How the pigment stripes form in snapdragon (Antirrhinum majus) flowers : a study of the molecular mechanism of venation pigmentation patterning in flowers : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Molecular Biology at Massey University, Palmerston North, New Zealand

Floral stripes are a common pigmentation pattern in plants. Defining the molecular mechanisms of the striped pattern formation will aid understanding of how a gene can be differentially regulated across a population of similar cells. In the venation phenotype of Antirrhinum majus, the anthocyanin pigment is typically confined to the adaxial epidermal cells overlaying the petal veins. To explore how this pattern forms this study focused on the expression and regulation of Venosa, a Myb regulator of anthocyanin biosynthesis. Pigment complementation experiments demonstrated that the lack of a MYB factor caused the lack of pigment in the cells outside the venation pigmentation domain. An allele of Venosa was isolated and identified. It was a mutant version of functional Venosa due to the central part being replaced by a transposon. Phenotype / genotype analysis indicated that the venation pigmentation patterning was due to the functional Venosa. In situ mRNA hybridisation showed that Venosa was expressed from the xylem to the adaxial epidermis, and was controlled spatially and quantitatively by a signal associated with the petal veins. Venosa expression provided the longitudinal axis for venation pigmentation stripes, and determined the location and intensity of the pigmented cells. Because another factor required for pigmentation, a bHLH factor, is specifically expressed in epidermal cells and it provides the transverse axis. The pigmented stripes are the cross expression domain of these two kinds of factors. The transcriptional controlling property of a 2.4 kb (relative to the ATG) promoter region of the Venosa gene was analysed. The -900 bp fragment was characterised in detail using 5'-end deletion mutagenesis. A heterologous host, tobacco, was used for analysis in stable transgenics. The homologous host, Antirrhinum, was used for transient assays. The efficacy and efficiency of different reporter genes (intron-containing GUS, GFP, Venosa cDNA and genomic Venosa) and enhancement systems (transcriptional enhancer, translational enhancer, inhibitor of post transcriptional gene silencing and a two-step signaling amplification system) for the detection of low-level reporter gene expression were also tested. The strength of expression correlated to the length of the promoter fragment, and expression was detected using deletions down to -500 bp, although only weak expression was found. This expression was flower specific but not vein related in both plant hosts. No expression was detected in petals of either host with fragments shorter than -500 bp. The results suggest that the fragment from -380 bp to -900 bp positively affected Venosa expression at the transcriptional level, but might not be sufficient to define venation. A possibility is that the venation controlling property is negatively controlled at the epigenetic level, such as DNA methylation status and / or chromatin structure. The role of gibberellin and sugar in the pigment and venation patterning formation of Antirrhinum was studied. The results suggest that gibberellin is not required for pigmentation or venation patterning. Convincing evidence on the role of sugar signaling could not be obtained from the experiments, due to the difficulty in separating the impact on pigmentation from other functions of sugars in petal development. In addition, the in situ analysis detected the expression of a gene probably related to aurone biosynthesis that may be a regulatory gene of this biosynthetic pathway

The Vector Construction of Anthocyanins as a Visual Marker Gene and Its Transient Expression in Maize Immature Embryos

采用花青素调控因子C1/Bperu分别与玉米胚特异性启动子Glb1和组成型启动子CaMV35S构建成植物转化载体pGlb1CB和p35SCB，并利用基因枪转化方法，将重组表达载体转入玉米幼胚。显微观察结果证实，这两个载体均能在玉米幼胚细胞中瞬时表达。用花青素作为标记基因不仅可以在一定程度上减少公众对转基因生物安全性方面的担忧，而且可以帮助直观地从转化当代和后代种子中通过颜色标记筛选到转化籽粒，从而可以大大简化筛选程序，提高效率，节约检测成本

A Small Family of MYB-Regulatory Genes Controls Floral Pigmentation Intensity and Patterning in the Genus Antirrhinum

The Rosea1, Rosea2, and Venosa genes encode MYB-related transcription factors active in the flowers of Antirrhinum majus. Analysis of mutant phenotypes shows that these genes control the intensity and pattern of magenta anthocyanin pigmentation in flowers. Despite the structural similarity of these regulatory proteins, they influence the expression of target genes encoding the enzymes of anthocyanin biosynthesis with different specificities. Consequently, they are not equivalent biochemically in their activities. Different species of the genus Antirrhinum, native to Spain and Portugal, show striking differences in their patterns and intensities of floral pigmentation. Differences in anthocyanin pigmentation between at least six species are attributable to variations in the activity of the Rosea and Venosa loci. Set in the context of our understanding of the regulation of anthocyanin production in other genera, the activity of MYB-related genes is probably a primary cause of natural variation in anthocyanin pigmentation in plants

Methods for transient assay of gene function in floral tissues

<p>Abstract</p> <p>Background</p> <p>There is considerable interest in rapid assays or screening systems for assigning gene function. However, analysis of gene function in the flowers of some species is restricted due to the difficulty of producing stably transformed transgenic plants. As a result, experimental approaches based on transient gene expression assays are frequently used. Biolistics has long been used for transient over-expression of genes of interest, but has not been exploited for gene silencing studies. <it>Agrobacterium</it>-infiltration has also been used, but the focus primarily has been on the transient transformation of leaf tissue.</p> <p>Results</p> <p>Two constructs, one expressing an inverted repeat of the <it>Antirrhinum majus </it>(Antirrhinum) chalcone synthase gene (<it>CHS</it>) and the other an inverted repeat of the Antirrhinum transcription factor gene <it>Rosea1</it>, were shown to effectively induce <it>CHS </it>and <it>Rosea1 </it>gene silencing, respectively, when introduced biolistically into petal tissue of Antirrhinum flowers developing <it>in vitro</it>. A high-throughput vector expressing the Antirrhinum <it>CHS </it>gene attached to an inverted repeat of the <it>nos </it>terminator was also shown to be effective. Silencing spread systemically to create large zones of petal tissue lacking pigmentation, with transmission of the silenced state spreading both laterally within the affected epidermal cell layer and into lower cell layers, including the epidermis of the other petal surface. Transient <it>Agrobacterium</it>-mediated transformation of petal tissue of tobacco and petunia flowers <it>in situ </it>or detached was also achieved, using expression of the reporter genes <it>GUS </it>and <it>GFP </it>to visualise transgene expression.</p> <p>Conclusion</p> <p>We demonstrate the feasibility of using biolistics-based transient RNAi, and transient transformation of petal tissue via <it>Agrobacterium </it>infiltration to study gene function in petals. We have also produced a vector for high throughput gene silencing studies, incorporating the option of using T-A cloning to insert the gene sequence of interest. These techniques should allow analysis of gene function in a much broader range of flower species.</p