Junwei Zhang

Extraction of high-quality tissue-specific RNA from London plane trees (Platanus acerifolia), permitting the construction of a female inflorescence cDNA library

The London plane tree (Platanus acerifolia Willd.) has global importance as an urban landscaping tree and is the subject of genetic-improvement programs for productive sterility, disease and/or insect resistance. Molecular analysis techniques are crucial to such programs, but may be impeded by specific difficulties encountered during nucleic acid isolation. A detailed RNA isolation and purification protocol, based on established cetyltrimethyl-ammonium bromide (CTAB) extraction techniques combined with additional purification steps using butanol and the ionic detergent CTAB, which overcomes these problems in the woody species P. acerifolia, was conducted. In short, phenolic compounds are bound to soluble polyvinylpyrrolidone and then separated out through LiCl precipitation of the RNA. Subsequently, protein- and carbohydrate-contaminants are removed by chloroform partitioning followed by LiCl-mediated precipitation. The resulting isolates of RNA were found to be of sufficient quality for successful use in reverse transcription PCR analysis. Furthermore, RNA isolates from female inflorescences were used for the construction of a cDNA library. This library was found to contain several full-length cDNA clones of MADS-box genes, consistent with the library being representative of inflorescence expression profiles.

Phylogenetic and evolutionary analysis of A-, B-, C- and E-class MADS-box genes in the basal eudicot Platanus acerifolia

London plane tree (Platanus acerifolia Willd.) is an important member of the Platanaceae family, being popular as an urban landscaping tree. Here, we report the isolation of five MADS-box genes from the basal angiosperm, Platanus acerifolia. Sequence and phylogenetic analyses identified FRUITFUL-like, APETELA3-like, AGAMOUS-like, SEPALLATA1-like and SEPALLATA3-like sequences and, hence, we term the respective Platanus acerifolia genes as PlacFUL, PlacAP3, PlacAG, PlacSEP1 and PlacSEP3. From these identities we infer that they represent candidate A-, B-, C-class and two E-class genes, respectively. The conserved MIK or MIKC domains from the nucleotide and protein sequences of PlacFUL, PlacAP3, PlacAG, PlacSEP1 and PlacSEP3 were analyzed using the maximum-likelihood, MrBayes and neighbor-joining methods. The results confirmed P. acerifolia as a basal eudicot. Expression pattern was determined by reverse transcriptase PCR, which showed all paralogous genes have distinct expression patterns, suggesting that they had undergone functional divergence.

Identification and expression analysis of nuclear factor Y families in Prunus mume under different abiotic stresses

The nuclear factor Y (NF-Y) is one of the largest transcription factor families in plants consisting of NF-YA, NF-YB, and NF-YC subunits. It could play important roles in various processes such as flowering time, seed development, and response to drought. In this study, 6 NF-YA, 13 NF-YB, and 8 NF-YC proteins were identified and characterized in Prunus mume. Analyses of a conserved domain indicated that the PmNF-Y subunits shared an elevated degree of homology with the corresponding Arabidopsis NF-Y ones. Phylogenetic analysis showed that each NF-Y subunit family from Prunus mume and Arabidopsis could be divided into 4 or 2 clades based on their full-length proteins. The gene expression patterns of all 27 PmNF-Y genes were examined under abscisic acid (ABA), osmotic, salt, and H2O2 treatments using real-time quantitative PCR analyses. PmNF-YA1/2/4/5/6, PmNF-YB3/4/8/10/11/13, and PmNF-YC1/2/4/5/6/8 were found to be up-regulated under the ABA and osmotic treatments. PmNF-YA1/2/3/4/5/6, PmNF-YB1/3/8/10/11/13, and PmNF-YC1/2/5/6/8 were obviously induced by the H2O2. In addition, only PmNF-YA2 and PmNF-YB3 expressions were enhanced under the salt stress. These findings could provide an entry point to investigating the roles of PmNF-Y genes during abiotic stress responses.

Cloning and characterization of paleoAP3-like MADS-box gene in London plane tree

We isolated PaAP3, a homolog of the class B MADS-box transcription factor gene APETALA3 (AP3), from the monoecious plant London plane tree (Platanus acerifolia Willd.). PaAP3 encodes a protein that shares good levels of identity with class B genes from Arabidopsis thaliana (35 and 51 % identity with PISTILLATA (PI) and AP3, respectively), and also with class B genes of other woody species (59 % identity with PTD from Populus trichocarpa and 66 % with TraAP3 from Trochodendron aralioides). Reverse transcription polymerase chain reaction showed that PaAP3 was expressed in both the female and male flowers of P. acerifolia, but almost no signal was detected in the vegetative tissues or mature embryos. The PaAP3 expression in male flowers showed a relationship with developmental stage. There was a small transient increase during differentiation of the flower primordia in June, but maximal levels occurred during December when flower development appeared arrested. Increased PaAP3 expression was also detected in March of the following year, corresponding to meiotic divisions of the microspore mother cells, but this was lost by April when the pollen was mature.

Structural and expression analyses of three PmCBFs from Prunus mume

C-repeat binding factor (CBF), also called the dehydration-responsive element binding factor 1 (DREB1), can be induced by low-temperature (LT), and plays an important role in abiotic stress tolerance in higher plants. In present study, two new homologous genes of CBF from Prunus mume (PmCBFb and PmCBFc) have been identified and characterized. The complete coding sequences of PmCBFb and PmCBFc were 714 and 723 bp, respectively. They encoded putative proteins of 237 and 240 amino acids. Neither of them had introns. Genome PCR sequencing showed that PmCBFb was arranged in tandem with PmCBFa (another CBF/DREB1 homolog in P. mume) within a region of nearly 4 kb. Promoter prediction analyses indicated that multiple types of cis-elements related to abiotic stress and irradiance existed in the putative promoter region of PmCBFb. LT treatment of seedlings showed that the expression of PmCBF genes were induced by 2 °C within 30 min, and their expression reached a peak after 8–12 h. In addition, PmCBFa and PmCBFb appeared more sensitive to LT than PmCBFc. However, the exact roles of PmCBF genes in plant cold tolerance need to be further investigated.