis.

The lower seed setting is one of the major hindrances which face grain yield in rice. One of the main reasons to cause low spikelet fertility (seed setting) is male sterility or pollen abortion. Notably, pollen abortion has been frequently observed in advanced progenies of rice. In the present study, 149 BC₂F₆ individuals with significant segregation in spikelet fertility were generated from the cross between N040212 (indica) and Nipponbare (japonica) and used for primary gene mapping. Three QTLs, qSS-1, qSS-7 and qSS-9 at chromosomes 1, 7 and 9, respectively, were found to be associated with seed setting. The recombinant analysis and the physical mapping information from publicly available resources exhibited that the qSS-1, qSS-7 and qSS-9 loci were mapped to an interval of 188, 701 and 3 741 kb, respectively. The seed setting responsible for QTL qSS-1 was further fine mapped to 93.5 kb by using BC₂F₇ population of 1 849 individuals. There are 16 possible putative genes in this 93.5 kb region. Pollen vitality tests and artificial pollination indicated that the male gamete has abnormal pollen while the female gamete was normal. These data showed that low seed setting rate relative to qSS-1 may be caused by abnormal pollen grains. These results will be useful for cloning, functional analysis of the target gene governing spikelet fertility (seed setting) and understanding the genetic bases of pollen sterility.

MicroRNAs (miRNAs) are endogenous small RNAs that play important regulatory roles in the growth and development processes of plants and animals. In this study, we examined the expression profiles of pollen miRNAs from a maize S type cytoplasmic male sterile line and its fertility restored line. In total, 100 known miRNAs belonging to 20 families and 81 novel miRNAs belonging to 44 families were identified. Two and seven known miRNAs had significant expression difference between the two lines at the level of P-value<0.01 and 0.011.5 fold expression difference were verified by stem-loop RT-qPCR. Gene Ontology analysis of miRNA target genes revealed that these genes mainly participated in the transcriptional regulation processes.

Different amino acids have been shown to affect feed intake when injected directly into the central nervous system of birds. In the present study, we investigated the effects of L-glutamine and L-alanine on feed intake and the mRNA expression levels of hypothalamic neuropeptides involved in feed intake regulation in broiler chicks. L-Glutamine or Lalanine was intra-cerebroventricularly (ICV) administered to 4-d-old broiler chicks and the feed intake were recorded at various time points. Quantitative PCR was performed to determine the hypothalamic mRNA expression levels of neuropeptide Y (NPY), agouti related protein (AgRP), pro-opiomelanocortin (POMC), melanocortin receptor 4 (MC4R) and corticotropin releasing factor (CRF). Our results showed that ICV administration of L-glutamine (0.55 or 5.5 μmol) significantly increased feed intake up to 2 h post-administration period and the hypothalamic NPY mRNA expression levels, while it markedly decreased hypothalamic POMC and CRF mRNA expression levels. In contrast, ICV administration of L-alanine (4 μmol) significantly decreased feed intake for the first 0.5 h post-administration period, and reduced the hypothalamic AgRP mRNA expression levels, while it remarkablely enhanced the mRNA expression levels of MC4R and CRF. These findings suggested that L-glutamine and L-alanine could act within the hypothalamus to influence feed intake in broiler chicks, and that both orexigenic and anorexigenic neuropeptide genes might contribute directly to these effects.