Plants possess the ability to generate new leaves continuously throughout their entire lifespan due to the persistent activity of stem cells in vegetative meristems. During the reproductive phase, the floral meristem (FM) gives rise to all of the floral organ primordia in sequential whorls or spirals, which will further develop into mature organs that compose the flower. Distinct from the vegetative meristem, however, the stem cell activity of the FM will be terminated at a specific time point during primordium initiation, since each flower only needs a finite number of floral organs.
Floral meristem termination (FMT)
is a well-regulated process that ensures the correct number of healthy primordia are produced in a flower, and variation in the timing of FMT provides one of the most important sources of generating floral morphological diversity.
* Organs of the same whorl are represented by the same color. (white=sepals, pink=petals, grey=stamens, blue=carpels).
Surprisingly, how the timing of FMT is fine-tuned at a developmental and evolutionary level is still very poorly understood.
Almost all of our knowledge of FM proliferation has been gained in the context of artificial selection (e.g. in agricultural crops), which emphasizes dramatic allelic effects; mutations in these candidate genes usually lead to a total cessation or massive over-proliferation of stem cell activity. Our understanding of the regulation of FM proliferation is hindered by the fact that all current established model systems (and their close relatives) invariably have only four whorls of floral organs, providing no ideal starting point to investigate the natural variation in the timing of FMT. Nonetheless, flowers with only four whorls of organs do not represent the floral morphological diversity we see in nature.
Under these circumstances, Aquilegia is a well-suited system for investigating FMT. Aquilegia species have both a high degree of sequence similarity and interfertility, and their flowers have identical numbers of all floral organs except for stamens.
In order to develop crucial tools and database, I’m taking four approaches to understand FMT in Aquilegia at a cellular, molecular, transcriptomic, and genomic levels:
1. How do the parameters of cell division/expansion change during FMT in Aquilegia?
This project is a collaboration with Dr. Stephanie Conway and supported by the Emerging Research Organisms Grant from the Society of Developmental Biology.
To fully characterize cellular behavior during FMT in real time at high resolution, we have developed a live confocal microscopy protocol. We are now able to remove an early stage FM from a plant, grow it on a culture dish, and image it every 24-36 hours for many days until FMT occurs. We are able to track cell divisions that occurs in the FM during this window, and quantify the orientation, rate, and spatial pattern of cell divisions and expansion using MorphoGraphX.
This project will help me to fully capture the dynamic cellular process during FMT and understand its underlying patterns. The techniques we developed for this project can also be applied to experiments such as tracking gene expression in future studies.
2. Candidate gene approach: are previously identified genetic pathways involved in FMT in Aquilegia?
Previous studies in Arabidopsis thaliana have characterized a genetic pathway that contribute to FMT. Particularly, the elegant timer model of the AG-KNU-WUS pathway ensured there are only four whorls of organs that are produced in an Arabidopsis flower.
However, flowers with only four whorls comprise a very small portion of floral morphological diversity in nature. For instance, organ whorl numbers vary from 11 to 20 in Aquilegia flowers.
In order to assess the conservation of the A. thaliana pathway, I conducted functional studies of relevant candidate genes using virus-induced gene silencing (VIGS) in Aquilegia coerulea. My results suggest that this pathway is not conserved in Aquilegia and I am currently in the progress of characterizing the novel functions of these genes in floral development.
3. How do the transcriptional dynamics change before, during, and after FMT in Aquilegia?
In order to identify Aquilegia genes that are involved in the early floral development, including the process of FMT, I have extracted RNA from FMs of A. coerulea, which has a well annotated genome sequence, at four different developmental stages, with 8 biological replicates per stage.
With the help of differential gene expression analysis and the weighted gene co-expression network analysis, this project will produce a powerful dataset that will enable me to genome-wide analysis of transcriptional dynamics across the entire process of FMT, identifying genes and co-expressed genetic modules that are differentially expressed at key points in the FMT process.
4. What are the genomic regions controlling the natural variation in Aquilegia FMT?
A. canadensis and A. brevistyla are two sister species in North America that are both stunningly beautiful. There are a number of floral traits vary between them, and one of them being that their flowers have identical numbers of all floral organs except for stamens. Therefore, variation in the timing of FMT can be well represented by the variation of stamen whorl numbers in Aquilegia flowers.
Our collaborator Dr. Evangeline Ballerini crossed these two species as parents, self-pollinated the F1, and generated a beautiful F2 population. In total, I counted the stamen whorl numbers of 4265 flowers of 364 F2 individuals, 357 flowers of 27 A. brevistyla individuals, and 197 flowers of 16 A. canadensis individuals, and their mean stamen whorl numbers are 8.05, 7.16, and 9.15, respectively. I have detected five major QTL on chromosomes 2, 3, 4, 5 and 6 that contribute to stamen whorl variation, with the QTL on chromosome 4 exhibiting the highest LOD score of 11.8. By combining these results with those from the transcriptomics project, I’m hoping to identify critical genes that are controlling stamen whorl number, which is the proxy for variation in FMT.