Jiaqi 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.

Isolation and Functional Analyses of a Putative Floral Homeotic C-Function Gene in a Basal Eudicot London Plane Tree (Platanus acerifolia)

The identification of mutants in model plant species has led to the isolation of the floral homeotic function genes that play crucial roles in flower organ specification. However, floral homeotic C-function genes are rarely studied in basal eudicots. Here, we report the isolation and characterization of the AGAMOUS (AG) orthologous gene (PaAG) from a basal eudicot London plane tree (Platanus acerifolia Willd). Phylogenetic analysis showed that PaAG belongs to the C- clade AG group of genes. PaAG was found to be expressed predominantly in the later developmental stages of male and female inflorescences. Ectopic expression of PaAG-1 in tobacco (Nicotiana tabacum) resulted in morphological alterations of the outer two flower whorls, as well as some defects in vegetative growth. Scanning electron micrographs (SEMs) confirmed homeotic sepal-to-carpel transformation in the transgenic plants. Protein interaction assays in yeast cells indicated that PaAG could interact directly with PaAP3 (a B-class MADS-box protein in P. acerifolia), and also PaSEP1 and PaSEP3 (E-class MADS-box proteins in P. acerifolia). This study performed the functional analysis of AG orthologous genes outside core eudicots and monocots. Our findings demonstrate a conserved functional role of AG homolog in London plane tree, which also represent a contribution towards understanding the molecular mechanisms of flower development in this monoecious tree species.

Four SQUAMOSA PROMOTER BINDING PROTEIN-LIKE homologs from a basal eudicot tree (Platanus acerifolia) show diverse expression pattern and ability of inducing early flowering in Arabidopsis

Key messageFourSPL3/4/5orthologous genes fromPlatanus acerifoliawere identified, and their structure, expression patterns, role in controlling phase change and ability of inducing early flowering inArabidopsiswere analyzed.AbstractPlant flowering behavior is controlled by multiple pathways. The Arabidopsis thalianaSQUAMOSA PROMOTER BINDING PROTEIN-LIKE3/4/5 (AtSPL3/4/5) genes are involved in regulating vegetative phase change. Recent studies have shown that AtSPL3/4/5 genes are directly involved in inducing expression of three flowering integrator genes namely, LEAFY (LFY), FRUITFULL (FUL) and APETALA1 (AP1), thereby triggering flowering. This regulatory integration reveals a commonality of the flowering response with various other plant developmental pathways. We have isolated four SPL3/4/5 orthologous genes (PaSPL3a/b/c/d) from a basal eudicot London plane tree (Platanus acerifolia Wild), a woody species with a long juvenile phase. We investigated the role of these Platanus genes in flowering and detected expression levels between trees in vegetative and reproductive phase. Real-time PCR analysis demonstrated diverse expression patterns for the four PaSPL3 genes throughout the various P. acerifolia tissues, but all four were highly expressed in developing flower bud tissues. Expression of PaSPL3a/b/c in leaves changed according to the season and was regulated by temperature. Furthermore, three of the orthologous were found to induce an early flowering phenotype when overexpressed in Arabidopsis. The findings of this study indicate that PaSPL3 genes may participate in early stage inflorescence development in London Plane. However, there appears to be some divergence in the function of these genes in the control of phase change in London plane as compared to that within Arabidopsis.

Genome-wide identification and characterization of the SBP-box gene family in Petunia

BackgroundSQUAMOSA PROMOTER BINDING PROTEIN (SBP)-box genes encode a family of plant-specific transcription factors (TFs) that play important roles in many growth and development processes including phase transition, leaf initiation, shoot and inflorescence branching, fruit development and ripening etc. The SBP-box gene family has been identified and characterized in many species, but has not been well studied in Petunia, an important ornamental genus.ResultsWe identified 21 putative SPL genes of Petunia axillaris and P. inflata from the reference genome of P. axillaris N and P. inflata S6, respectively, which were supported by the transcriptome data. For further confirmation, all the 21 genes were also cloned from P. hybrida line W115 (Mitchel diploid). Phylogenetic analysis based on the highly conserved SBP domains arranged PhSPLs in eight groups, analogous to those from Arabidopsis and tomato. Furthermore, the Petunia SPL genes had similar exon-intron structure and the deduced proteins contained very similar conserved motifs within the same subgroup. Out of 21 PhSPL genes, fourteen were predicted to be potential targets of PhmiR156/157, and the putative miR156/157 response elements (MREs) were located in the coding region of group IV, V, VII and VIII genes, but in the 3’-UTR regions of group VI genes. SPL genes were also identified from another two wild Petunia species, P. integrifolia and P. exserta, based on their transcriptome databases to investigate the origin of PhSPLs. Phylogenetic analysis and multiple alignments of the coding sequences of PhSPLs and their orthologs from wild species indicated that PhSPLs were originated mainly from P. axillaris. qRT-PCR analysis demonstrated differential spatiotemperal expression patterns of PhSPL genes in petunia and many were expressed predominantly in the axillary buds and/or inflorescences. In addition, overexpression of PhSPL9a and PhSPL9b in Arabidopsis suggested that these genes play a conserved role in promoting the vegetative-to-reproductive phase transition.ConclusionPetunia genome contains at least 21 SPL genes, and most of the genes are expressed in different tissues. The PhSPL genes may play conserved and diverse roles in plant growth and development, including flowering regulation, leaf initiation, axillary bud and inflorescence development. This work provides a comprehensive understanding of the SBP-box gene family in Petunia and lays a significant foundation for future studies on the function and evolution of SPL genes in petunia.

