Yushan Qiao

Genome-wide analysis of the AP2/ERF gene family in Populus trichocarpa.

Populus is a model system for investigating the wood development, crown formation, and disease resistance in perennial plants. AR2/ERF is a large family of transcription factors in plant, encoding transcriptional regulators with a variety of functions involved in the developmental and physiological processes. Here, starting from database of Populus genome, we identified 200 AP2/ERF genes by in silico cloning method using the AP2/ERF conserved domain amino acid sequence of Arabidopsis thaliana as probe. Based on the number of AP2/ERF domains and the function of the genes, those AP2/ERF genes from Populus were classified into four subfamilies named the AP2, DREB, ERF, RAV, and a soloist. Among these genes, the number genes of total AP2/ERF family genes, DREB subfamily, and ERF subfamily from Populus trichocarpa were about 1.4-1.6-fold than those from A. thaliana. The rates were very similar for the putative homologs between Populus and Arabidopsis.

Analysis of Brassica rapa ESTs: gene discovery and expression patterns of AP2/ERF family genes

Chinese cabbage (Brassica rapa subsp. pekinensis) is among the most important vegetables and is widely cultivated in world. Genes in the AP2/ERF family encode transcriptional regulators that serve a variety of functions in the plants. Expressed sequence tags (ESTs) are created by partially sequencing randomly isolated gene transcripts and have proved valuable in molecular biology. Starting from the database with 142 947 ESTs of B. rapa, 62 putative AP2/ERF family genes were identified by in silico cloning using the conserved AP2/ERF domain amino acid sequence of Arabidopsis thaliana as a probe. Based on the number of AP2/ERF domains and functions of the genes, the AP2/ERF transcription factors from B. rapa were classified into four subfamilies (DREB, ERF, AP2 and RAV). Using large-scale available EST information as a source of expression data for digital expression profiling, differentially detected genes were identified among diverse plant tissues. Roots contained the largest number of transcripts of the AP2/ERF family genes, followed by leaves and seeds. Only a few of the 62 AP2/ERF family genes were detected in all tissues: most were detected only in some tissues but not in others. The maximum detected was that of BraERF-B2-5, and it was recorded from seed tissue.

Direct Isolation of High-Quality Low Molecular Weight RNA of Pear Peel from the Extraction Mixture Containing Nucleic Acid

Low molecular weight RNA (LMW RNA) is generally obtained either from the total RNA or from total nucleic acids solution. Many steps and chemical reagents are involved in traditional methods for LMW RNA isolation where degradation of LMW RNA often occurs, especially for plant materials with high levels of secondary catabolites. In this study, an efficient method was developed to directly isolate pure LMW RNA from pear peel, a material rich in polyphenolics that is covered with a layer of wax. The method was based on polyethylene glycol (PEG) precipitation combining CTAB buffer which is often used to isolate RNA from polysaccharide-rich and polyphenolics-rich materials. The entire procedure could be completed within 6 h and many samples could be processed at the same time. Few and common chemicals are used with this method. Hence, it could be used as an ordinary method in the laboratory. The developed method was further tested by isolating LMW RNA from Arabidopsis. Using the isolated LMW RNA samples, microRNAs were successfully detected and characterized.

Transcription factors and anthocyanin genes related to low-temperature tolerance in rd29A:RdreB1BI transgenic strawberry.

Dehydration-responsive element-binding (DREB) transcription factors play critical roles in plant stress responses and signal transduction. To further understand how DREB regulates genes expression to promote cold-hardiness, Illumina/Solexa sequencing technology was used to compare the transcriptomes of non-transgenic and rd29A:RdreB1BI transgenic strawberry plants exposed to low temperatures. Approximately 3.5 million sequence tags were obtained from non-transgenic (NT) and transgenic (T) strawberry untreated (C) or low-temperature treated (LT) leaf samples. Over 1000 genes were differentially expressed between the NT-C and T-C plants, and also the NT-C and NT-LT, as well as the T-C and T-LT plants. Analysis of the genes up-regulated following low-temperature treatment revealed that the majority are linked to metabolism, biosynthesis, transcription and signal transduction. Uniquely up-regulated transcription factors as well as anthocyanin biosynthetic pathway genes are discussed. Accumulation of anthocyanin in the stolon and the base of the petiole differed between non-treated NT and T plants, and this correlated with gene expression patterns. The differentially expressed genes that encode transcription factors and anthocyanin enzymes may contribute to the cold hardiness of RdreB1BI transgenic strawberry. The transcriptome data provide a valuable resource for further studies of strawberry growth and development and DREB-mediated gene regulation under low-temperature stress.

