Investigating the role of Ycf48 in the biogenesis of Photosystem II

Abstract

Photosystem II is a large, multi-subunit pigment-protein super-complex capable of splitting water to generate protons and electrons ultimately for the reduction of carbon dioxide into sugars: and as a by-product of this reaction molecular oxygen is produced. Photosystem II is composed of more than 20 protein subunits and in addition an increasing number of protein factors have been discovered that are required for biogenesis or assembly of the photosystem; however, these assembly factors are not present in the mature complex. Light-induced water-splitting by Photosystem II results in “photodamage” and therefore the photosystem continually undergoes a self repair process. In this project the roles of five hydrophilic protein assembly factors in Photosystem II biogenesis and repair have been investigated. The protein factors investigated were: Ycf48, PsbP, Psb27, Psb28, and Psb28-2. For this study a number of knockout mutants were utilised and the following mutants: ΔYcf48, ΔPsbP, ΔPsbP:ΔYcf48, ΔPsb27, ΔPsb27:ΔYcf48, ΔPsb28, ΔPsb28:ΔYcf48, ΔPsb28-2, ΔPsb28-2:ΔYcf48, ΔPsb28:ΔPsb28-2, and ΔPsb28:ΔPsb28-2:ΔYcf48 have been compared to wild type. The ΔPsbP mutant was largely indistinguishable from wild type, with similar photoautotrophic growth and oxygen evolution rates. However, the ΔPsbP mutant did have lower levels of light-harvesting phycobilisomes and carotenoid pigments than wild type. The ΔPsbP:ΔYcf48 strain was found to have increased carotenoids and reduced chlorophyll α, suggesting an enhanced susceptibility to light stress in this strain. However, photoautotrophic growth, oxygen evolution and 77 K fluorescence emission with a 440 nm chlorophyll-specific excitation (a probe of the relative assembly levels of Photosystem II) all indicated the ΔPsbP:ΔYcf48 strain resembled the ΔYcf48 single mutant. However, using a 580 nm excitation wavelength that excites the bilin pigments in the light-harvesting phycobilisomes 77 K fluorescence emission suggested an altered phycobilisome-Photosystem II interaction in the ΔPsbP:ΔYcf48 mutant. The ΔPsb27 strain had a similar doubling time to wild type. However, the ΔPsb27 mutant's oxygen evolution rate and total photosystem level were both higher than wild type. An increased Photosystem II to Photosystem I ratio in the ΔPsb27 mutant appeared to be responsible for these differences. The ΔPsb27:ΔYcf48 strain was unable to grow photoautotrophically. The ΔPsb27:ΔYcf48 mutant had a reduced oxygen evolution rate, and reduced total levels of photosystems compared to wild type. The ΔPsb27:ΔYcf48 strain also had the increased Photosystem II to Photosystem I ratio found in the ΔPsb27 strain. Removal of either or both of the Psb28 proteins, Psb28 and Psb28-2, had minimal effects on physiological fitness. However, removal of either or both of the Psb28 proteins in combination with the removal of ΔYcf48 resulted in strains unable to grow photoautotrophically. Similar to the ΔPsb27:ΔYcf48 mutant, the ΔPsb28:ΔYcf48, ΔPsb28-2:ΔYcf48, and ΔPsb28:ΔPsb28-2:ΔYcf48 mutants had decreased total photosystem levels, and an increased Photosystem II to Photosystem I ratio. These results suggest either an increased turnover or slow repair cycle is operating in these non-photoautotrophic strains. Results are discussed with regard to what is currently known about these assembly factors and the strains in which they were constructed.