Functional conservation and divergence of five SEPALLATA-like genes from a basal eudicot tree, Platanus acerifolia

Main conclusionFiveSEP-like genes were cloned and identified fromPlatanus acerifoliathrough the analysis of expression profiles, protein–protein interaction patterns, and transgenic phenotypes, which suggested that they play conservative and diverse functions in floral initiation and development, fruit development, bud growth, and dormancy.SEPALLATA (SEP) genes have been well characterized in core eudicots and some monocots, and they play important and diverse roles in plant development, including flower meristem initiation, floral organ identity, and fruit development and ripening. However, the knowledge on the function and evolution of SEP-like genes in basal eudicot species is very limited. Here, we cloned and identified five SEP-like genes from London plane (Platanus acerifolia), a basal eudicot tree that is widely used for landscaping in cities. Sequence alignment and phylogenetic analysis indicated that three genes (PlacSEP1.1, PlacSEP1.2, and PlacSEP1.3) belong to the SEP1/2/4 clade, while the other two genes (PlacSEP3.1 and PlacSEP3.2) are grouped into the SEP3 clade. Quantitative real-time PCR (qRT-PCR) analysis showed that all PlacSEPs, except PlacSEP1.1 and PlacSEP1.2, were expressed during the male and female inflorescence initiation, and throughout the flower and fruit development process. PlacSEP1.2 gene expression was only detected clearly in female inflorescence at April. PlacSEP1.3 and PlacSEP3.1 were also expressed, although relatively weak, in vegetative buds of adult trees. No evident PlacSEPs transcripts were detected in various organs of juvenile trees. Overexpression of PlacSEPs in Arabidopsis and tobacco plants resulted in different phenotypic alterations. 35S:PlacSEP1.1, 35S:PlacSEP1.3, and 35S:PlacSEP3.2 transgenic Arabidopsis plants showed evident early flowering, with less rosette leaves but more cauline leaves, while 35S:PlacSEP1.2 and PlacSEP3.1 transgenic plants showed no visible phenotypic changes. 35S:PlacSEP1.1 and 35S:PlacSEP3.2 transgenic Arabidopsis plants also produced smaller and curled leaves. Overexpression of PlacSEP1.1 and PlacSEP3.1 in tobacco resulted in the early flowering and producing more lateral branches. Yeast two-hybrid analysis indicated that PlacSEPs proteins can form homo- or hetero-dimers with the Platanus APETALA1 (AP1)/FRUITFULL (FUL), B-, C-, and D-class MADS-box proteins in different interacting patterns and intensities. Our results suggest that the five PlacSEP genes may play important and divergent roles during floral initiation and development, as well as fruit development, by collaborating with FUL, B-, C-, and D-class MADS-box genes in London plane; PlacSEP1.3 and PlacSEP3.1 genes might also involve in vegetative bud growth and dormancy. The results provide valuable data for us to understand the functional evolution of SEP-like genes in basal eudicot species.

Functional analysis of the promoters of B-class MADS-box genes in London plane tree and their application in genetic engineering of sterility

Breeding flowerless and/or fruitless varieties are highly desirable for London plane tree because it can prevent pollen- and fruit-mediated environmental contamination. Floral tissue-specific cell ablation is an efficient method to create such sterile plants. Here we isolated and characterized APETALA3 (AP3)-like and PISTILLATA (PI)-like genes and the promoters of PaAP3 and PaPI, in London plane tree respectively. The promoter fragments were fused to GUS (β-glucuronidase) and BARNASE gene, respectively, and transformed into tobacco plants. In pPaAP3::GUS transgenic plants, the GUS activity could be detected in various organs, including leaves, stems and all floral organs. Furthermore, most tobacco plants transformed with pPaAP3::BARNASE were dead and the survivals showed abortion of inflorescence. In contrast, heterologous expression of pPaPI::GUS in tobacco plants led to specific GUS activity in the inner three whorls of flowers. Accordingly, tobacco plants transformed with pPaPI::BARNASE lack petal, stamen and pistil, with only sepal left. The results suggest that sterile lines of P. acerifolia may be obtained by genetic engineering with pPaPI::BARNASE construct, which might solve the problems of shedding fruit hairs and disseminative pollens, reducing air pollution and reducing the allergens that harmful to human health.