| Flowers consist of four concentric whorls of organs from center to edge:pistils, stamens, petals, and sepals. Precisely coordinated growth of stamens and pistils determines the fertility. Stamen development is involved in a series of pectin degradation events, which needs the participation of an important pectin digesting emzyme, namely polygalacturonases (PGs). PGs have been shown to take part in lots of processes in plant development, including fruit ripening, organ abcission and pollen maturation. PGs belong to one of the most largest hydrolase gene family. However, so far, there’s no consensus on a scientific classification of plant PG genes, and there’s still lack of knowledge on what mechanism contributes to the differences in PG family structure between grasses and dicots. Although it has been suggested that hormones can regulate stamen development and that expression of PG genes can also be regulated by hormones in other tissues, few researches focused on exploring whether hormones can regulate PG genes’expression during stamen development. In addition, researches that have characterized PGs’biological functions in plant development are still limited. Therefore, the evolution of PG family members in Brassica campestris (syn. Brassica rapa) was analyzed and a comparative analysis of evolution, expression and regulatory elements of PGs in grasses and dicots was made. Furthermore, PG genes associated with stamen development of Arabidopsis were screened and the influence of hormones on these PG genes were explored. Finally, the expression and biological functions of two duplicated PG genes, namely BrPG22-1 and BrPG22-2, which were potential genes involved in stamen development in B. campestris, were analyzed. Results were summarized as follows:(1) Comprehensive analysis of phylogeny, gene structures, physico-chemical properties and coding sequence evolution demonstrated that plant PGs should be classified into seven divergent clades and each clade’s members had specific sequence and structure characteristics, and/or were under specific selection pressures. Genomic distribution and retention rate analysis implied duplication events and biased retention contributed to PG family’s expansion. Promoter divergence analysis using "shared motif method" revealed a significant correlation between regulatory and coding sequence evolution of PGs, and proved Clades A and E were of ancient origin. Quantitative real-time PCR analysis showed that expression patterns of PGs displayed group specificities in Brassica rapa. Particularly, nearly half of PG family members, especially those of Clades C, D and F, closely relates to reproductive development. Most duplicates showed similar expression profiles, suggesting dosage constraints accounted for preservation after duplication. Promoter-GUS assay further indicated PGs’extensive roles and possible redundancy during reproductive development.(2) A comprehensive analysis of the evolution, expression and cis-regulatory element of PGs in grasses and dicots was made. A total of 577 PGs identified from five grasses and five dicots fell into seven clades. Evolutionary analysis demonstrated the distinct differences between grasses and dicots in patterns of gene duplication and loss, and evolutionary rates. Grasses generally contained much fewer Clades C and F members than dicots. This disparity was the result of less duplications and more gene losses in grasses. More duplications occurred in Clades D and E, and expression analysis showed that most of Clade E members were expressed ubiquitously at a high overall level and Clade D members were closely related to male reproduction in both grasses and dicots, suggesting their biological functions were highly conserved across species. In addition to the general role in reproductive development, PGs of Clades C and F specifically played roles in root development in dicots, shedding light on organ differentiation between the two groups of plants. Regulatory element analysis of Clades C and F members implied that possible functions of PGs in specific biological responses contributed to their expansion and preservation. This work can improve the knowledge of PGs in plants generally and in grasses specifically, and is benefical to functional studies.(3) A total of 14 PG genes were identified as members associated with stamen development according to the microarray data of Arabidopsis thaliana. Results of phylogenetic analysis demonstrated that most of these genes belonged to Clade D. Promoter-GUS assay further showed the expression dynamics of these PG genes during stamen development. It was found that most of these genes were expressed in tapetum and mature pollens. Using GA3 and NAA to treat the seedlings of the transgenic Arabidopsis plants with PG promoter-GUS fusion vectors, results demonstrated that GA and Auxin can control the expression of PG genes associated with stamen development.(4) Based on the genomic information from the BRAD database, the DNA and cDNA sequences of BrPG22-1 and BrPG22-2 were cloned. The amino acid sequence identity of BrPG22-1 and BrPG22-2 was as high as 92.0%. Results from transmembrane structure prediction and subcellular localization analysis showed that both the PGs were secretory proteins. Expression profiles of BrPG22-1 and BrPG22-2 in B. campestris were further investigated by RT-PCR and promoter-GUS assay. Results showed that the two genes were both highly expressed in stamen at early stages, supporting their roles in stamen development, BrPG22-1 was further expressed in filaments and ovules, indicating that it had undergone neofunctionali-zation. The functional interruption of BrPG22-1 and BrPG22-2 by antisense RNA technology had no effect on the morphology, suggesting that there might be functional redundancy from other PG family members. |
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