Comparative proteomic analysis of rd29A:RdreB1BI transgenic and non-transgenic strawberries exposed to low temperature.

Low-temperature stress is one of the major abiotic stresses in plants worldwide, and the dehydration responsive element binding protein (DREB) transcription factor induces expression of genes involved in environmental stress tolerance in plants. A proteomic approach based on two-dimensional gel electrophoresis (2-DE) and subsequent mass spectrometric identification was used to study the changes in the leaf proteome profiles of rd29A:RdreB1BI transgenic and non-transgenic strawberries exposed to low-temperature conditions. By comparing the proteomic profiles, we located 21 protein spots that were reproducibly up- or down-regulated by more than twofold between transgenic and non-transgenic strawberries. Eight identified proteins function in energy and metabolism, four in biosynthetic processes, four were stress and defense related, three spots were identified as cold-stress related expressed sequence tags (ESTs), and two were unknown proteins. The change patterns of low-temperature tolerance proteins, including photosynthetic proteins (RuBisCO large subunit and RuBisCO activase), cytoplasmic Cu/Zn-superoxide dismutase (Cu/Zn-SOD), late embryogenesis abundant protein 14-A (Lea14-A), eukaryotic translation initiation factor 5A (eIF5A), and cold-stress related ESTs, were differentially regulated between non-transgenic and rd29A:RdreB1BI transgenic strawberries. They are likely important gene products in the regulatory network of the RdreB1BI gene. Consequently, this study provides the first characterization of the transgenic strawberry proteome and the predicted target proteins of the RdreB1BI gene by using proteomic approaches.

Variation in cytosine methylation in Clementine mandarin cultivars

Summary Clementines are an important group of citrus cultivars. However, little is known about their genetic diversity. In this study, methylation-sensitive amplification polymorphism (MSAP), based on the application of isoschizomers (Hpa II and Msp I), was used to analyse cytosine methylation patterns in 18 Clementine cultivars. Conventional AFLP analysis showed only two polymorphic bands out of a total of 1,822 AFLP bands generated using 28 primer pairs. Three types of bands were generated by MSAP using 27 pairs of primers. Type I were present in both Eco RI + Hpa II and Eco RI + Msp I gels. Type II or Type III were present only in Eco RI + Hpa II, or Eco RI + Msp I gels, respectively. The total numbers of these three Types of bands were 1,377, 98, and 93, respectively. Among these three Types of bands, the number of polymorphic bands were 76 (5.5%), 30 (30.6%), and 15 (16.1%), respectively. Of these, non-reproducible polymorphic bands in duplicated samples were 27 (35.5%), 18 (60.0%), and 9 (60.0%). Each Clementine cultivar had specific and relatively fixed methylated CCGG sites, which could be revealed from their specific MSAP patterns. The reproducible polymorphic bands in duplicated samples were used to analyse the extent of variation in methylation. The diversity of the cultivars revealed by the resulting dendrogram suggests that DNA methylation may be one of the main factors accounting for the difference in agronomic traits between Clementine cultivars.

Effect of ethylene and 1-MCP on post-harvest physiology and on expression of the ethylene receptor genes PpETR3 and PpERS2 in pear (Pyrus pyrifolia Nakai 'Kikusui') fruit.

Summary Exogenous ethylene and 1-MCP were investigated for their potential effects on fruit quality, post-harvest physiology, and expression of the ethylene receptor genes PpETR3 and PpERS2 in ‘Kikusui’ pear fruit. The results showed that exogenous ethylene treatment promoted a reduction in fruit firmness and soluble solids content (SSC), an increase in the activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) at a later stage of fruit storage, improved the rates of respiration and ethylene release, and up-regulated expression of the PpERS2 gene from 0 – 9 d after harvest, but did not affect expression of PpETR3. However, the effects of 1-MCP on fruit quality and post-harvest physiology were contrary to those of ethylene. 1-MCP up-regulated expression of the PpETR3 gene from 0 – 9 d and down-regulated expression of PpERS2 from 6 – 15 d after harvest. These data indicate that exogenous ethylene or 1-MCP could accelerate or inhibit pear fruit ripening during storage, respectively. At different stages of storage, ethylene and 1-MCP had different effects on the expression of PpETR3 and PpERS2. Moreover, the expression patterns of PpETR3 and PpERS2 during fruit ripening suggest that these two ethylene receptors have important functions during ethylene signal transduction.

