Evolución y desarrollo del sistema apertural del polen en eudicotiledóneas basalesanálisis del gen inaperturate pollen1 e identificación de genes candidatos determinantes de la morfología apertural

Resumo

The apertures of the pollen grains are areas where the pollen wall is weakened or absent and these play a major role in pollen survival and reproductive success. One of the great mysteries in the field of plant evolution and development is the genetic determinism of the enormous variety of aperture systems (shape, number, and position of apertures) in angiosperms (Furness and Rudall, 2004; Zhou and Dobritsa, 2019). In recent years, have followed several biochemical discoveries represent major advances: INAPERTURATE POLLEN 1 (INP1) protein, which is essential for aperture formation in Arabidopsis thaliana, Zea mays, and Oryza sativa (Dobritsa and Coerper, 2012; Li et al., 2018; Zhang et al., 2020); D6 PROTEIN KINASE LIKE 3 (D6PKL3), which participates in the formation and determinism of aperture number in Arabidopsis (Lee et al., 2018); and ELMOD family members, which are involved in aperture number determinism (ELMOD_A and B) and possibly aperture shape (ELMOD_E) (Zhou et al., 2021). However, whether INP1, despite its sequence diversity, conserves its function in other taxonomic groups is unknown, and the processes involved in the formation of the different apertural systems are poorly understood. Studies on the genetic determinism of apertures in the family Papaveraceae, due to its great variety of aperture systems and its phylogenetic position as one of the most basal lineages of eudicots (Wang et al., 2009), would help answer these questions. This doctoral thesis examines the involvement of INP1 in the formation of apertures in Eschscholzia californica, a member of Papaveraceae. Also, genes possibly involved in the formation of the apertures and in the determinism of their shape are investigated. In Chapter 1, a functional study is made of EcINP1 in Eschscholzia californica, and genes that could be involved in the formation of the apertures are proposed. The temporal and spatial expression of EcINP1 is analysed, and its function validated by generating mutants through virus-induced gene silencing (VIGS), while the germination rate and transcriptome of wild-type plants are compared with those of inp1 mutants. EcINP1 is maximally expressed in anthers at the tetrad stage of pollen development and its role as an essential factor for aperture formation is conserved. In Eschscholzia californica the apertures are unnecessary for pollen germination. In addition, this study found 971 differentially expressed genes (DEGs) between wild-type and EcINP1-mutants, highlighting those possibly involved in aperture formation, such as the E. californica homologues of NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1), D6 PROTEIN KINASE LIKE 3 (D6PKL3), and PROTEIN KINASE ASSOCIATED WITH BRX (PAX). These genes showed expression patterns that coincide with those of EcINP1, supporting the contention of an involvement in aperture formation and/or possible interaction of their products with the EcINP1 protein. In Chapter 2, genes that could be involved in determining aperture shape were identified. Sequencing was performed for the transcriptome of four species of Papaveraceae, three of which do not have their reference genome available, so a de novo reference assembly was generated for each species. The resulting de novo assemblies were filtered and annotated. Afterwards, the transcriptome of two species with colpate pollen was compared with that of two others having porate pollen and an analysis was made of the genes differentially expressed between the two groups. At total of 531 DEGs were found, among which ELMOD_E, the only gene described so far as a possible determinant of aperture shape (Zhou et al., 2021), was not found, implying that ELMOD_E may not regulate the change from colpo to pore in Papaveraceae. By contrast, INP1 was found to be differentially expressed. On the other hand, among the DEGs appeared genes involved in processes thought to be related to aperture formation, such as genes related to callose synthesis or degradation, including the transcription factor DYSFUNCTIONAL TAPETUM 1 (DYT1) or to the organization of cytoskeletal elements. References Dobritsa AA, Coerper D. 2012. The novel plant protein INAPERTURATE POLLEN1 marks distinct cellular domains and controls formation of apertures in the Arabidopsis pollen exine. The Plant Cell 24: 4452-4464. Furness CA, Rudall PJ. 2004. Pollen aperture evolution–a crucial factor for eudicot success?. Trends in plant science 9: 154-158. Lee BH, Weber ZT, Zourelidou M, et al. 2018. Arabidopsis protein kinase D6PKL3 is involved in the formation of distinct plasma membrane aperture domains on the pollen surface. The Plant Cell 30: 2038-2056. Li P, Ben-Menni Schuler S, Reeder SH, Wang R, Suárez Santiago VN, Dobritsa AA. 2018. INP1 involvement in pollen aperture formation is evolutionarily conserved and may require species-specific partners. Journal of Experimental Botany 69: 983-996. Wang W, Lu AM, Ren Y, Endress ME, Chen ZD. 2009. Phylogeny and classification of Ranunculales: evidence from four molecular loci and morphological data. Perspectives in Plant Ecology, Evolution and Systematics 11: 81-110. Zhang X, Zhao G, Tan Q. 2020. Rice pollen aperture formation is regulated by the interplay between OsINP1 and OsDAF1. Nature Plants 6: 394-403. Zhou Y, Dobritsa AA. 2019. Formation of aperture sites on the pollen surface as a model for development of distinct cellular domains. Plant Science 288: 110222. Zhou Y, Amom P, Reeder SH, Lee BH, Helton A, Dobritsa AA. 2021. Members of the ELMOD protein family specify formation of distinct aperture domains on the Arabidopsis pollen surface. Elife 10: e71061.