Identifying the wall-degrading enzymes responsible for microspore release from the pollen tetrad

Abstract

Dissolution of the pollen tetrad walls after meiosis, and subsequent release of the haploid microspores, is essential for pollen development in many species, including Arabidopsis thaliana. The thick wall of the tetrad consists of callose (β-1,3-glucan), with a primary cell wall of cellulose, hemicellulose and pectin surrounding the whole structure. Despite the importance of microspore release, not all the wall-degrading enzymes involved have been identified in A. thaliana.18 endo-β-1,3-glucanase genes were identified in A. thaliana that are expressed in buds at microspore release. Single and multiple-gene T-DNA insertion lines showed no detectable phenotype, indicating possible gene redundancy. GFP-tagging of the two most specifically expressed genes (AtA6 and AtA6F) showed AtA6 is present in the locule at microspore release, but AtA6F is not. Early ectopic expression of AtA6 and AtA6F showed they have enzymatic activity, but may not be able to directly target the callose wall of the tetrad.Although endo-β-1,3-glucanases are likely to be most important for callose degradation, there are exo-β-1,3-glucanase encoding genes expressed at microspore release. Two GH3 exo-β-1,3-glucosidases are highly expressed at microspore release and so may play a role in tetrad dissolution. However, the GH5-14 exo-β-1,3-glucosidases found in most plant species are absent from A. thaliana and all other sequenced members of the Brassicales.Three genes (named the ‘QUARTET’ genes) are important for degradation of the pectin component of the outer wall. Here, analysis of double and triple quartet T-DNA insertion lines indicated redundancy between the QRT genes. Degradation of only the pectin component of the wall may be sufficient for microspore release, however, six endo-β-1,4-glucanase genes are expressed in the anther at microspore release, and may target cellulose or hemicellulose in the tetrad cell wall. T-DNA insertion line analysis showed no phenotypic differences compared to wild-type, indicating that these endo- β-1,4-glucanases may not play a major role in microspore release.

Date of Award	12 Feb 2016
Original language	English
Awarding Institution	University of Bath
Supervisor	Susan Crennell (Supervisor), James Doughty (Supervisor) & Roderick Scott (Supervisor)