Phenotypic And Genetic Analysis Of An Abnormal Flower Development Line (AFDL) In Arabidopsis

Molecular genetic mechanisms of flower development are the core and hotspot of plant developmental biology. Currently, we have obtained a considerable knowledge on the molecular mechanisms of flower development, which are found elaborate and complex. However, there are still unknow pathways need to be discovered and studied. We obtained a mutant exhibiting an abnormal flower development (named AFDL) when screening Arabidopsis mutants (Atlg52910) associated with the development of vascular tissues. In this study, cDNA microarrays and Real-time PCR are utilized to provide insight into the changes in genes expression that occur during flower development of AFDL, in comparison to the wild-type.The results are summarized as following:1. Homo- and heterozygosity identification was performed on AFDL line, and the results showed insertional mutant of the Atlg52910 gene is inconsistent with the AFDL phenotype. The RNAi and complementation constructs of this gene were introduce into widetype and AFDL line respectively, the phenotypes of AFDL did not appear in RNAi transgenic plants, and wildtype phenotype was not observed on the transgenic AFDL plants. This further provided evidence that the phenotype of AFDL was not caused by the mutation of the Atlg52910 gene. Furthermore, the result of TAIL-PCR analysis did not find out the other Ds insertion sites, which suggested AFDL were not caused by any Ds insertional mutations. The phenotype of the AFDL were stable after multi-generation, suggesting it has a genetic basis. In addition, we had analyzed the segregation of one AFDL plant by selfing and found out that it was not caused by a single locus, due to that the ratio of the plants with abnormal and normal phenotypes deviated from Mendel's law.2. The phenotypic analysis is suggested that AFDL and ap1 mutants have more similarities. Convertion of flower meristem into inflorescence meristem, great changes of the shape of the first and second whorl flower organ, convertion of the first sepal whorl into leaf-like structures, multiple flowers towarding axillary, absence of the petal were all observed in both lines. The microarray data showed that the transcript level of AP1 is 128 times lower in AFDL flower in comparison to that in wildtype and this was further confirmed by the Real-time PCR results, indicating the AFDL had lowered AP1 expression, giving rise to the similar phenotype to apl mutant.3. AFDL and ap1 mutants also had obviously differences. (1) There is a higher level of convertion of flower meristem into inflorescence meristem in AFDL because more inflorescences and cauliflower-like flowers were observed. The phenotypes much resembled to that of ap1/cal double mutants. (2) AFDL plants appeared weak, moderate, severe phenotype according to the extents. Sometimes, there were different extents in the same line. (3) Changes of the fourth floral whorl were observed, more pronounced to that of the first and second whorl. (4) Non-deterministic meristem was out-of-order in flower development. All results indicate AFDL and the AP1 mutant is different.4. API sequence analysis showed that the promoter, coding sequences and intron splice sites of API genomic sequence in AFDL plants were the same to the wildtype. Real-time PCR results at the mRNA transcription level further indicated that API existed and expressed. These results indicated that API didn't lost in AFDL plants and the changes of its expression might be regulated by other factors.5. Later flowering phenotype were observed in AFDL plants. The Real-time PCR analysis of integrator genes in the four pathways of flowering regulation indicated that FLC and FLM over-expressed in leaf, thus it might be the reasons to cause later flowering in AFDL plants.6. The complicated phenotypes of AFDL suggest a comprehensive regulation mechanism based on the involvement of many genes in AFDL phenotype formation. The microarray data indicates a large number of genes expressed dynamically in response to internal and external environment stimuli. The down-regulation of API of AFDL may be related to its sensitive and reactive to these factors.In conclusion, the AFDL strain identified in this study is phenotypically similar to apl mutants with noticeable deviations. The abnormal flower development of AFDL was mainly caused by significantly reduced expression of API, but also is attributable to crosstalk among key genes in the regulatory network controlling the transition of vegetative growth to the flowering phase as well as flower development. AFDL exhibits variation in flowering with complex flower structures and in gene expression in response to stress conditions, thus merits further molecular characterization to understand better the regulatory molecular network. Therefore, AFDL may serve as a good model to study the complex regulation of flower development, especially interactions of regulatory genes in response to various endogenous signals and environmental cues.