Overexpression of the Malus hupehensis MhTGA2 Gene, a Novel bZIP Transcription Factor for Increased Tolerance to Salt and Osmotic Stress in Transgenic Tobacco

Basic leucine zipper (bZIP) proteins form one of the largest families of transcription factors, and their individual contributions in a particular regulatory network remain difficult to assess. Here, we identified and functionally characterized the Malus hupehensis bZIP transcription factor designated MhTGA2 from M. hupehensis leaves treated with salicylic acid (SA). The MhTGA2 protein was localized in the nucleus. The expression of MhTGA2 was higher in leaves than in stems and roots. This gene was significantly induced by SA, methyl jasmonate, and 1-aminocyclopropane-1-carboxylic acid and weakly induced by abscisic acid. The gene was clearly induced by low temperature (4°C) and NaCl and weakly induced by polyethylene glycol but not induced by apple ring spot pathogens. Overexpression of the MhTGA2 gene in transgenic tobacco plants conferred enhanced resistance to NaCl at the stage of seed germination and in seedlings, and it conferred resistance to mannitol at the stage of seed germination. Furthermore, overexpression of MhTGA2 led to higher expression levels of pathogenesis-related proteins and osmotic-stress-related genes in transgenic tobacco than in wild-type plants. These results suggest that the MhTGA2 transcription factor might be active in systemic acquired resistance and might function as a possible regulator of enhanced environmental-stress tolerance. MhTGA2 is thus a potentially constructive candidate gene for engineering salt and osmotic stress tolerance in cultivated plants.

Interval mapping for red/green skin color in Asian pears using a modified QTL-seq method

Pears with red skin are attractive to consumers and provide additional health benefits. Identification of the gene(s) responsible for skin coloration can benefit cultivar selection and breeding. The use of QTL-seq, a bulked segregant analysis method, can be problematic when heterozygous parents are involved. The present study modified the QTL-seq method by introducing a |Δ(SNP-index)| parameter to improve the accuracy of mapping the red skin trait in a group of highly heterozygous Asian pears. The analyses were based on mixed DNA pools composed of 28 red-skinned and 27 green-skinned pear lines derived from a cross between the ‘Mantianhong’ and ‘Hongxiangsu’ red-skinned cultivars. The ‘Dangshansuli’ cultivar genome was used as reference for sequence alignment. An average single-nucleotide polymorphism (SNP) index was calculated using a sliding window approach (200-kb windows, 20-kb increments). Nine scaffolds within the candidate QTL interval were in the fifth linkage group from 111.9 to 177.1 cM. There was a significant linkage between the insertions/deletions and simple sequence repeat markers designed from the candidate intervals and the red/green skin (R/G) locus, which was in a 582.5-kb candidate interval that contained 81 predicted protein-coding gene models and was composed of two subintervals at the bottom of the fifth chromosome. The ZFRI 130-16, In2130-12 and In2130-16 markers located near the R/G locus could potentially be used to identify the red skin trait in Asian pear populations. This study provides new insights into the genetics controlling the red skin phenotype in this fruit.

Dynamic Changes in Phenolics and Antioxidant Capacity during Pecan (Carya illinoinensis) Kernel Ripening and Its Phenolics Profiles

Pecan (Carya illinoinensis) kernels have a high phenolics content and a high antioxidant capacity compared to other nuts—traits that have attracted great interest of late. Changes in the total phenolic content (TPC), condensed tannins (CT), total flavonoid content (TFC), five individual phenolics, and antioxidant capacity of five pecan cultivars were investigated during the process of kernel ripening. Ultra-performance liquid chromatography coupled with quadruple time-of-flight mass (UPLC-Q/TOF-MS) was also used to analyze the phenolics profiles in mixed pecan kernels. TPC, CT, TFC, individual phenolics, and antioxidant capacity were changed in similar patterns, with values highest at the water or milk stages, lowest at milk or dough stages, and slightly varied at kernel stages. Forty phenolics were tentatively identified in pecan kernels, of which two were first reported in the genus Carya, six were first reported in Carya illinoinensis, and one was first reported in its kernel. The findings on these new phenolic compounds provide proof of the high antioxidant capacity of pecan kernels.