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The characterisation of WHIRLY1 functions in chloroplast development

Razak, Nurhayati Binti (2019) The characterisation of WHIRLY1 functions in chloroplast development. PhD thesis, University of Leeds.

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Chloroplast biogenesis requires coordinated expression of plastome and nuclear genes. The single-stranded DNA binding protein, WHIRLY1 (WHY1), which is localised in chloroplasts and nuclei has important but poorly characterised roles in this process. WHY1 functions in barley chloroplast development were assessed in the base, middle and tip sections of two RNAi-knockdown lines (W1-1 and W1-7) with less than 5% of the wild type protein. RNA-seq analysis revealed that transcripts encoding photosynthetic proteins were highly expressed in the embryos of the dry seeds of the W1-7 compared to the wild type. The greening of the developing leaves was delayed in the WHY1-deficient seedlings relative to the wild type, with slower pigment accumulation and attainment of photosynthetic capacity in the WHY1-deficient leaves. However, the leaves of all lines reached a similar stage of chloroplast development at 14 days after germination. Transcript and metabolite profiling analysis showed changes in RNA and amino acid metabolism, TCA cycle, photosynthesis and photorespiration, particularly in the basal sections of the WHY1-deficient leaves. The expression of the plastid-encoded ribosomal genes was greatly increased in the WHY1-deficient lines, including transcripts involved in RNA processing such as pentatricopeptide repeat proteins, redox-associated proteins and transcription factors of the MYB, bHLH and WRKY families. The levels of transcripts encoding FAR1, Val-tRNA synthetase and chloroplast 50S and 30S ribosomal subunits were significantly higher in the basal sections of the W1-7 leaves than the wild type. The WHY1-deficient leaves had twice the amount of plastid DNA as the wild type. Nevertheless, plastome-encoded transcripts and proteins were significantly lower than the wild type. Conversely, the levels of nuclear-encoded photosynthetic transcripts and proteins were significantly higher that the wild type. Developing WHY1-deficient leaves showed aberrant splicing of plastid ribosomal RNAs of 23S and 4.5 ribosomal RNAs. The Arabidopsis WHY1 protein interacted with the RH22, which is required for the splicing of chloroplast rRNAs. The LEA5 protein was also shown to interact with RH22 in the chloroplasts. WHY1 therefore has multiple roles in chloroplasts. In particular, plastid-encoded ribosomal transcripts are not effectively translated into ribosomal subunits in the absence of WHY1 during early leaf development. WHY1 is required for the transcription and translation of plastome genes that are required for the transition from plastids to chloroplasts in the developing barley leaf.

Item Type: Thesis (PhD)
Academic Units: The University of Leeds > Faculty of Biological Sciences (Leeds)
The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Biology (Leeds)
Identification Number/EthosID: uk.bl.ethos.792024
Depositing User: Dr Nurhayati/N Razak
Date Deposited: 27 Nov 2019 16:54
Last Modified: 18 Feb 2020 12:51
URI: http://etheses.whiterose.ac.uk/id/eprint/25457

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