shi, shuya
(2024)
Analysis of the function of Arabidopsis MYB99 in pollen formation.
PhD thesis, University of Nottingham.
Abstract
The pollen wall is an important structure which is critical for pollen formation that protects pollen from adverse conditions. The formation of the pollen wall is linked to the developmental cycle of the tapetum in Arabidopsis, with substances released from the tapetum as it degrades to complete pollen wall formation. Tapetum development is strictly regulated by a transcription factor (TF) cascade. MYB99 acts at end of the cascade and is involved in tapetum development and pollen wall formation. MYB99 is involved in the phenylpropanoid pathway in the tapetum by regulation of secondary metabolites (e.g., flavanols). However, MYB99 function is not clear as myb99 mutants do not show a defined phenotype and appear as wild type.
In this study, MYB99 mutants (myb99-1 and myb99-3) have been identified. The pollen of two null mutants showed UV sensitivity and high autofluorescence compared with wild type. Then, a dominant gain of function mutants was made for MYB99 using SRDX and VP64 systems driven by tapetum-specific high-level expression promoters (MEE48 and ATA20). Transformants generated containing the proMEE48:MYB99-SRDX and proATA20:MYB99-SRDX constructs were male sterile. To further evaluate which stage of tapetum development caused the sterile phenotype, transgenic dominant gain of function mutants was generated for MYB99 driven by tapetal specific promoters (AMS, MYB80, MS1, GRP17, A6L, and MYB99). The phenotype of the proMYB99: MYB99-SRDX transgenic line showed white and sticky pollen, which suggests that MYB99 controls pollen wall pigment associated with the phenylpropanoid pathway.
The proMYB99-MYB99-SRDX phenotype was further analyzed, showing white pollen, defective pollen wall and a rate of pollen germination that was more susceptible to UV damage than the wild type. Based on the results of RNA sequencing, luciferase assay, complementation assay, HPLC for detecting pollen coating components and FTIR for detecting the pollen exine, it appears that MYB99 regulates the glycosylation of flavonoids by UGT72E2 that encodes for coniferyl alcohol glucosyltransferase, which regulates the production of pollen wall colour. MYB99 appears to indirectly regulate GOXL4 that encodes for a galactose oxidase. MYB99 directly regulates Chalcone synthase (CHS), which controls flavonoids production in the process of pollen wall formation, regulating the production of flavonoids in the pollen wall. FTIR analysis suggests that regulation of MYB99 affects abundance of aromatic substituents in the exine.
The predicted redundant genes of MYB99 and the homologous genes of MYB99 were analyzed and it appears that MYB80, MS1, MYB20, MYB42, MYB43, and MYB85 are not functionally redundant with MYB99.
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